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ANIMAL TISSUE TECHNIQUES
A Series of Biology Books
EDITORS

George W. Beadle (1946-1961), Ralph Emerson, Douglas M. Whitaker

Laboratory Studies in Biology:

Observations and their Implications
Chester A. Lawson, Ralph W. Lewis,
Mary Alice Burmester, Garrett Hardin

Experiments in General Biology

Graham DuShane, David Regnery

General Genetics
Adrian M. Srb, Ray D. Owen

Principles of Human Genetics

(Second Edition)
Curt Stern

An Introduction to Bacterial Physiology

(Second Edition)
Evelyn L. Oginsky, Wayne W. Umbreit

Principles of Plant Physiology

James Bonner, Arthur W. Galston

Plants in Action: A Laboratory

Manual of Plant Physiology
Leonard Machlis, John G. Torrey

Comparative Morphology of Vascular Plants

Adriance S. Foster, Ernest M. Gifford, Jr.

Taxonomy of Flowering Plants

C. L. Porter

Biology: Its Principles and Implications

Garrett Hardin

Growth, Development, and Pattern

N. J. Berrill

Animal Tissue Techniques

Gretchen L. Humason
ANIMAL
TISSUE
TECHNIQUES
Gretchen L. Humason
LOS ALAMOS SCIENTIFIC LABORATORIES

= PY
SAN FRANCISCO IE = .
AND LONDON E

W. H. FREEMAN AND COMPANY

© Copyright 1962 by W. H. Freeman and Company

The publisher reserves all rights to reproduce this book in

whole or in part, with the exception of the right to use short
quotations for review of the book.

Printed in the United States of America

Library of Congress Catalog Card Number: 61—17383 (C2)

Preface

In the area of histological studies the requirements for a useful

laboratory handbook and textbook are both diverse and exceedingly
complex. The needs of beginning students in premedical courses, of
medical technicians in training, of zoology majors, and of other stu-
dents are similar but not identical. The needs of research assistants
and of experienced tissue technicians at work in the field resemble,
but are not identical with, the needs of the students. ‘The author has
tried to keep these diversities in mind while pursuing her two major
goals: (1) to familiarize the student with basic—standard—procedures
and introduce him to specialized technics, and (2) to furnish the work-
ing technician with a prop during the first job and an occasional
reference point in later work, until consulting specialized references
and journals has become a habit.
Basic procedures are applicable to both normal and _ pathological
tissues in zoological and medical fields. Because most histological re-
actions follow a logical and specific sequence, a simplified discussion
of principles has been written for many of the technics. Then perhaps
an understanding of each step can prevent mistakes during first trials
of a method. There are an infinite number of problems and blunders
which can plague a tissue technician. Suggestions concerning such
stumbling blocks and some of their remedies may forewarn the novice
and be of assistance to others.
This book is not intended to form a complete “reference”; the
experienced worker knows of numerous such tomes as well as journals
that specialize in biological subjects. However, special methods of
wide usage and exceptional merit are included, particularly those
\
vi Preface

methods that are not overly complicated or unpredictable. It is hoped

that the technician will watch the literature for modifications and im-
provements of “standard” technics; in this way his work can be kept
up to date and perhaps simplified. Methods for fixation are fairly well
established, and only occasional variations appear from time to time.
The section on fixation presented here is as modern as the author can
make it, and it includes a brief description of the chemicals employed.
The perfecting of old staining technics, as well as the development
of new ones, continues to appear in the literature; an attempt has been
made to incorporate the latest procedures and modifications in this
book. The discussion of fixation and its solutions and of dyes and their
action is given for the benefit of those who cannot, or will not, seek out
the source material.
Certain important fields are not covered in this book either because
they are not widely used methods or because they are considered too
highly specialized. A few of these fields are: vital staining—the selec-
tive dyeing of certain isolated cells while alive; tissue transplantation
—transferring a tissue from its normal location to a different one,
where it can be observed more easily; tissue culture methods—growing
cells in artificial habitats; tracer technics—using radioactive isotopes
which can be traced in the body; and microincineration—the study
of minerals in the tissue. However, three fields of increasing popu-
larity and importance are introduced—autoradiography, histochemical
methods, and electron microscopy. ‘The coverage on these three sub-
jects is held to a minimum, but appended references and bibliography
can help a technician find more extensive source material.
A logical arrangement of special staining methods is hard to come
by, so the author has followed her own inclinations. Some sections
are organized by related tissues, others by related methods. The latter
was considered desirable for such processes as silver impregnation,
metachromasia, and methods using Schiff’s reagent. It has been im-
possible to draw a hard and fast line, but it is hoped that cross-
referencing and indexing will provide the answers. The author has
had personal experience with most of the methods, and has also tried
to include modifications which might appeal to other adventurous
technicians.
The book is divided into four parts: 7. General procedures and con-
siderations which every tissue technician should know and understand.
This part will be of particular value in a beginners’ course; I7. Special
staining methods for most tissues. The idea here is that an instructor
can choose a few favorites for teaching purposes to round out a course,
Preface Vil

while the professional technician can find most of the special methods
required on the job; ///. Special procedures, special in the sense that
they are not common to every lab, but are important to many. Dis-
cussion is far from complete for many of them, but references are
appended. JV. Laboratory aids, general information handy to have in
any lab. The list of references includes those occurring in the text
and others whose titles may lead an occasional technician to otherwise
undiscovered source material.

Acknowledgments
An invaluable personal satisfaction has been derived by the author
from her association with students, no matter what their caliber. Stu-
dents force a teacher to develop tolerance and patience, qualities
essential in this temperamental profession. Students also are a blessing,
because there is no surer way to master a subject than to teach it.
One student in particular should receive credit for her encouragement
and her prodding toward this book—Miss Marlies Natzler of the Uni-
versity of California at Los Angeles.
Acknowledgment is due the artist, Marvin Linke, for his quick
understanding of the subjects he was asked to illustrate.
Gratitude is extended to the Zoology Department of the University
of California at Los Angeles for the lessons the author learned while
a student, a departmental technician, and a lecturer, and to Dr. C. C.
Lushbaugh of Los Alamos for guidance in pathology.

August 1961 Gretchen L. Humason

 Standard References in Histology

Every technician should have at least a nodding

acquaintance with the following text and reference books.

BAKER, J. R., Principles of Biological Mi- GuRR, EpwArRD, A Practical Manual of

crotechnique, Methuen and Company, Medical and Biological Staining Tech-
London, 1958. niques, Interscience Publishers, N.Y.,
1956.
Conn, H. J., Biological Stains, Biotech
Publications, Geneva, N.Y., 1953.
, Methods of Analytical Histology
and Histochemistry, Leonard Hill, Ltd.,
, DARROW, MARY, and EMMEL,
Victor, Staining Procedures, The Wil- London, 1958.
liams and Wilkins Company, Baltimore, , Encyclopaedia of Microscopic
1960. Stains, Edward Gurr, London, 1960.
Cowpry, E. V., Laboratory Technique in KRAJIAN, ARAM A., and GRADWOHL,
Biology and Medicine, The Williams and R. B. H., Histopathological Technic,
Wilkins Company, Baltimore, 1952. C. V. Mosby Company, St. Louis, 1952.
CuLuLinc, C. F. A., Handbook of Histo-
pathological Technique, Butterworth Littie, R. D., Histopathologic Technic
and Company, London, 1957. and Practical Histochemistry, Blakiston
Company, 1954.
DAVENPORT, H. A., Histological and
Histochemical Techniques, W. B. Saun- McManus, J. F. A. and Mowry,
ders and Company, Philadelphia, 1960. ROBERT W., Staining Methods: Histo-
logic and Histochemical, Paul B. Hoeber,
Guick, DAvip, Techniques of Histo- and
1960.
Cytochemistry, Interscience Publishers,
NGY., 11949. MALLORY, FRANK B., Pathological Tech-
nique, W. B. Saunders Company, 1944.
GoMor!I,» GEORGE, Microscopic Histo-
chemistry, University of Chicago Press, PEARSE, A. G. E., Histochemistry, Theo-
N52: retical and Applied, Little Brown and
GRIDLEY, MARY FRANCES, Manual of
Company, Boston, 1960.
Histologic and Special Staining Technics,
Armed Forces Institute of Pathology, Romeis, B., Mikrosckopische Technick.,
Washington D.C., 1957. Leibniz Verlag, Miinchen, 1948.
 Contents

PART I. BASIC PROCEDURES

Chapter l.Fixation 9. .0 6 Bee ws we Oe 3

Chemicals Commonly Used in Fixing Solutions... 5
Maceration (a prefixation process) . . . . . . . +10
Maxineytae Wissue 2 -2 a eee we me a e Ul CD
Washine the, Pissue : 2 2 2 “31 3 2 » « =» « WU
Fixing Solutions. and ‘Their Uses> . 2@ 2 «;2 = = ts
Fixation, oy, Rertusion =~ >) a GMs «= 2 2 ae 22
Post-lixation: Treatments. © 6. + Wigeea s,s Ren “22
Decalewication:. 6, « . <ofa Res. e 2 ae lees

Ghapier 2, Dehydration . g. 2 «» .< Ma -» 2 BY, “ot

Preparation for Embedding . . . . . . . . . ~~ 30
Special Treatment for Small, Colorless Tissues . . 32

Chapter 3. Clearing, Infiltrating, and Embedding for Paraffin

Method |. . . cca) Se 3 ee OS
Cleariae® es es ee eC
Infiltration with Paraffin. . . . . . . . ... 35
Embedding with Paraffin. . Sy : ae 36
Embedding Cellular Contents of Body Plaids)...
1 = 38
Dehydration and Clearing Combinations . . . . . 39
dimuns schedule’, 2 2 « 2 Y ose = 2 = al
Automatic issue Processors.. 2 2940 1. «+ «= | «& 42

Chapter 4. Microtomes and Microtome Knives . |... .— 43

Microtomes Ae. e 2.0 Are er eee,” Zeke
Microtome Knives i: See nk 2. Meee
1X

SOIRG
 Contents

Chapter 5. Paraffin Method 5]

Sectioning 5]
Mounting 56

Chapter 6. Freezing Method 62

Fixation, Blocking, and Sectioning .
Mounting
Staining

Chapter 7. Nitrocellulose Method |

Dehydration and Infiltration .
Embedding
Sectioning hte
Hot Celloidin ‘Technic
Difficulties in Nitrocellulose Sectioning .
Staining and Mounting
Mounting Before Staining

Chapter 8. Specialized Embedding Technics

Water Soluble Wax Embedding and Sectioning
Double Embedding
Polyester Embedding .

Chapter 9. The Microscope |

The Compound Microscope .
The Operation of a Microscope . oil
Measuring Devices Used on a Microscope . OF
Specialized Microscopy 96

Chapter 10. Stains and Staining 104

Stains and Their Staining Action 104

Contents X1

Natural Dyes 105

Mordants 107
Synthetic Dyes . 109
Nature of Staining Action 11]
Standardization of Stains . Bh

Chapter 11. Staining Procedures 114

Cover Glass Mounting jieee
Mounting Media (mountants) 116
Aqueous Mounting Technics a

Chapter 12. Hematoxylin Staining 124

Single Solutions 124
Double Solutions 126
Double Solutions Never Mixed Before Use | eae)
Substitutes for Hematoxylin Solutions 128
Counterstains for Hematoxylin, Gallocyanin, and
Hematein 129
Hematoxylin Staining Procedures 130

PART II. SPECIFIC STAINING METHODS

Chapter 13. Connective Tissue |

Mallory Staining
Trichrome Staining
Picro-Ponceau Staining
Elastin Tissue Staining
Subcutaneous Tissue Staining
Bone Staining

Chapter 14. Silver Impregnation I |

Recticulum Technics .
X11 Contents

Chapter Ibs'Stlwen Tmpresnation IIe (ss | ee

Neusplosical Staining. «GS 2 a See ee
PSEROCVOES 4.8 A eg oe eee
Olisodendroglia and Microglia, =. . 2 gauss -. USE
Nissi" Siurbstamcey’ 2 «ee es eee, We
Neurofibrils? Sit. ile > RS Ee Ane
Nerve-GCelisandi Fibers: «.° ak, =. 2” See ele
Nerve Fibers; and Endings. Gaye) -. i242. a eee ec
Pericellular Pnd-Bulbs: .- 9a. 2. 2 0 2 eee UO
Desencrarme “xons "9, 8. ee
Motor ud 'Plates 7) 09 eg Ce a uc, e ee
Myebianemt 4 ee e See ce) lee

Chapter 16. Hematologic Elements and Related Tissues . . 218

RicodySmears* ) (2 .geeees ede, sec
Blood Tissue Elements and Inclusion Bodies. . . . 224
BonesyMarrow Stamimgiyt. oe. el,
Siainimegior Fibritiy gy. e) <2) so pt ke

Ghapter 1/7. "Pigments and Witnerals ..- . 3) (2 )) eeeeeeeee

Testmepiorslron see Ce 2) DADE 2 ). See oo
essmestor Galciuinsggpta: <a 9. se eee
lemoglobin Staming eee. . : 9. /s %)s15 aaeerZoo
Bile wiament stainiie sees. 2h) 2 e e
Miclanin’ and Lipofuscmystainine 2°) ee ee
Removal Ob Pisments: “ga .6 2. e ee L

Chapter Ls. Cytoplasmic Elemenisa:. )2 eo eee 240

Protem™ staming’ .°. voy. . . | eee 246
The Argentafin Reaction... ~ 4p ge eee 7
Fatstaming 1 \atov boty gr a, R PRIS
Golet Apparatus and Mitochondria, see Gee «6-299
Contents

Saccharides (carbohydrates)
Mucin Staining
Amyloid Staining .
Mast Cell Staining
Metachromasia
Pituitary and Pancreas

Chapter 19. Feulgen and PAS Technics and Related Reactions

Feulgen Reaction .
Periodic Acid-Schiff Reaction
Glycogen
Desoxyribonucleic Acid, Polysaccharides, and Proteins

Chapter 20. Microorganisms

Bacteria Staining
Fungi Staining .
Rickettsia Staining
Antigen Antibodies

PART III. HISTOCHEMISTRY AND

MISCELLANEOUS SPECIAL PROCEDURES

Chapter 21. Histochemistry |

Freezing-drying Method for Embedding
Ice Solvent Method
Acetone Fixation and Embedding
Alkaline Phosphatase .
Acid Phosphatase .
Amiunopeptidase
Esterases and Lipases .
Succinic Dehydrogenase
Dopa Oxidase Reaction
Arginine Test .
Mounting Media
XIV Contents

Chapter 22. Special Procedures? <- .OR Pe P e e

Extohative:Gytology™ <.-2 foe. awh So. = ee
SexeChromatin: .- <° £ geaghei *. Weuleeeaes 2 = aeue
Ghromosomes y ML ROR a. ee ea 2 Sn ee

Grapier 23. Special Procedures 1) Veet. so th eee. ail

Preparation of Invertebrates for Whole Mounts and
SECHGHS 4 as) Reet. <: 5 BAM ee en eee
PrepatationetiChick Embryds eg)... ee eee oe
Whole Mounts 625...) ome eee a) | ee ore
AvmaaliRanasites: “<5 2 Cee Ou

Chapter 2huspecial ProceduresT |

Eilmeounts 9 \ eet = 2 6 4 Ja oo
Cellulose “Tape Mountme . . -. « =, . |) = gameaod
PiastievViOumts . 76ers 29 es
MutoMadiography =. e6 . « «oe on, ole go
BlectromevicrOscopymes =. 5) 2 A ee ee

PART IV. SOLUTION PREPARATION

AND GENERAL LABORATORY AIDS

Chapter 25. Solution Prepanaion. 1) ony, Oe ae 407

Abbreviations and ‘Venmase 9. . Pe 7 ee ey
Stock Solutions RRO. OF: COTA LSI Rs eet hoe
Adhesive Solutions “Syme... 2. Sa MIENDPRPA RO ee ice te
Buiter Soluttiows’ : sae) . ) Sh CEEANREY Ree ele
Sidi Solubilities: <= i aeee. 3 fee

Chapter 26. ‘General LaboraionyeAids . 20. i ae ee | oe

Labeling and Cleaning Slides _. 1 sti) ea: «28
Restammine Faded Slides; {) 5... . ae geeiaee = 424
Two Different Stains on One Slide 1 WE) sie =D
Contents XV

Reclaimingo and Storingo Specimens 426

Preserving Gross Specimens in a Pliable Condition 427
Removing Laboratory Stains from Hands and Glassware 428

References 4sSs)I

Index 458
 ined
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7 pier yi! th) 4)
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 |
“er BASIC
PROCEDURES
|
|

|
 Chapter |

Fixation

When a tissue is removed from a living condition, several changes are

initiated in its cells. Bacteria begin to multiply and destroy them. Also
autolysis (self-digestion), a lysis or dissolution of the cells by contained
enzymes, sets in. The enzymes appear to reverse their action; instead of
synthesizing amino acids into cell proteins, they begin to split proteins
into amino acids. ‘These amino acids diffuse out of the cells, and as a re-
sult proteins no longer are coagulable by chemical reagents. The above
changes lead to so-called post-mortem conditions. For purposes of lab-
oratory examination it is necessary to treat the tissue to prevent these
post-mortem effects; it is also necessary to convert the cell parts into
materials that will remain insoluble during subsequent treatment, and
to protect the cells from distortion and shrinkage when subjected to
fluids such as alcohols and hot paraffin. Further important objectives
of tissue preparation are to improve the staining potential of tissue
parts and to change their refractive indices toward improved visibility.
The procedure used to meet the above requirements is called fixation
and the fluids are called fixatives or fixing solutions. Fixing solutions
should meet the following principal objectives:
Penetrate rapidly to prevent post-mortem changes.
Coagulate cell contents into insoluble substances.
=
No
oo Protect tissue against shrinkage and distortion during dehydration,
embedding and sectioning.
Allow cell parts to become selectively and clearly visible by means
of dyes and improved refractive indices.
4 Fixation (CHAP. 1)

In some cases the fixative may have a mordanting (combining insolu-

bly) effect on the tissue, thereby bringing the two together in a staining
action and assisting in the attachment of dyes and proteins to each other.
Tissues must be placed in fixatives as soon as possible after death. If,
however, delay is unavoidable, they should be placed in a refrigerator,
thus reducing autolysis and putrefaction to a minimum until the fixa-
tive can be applied.
A single chemical seldom possesses all of the requisite qualities of a
good fixative; a fixing solution therefore is rarely composed of only one
chemical. A familiar exception is formalin. Other reliable fixatives con-
tain one or more chemicals to coagulate the proteins of the cells, and
one or more chemicals to render the proteins insoluble without actu-
ally coagulating them. Coagulant fixatives change the spongework of
proteins into meshes through which paraffin can easily pass, thus form-
ing a tissue of proper consistency for sectioning. In addition, the pro-
tein linkages are strengthened by coagulants against breaking down
during later procedures. Used alone, however, coagulants have disadvan-
tages in that they may form too coarse a network for the best cytological
detail. Also, coagulation tends to induce the formation of artificial
structures (artifacts). Noncoagulant fixatives produce fewer artifacts
but if used alone have the disadvantage of giving the tissue a poor con-
sistency for embedding. It therefore follows that the ideal fixing fluid is
a combination of one or more protein coagulants and one or more non-
coagulants. The most efficient solutions are of this nature, and combine
if possible all types of action. This excludes those designed for the fixa-
tion of some specific cell component, such as chromosomes, glycogen,
mitochondria.
Because they contain ingredients which act upon each other, many
mixtures are most efficient when made up fresh. However, the individ-
ual parts usually can be made up as stock solutions and mixed together
immediately before use. Among the frequently used chemicals are form-
aldehyde, ethyl alcohol, acetic acid, picric acid, potassium dichromate,
mercuric chloride, chromic acid and osmium tetroxide. Since every
chemical possesses its own set of advantages and disadvantages, each so-
lution component should, whenever possible, compensate for a defect
in some other component. For example, in the case of the widely used
fixative, Bowin’s solution: 1. Formaldehyde fixes the cytoplasm but in
such a manner that it retards paraffin penetration. It fixes chromatin
poorly and makes cytoplasm basiphilic. 2. Picric acid coagulates cyto-
plasm so that it admits paraffin, leaves the tissue soft, fixes chromatin
and makes the cytoplasm acidophilic, but it shrinks and makes chroma-
Chemicals Commonly Used in Fixing Solutions 5

tin acidophilic. 3. Acetic acid compensates for both the latter defects.
No hard and fast rule exists concerning the choice of a fixative; gen-
erally selection is determined by the type of investigation. If there is
any question as to future plans for a tissue, formalin is a safe choice; it
permits secondary or post-fixation. Some suggestions can be included.
If over-all anatomy of the tissue is satisfactory, the routine fixatives can
be used: formalin, Susa, Zenker, Helly, or Bouin. Special fixatives for
cell inclusions are Carnoy, Flemming, Champy, Helly, Schaudinn,
Regaud, and others. For histochemistry the researcher is limited to
formalin, acetone, or ethyl alcohol.
Most fixing solutions are named after the person originating them;
Zenker and Bouin are examples. If the same man originated more than
one combination of chemicals, additional means of designating them
have been used. Flemming’s weak and strong solutions, Allen’s B3, B15
series, etc. Susa fluid was named by Heidenhain after the first two let-
ters of two words: swblimate and sdure.

Chemicals Commonly Used in Fixing Solutions

Acetic Acid, CH,COOH
Acetic acid is called glacial acetic acid because it is solid at tempera-
tures below 17°C. It can be considered one of the oldest fixatives on
record—in the 18th century vinegar (4—-10% acetic acid content) was
used to preserve Hydra. In modern technics it is rarely used alone but is
a frequent component of fixing solutions. Its efficient fixing action on
the nucleus and its rapid penetration make it an important part of good
fixatives. It fixes the nucleoproteins, but not the proteins of the cyto-
plasm. Acetic acid does not harden the tissue; actually it prevents some
of the hardening that may without it be induced by subsequent alcoholic
treatment. In some technics, however, acetic acid must be avoided be-
cause it dissolves out certain cell inclusions, such as Golgi and mito-
chondria. Some lipids are miscible with acetic acid, or actually soluble
in it. It neither fixes nor destroys carbohydrates.
Acids in general cause swelling in tissues—in collagen in particular—
by breaking down some of the cross-linkages between protein molecules
and releasing lyophil radicles which associate with water molecules. This
swelling in some cases may be a desirable property of acids, counteract-
ing and preventing some of the shrinkage caused by the majority of
fixing chemicals. In order to curtail continued swelling after fixation
6 Fixation (CHAP. 1)

with acetic or trichloracetic acid solutions, transfer the tissues to an al-

coholic washing solution in preference to water.

Acetone, CH,COCH,
Acetone is used only for tissue enzymes, such as phosphatases and
lipases. It is used cold and penetrates slowly. Only small pieces of tis-
sue will be fixed in this chemical.

Chromium Trioxide (chromic acid), CrO,

Crystalline chromium trioxide forms chromic acid when added to

water, usually a 0.5% solution. It is a valuable fixative, but rarely used
alone. It penetrates slowly, hardens moderately, causes some shrinkage,
forms vacuoles in the cytoplasm and often leaves the nuclei in abnormal
shapes. It is a fine coagulant of nucleoproteins and increases the stain-
ability of the nuclei. It oxidizes polysaccharides and converts them into
aldehydes—an action forming the basis of Bauer’s histochemical test
for glycogen and other polysaccharides. Better fixation, however, is ob-
tained with acetic acid, which will fix water-soluble polysaccharides;
later these can be post-treated with chromic acid. Fat can be made in-
soluble in lipid solvents by partial oxidation with chromic acid, but
the action can go too far. Potassium dichromate, which reacts in a simi-
lar fashion, is safer and is therefore more commonly used.
Excess chromic acid must be washed out, because later it can be re-
duced (undesirably, for our purpose) to green chromic oxide, Cr2Or.
Formalin and alcohol are reducing agents and must not be mixed with
chromic acid until immediately before use.

Ethyl Alcohol (ethanol), C,H,OH

Ethyl alcohol hardens tissue but causes serious shrinkage. It is a
strong cytoplasmic coagulant, but does not fix chromatin. Nucleic acid
is transformed into a soluble precipitate and is lost in subsequent solu-
tions and during staining. Alcohol cannot be a fixative for lipids be-
cause it does not make them insoluble in lipid solvents. It does not fix
carbohydrates, but neither does it extract mucins, glycogen, iron and
calcium. Alcohol seldom is used alone; occasionally it is used alone for
fixation of enzymes.
Chemicals Commonly Used in Fixing Solutions ~I

Formaldehyde, HCHO
Formaldehyde is a gas sold as a solution in water (approximately 40%
in content), in which form it is known as formalin. The use of the term
formol is incorrect, since a terminal -ol designates an alcohol or phenol.
Unless the author of a technic specifies that the dilution of his formalin
is in terms of actual formaldehyde content, dilutions are to be made
from the commercial product; i.e., a 10% solution would be 10 volumes
of formalin (40% formaldehyde saturated aqueous solution) to 90 vol-
umes of water.
Formaldehyde on standing over long periods of time may either pol-
ymerize to form paraformaldehyde, or oxidize into formic acid. A white
precipitate in a stock solution indicates polymerization; the solution
has been weakened. Cares (1945) suggests: shake the solution to suspend
the sediment. Pour into Mason jars and seal them tightly. Autoclave
at 15 lbs. for thirty minutes. Cool. This should produce a clear solution.
Dilute solutions (such as 10%) tend to oxidize more readily than do
stock solutions (40%). Marble chips may be left in the bottom of diluted
stock jars to keep the solution neutralized.
Formalin progressively hardens tissue, but is a mild coagulant of
proteins; in fact it is so weak that it might be considered a noncoagu-
lant. It neutralizes basic groups and increases the acidity of proteins.
In consequence, formalin-fixed proteins will stain well in basic dyes,
but less well in acidic dyes. Formalin has a moderate speed of penetra-
tion but its action is slow and somewhat incomplete unless tissues are
left in it for some time. It reacts most efficiently at PH 7.5-8. Although
formalin preserves the cells adequately it may not protect them com-
pletely, because it requires a long time to harden the tissue. Shrinkage,
therefore, can take place if dehydration, clearing and infiltration are
initiated before the hardening action is complete. Since these processes
are not employed in the freezing technic, formalin is a satisfactory fixa-
tive for this method.
Formalin is a good fixative for lipids; it does not dissolve lipoids or
fats. It does not fix soluble carbohydrates and it does dissolve glycogen
and urea.
The so-called formalin pigment may appear in tissues rich in blood.
This pigment is formed when hematein of the hemoglobin has escaped
from red blood corpuscles before or during death and reacts with the
formalin. It may be prevented by a short period of fixation in formalin
followed by a prolonged soaking in 5% mercuric chloride. Once
8 Fixation (CHAP. 1)

formed, however, the pigment can be removed in a solution of 1% po-

tassium hydroxide in 80% alcohol or picric acid dissolved in alcohol
(Baker, 1958). (See also page 244.)

Mercuric Chloride (corrosive sublimate), HgCl,

Mercuric chloride usually is applied as a saturated aqueous solution
(approximately 7%) and is acidic in action, owing to the release of Ht
and Cl~ ions in a water solution. It is a coagulant of proteins, both
cytoplasmic and nucleic. It penetrates reasonably well, but not as rap-
idly as acetic acid. It shrinks less than the other protein coagulants,
hardens moderately, and distorts the cells less than other fixatives. It
is excellent for mucin.
One disadvantage of mercuric chloride is that it deposits in the tissue
a precipitate of uncertain chemical composition. This precipitate is
crystalline, perhaps being mercurous chloride (needle shaped) and me-
tallic mercury (amorphous, irregular lumps). It may be removed by
iodine, the following reaction probably taking place: 2HgCl + I, =
HeCl, + Hels. Any metallic mercury also will be converted into mer-
curic iodide. The latter is soluble in alcohol, but the brown color of
iodine may remain in the tissue. This can be removed by prolonged
soaking in 70% alcohol, or more quickly by treatment with 5% sodium
thiosulfate, aqueous. A further disadvantage is that mercuric chloride
crystals inhibit adequate freezing, making it difficult to prepare good
frozen sections.
Most stains react brilliantly on tissue fixed in mercuric chloride.
Chromatin will stain strongly with basic stains and lakes; cytoplasmic
structures will react equally well with acidic or basic stains.

Osmium Tetroxide, OsO,

In solution, usually 1% aqueous, osmium tetroxide takes up a mole-
cle of water and becomes H,OsO;, erroneously called osmic acid. In
solution ionization is so minute that the pH is almost exactly that of
the distilled water used in making the solution. The substance is chem-
ically neutral, is not an acid and cannot be isolated. (Osmic acid would
be H2OsO,.) Baker (1958) suggests that its name might be hydrogen
per-persomate. The solution penetrates poorly and leaves tissue soft,
later becoming so friable in paraffin that tissues section badly. Osmic
acid preserves the cytoplasm and nuclei, but while it increases the stain‘
Chemicals Commonly Used in Fixing Solutions 9

ability of chromatin in basic dyes it reduces the stainability of the

cytoplasm. Fats and other lipids reduce osmium tetroxide, or combine
with it to form a dark compound. Thus fat sites become black and in-
soluble in absolute alcohol, cedar oil and paraffin; but they remain
soluble in xylene, toluene and benzene, and if left too long in xylene
or toluene (more than 5 minutes) they become colorless. Osmic acid is
not a fixative for carbohydrates.
When action is complete excess osmic must be washed out of the tis-
sue or it may be reduced during treatment in alcohol, forming an in-
soluble precipitate. Since osmium tetroxide is also easily reduced by
light and heat, it must be kept stored in a cool and dark place.

Tp ACO. AG. ta(INO;).O1

Picric acid is usually used in a saturated aqueous solution, 0.9-1.2%.

It is an excellent protein coagulant, forming protein picrates having a
strong affinity for acid dyes. However, it penetrates slowly, causes ex-
treme shrinkage, and offers no protection against subsequent shrink-
age. (There is no tendency to swell in this case, because of acidity.) The
shrinkage has been found to be close to 50% of the original tissue vol-
ume by the time it has undergone parafhn infiltration. It does not dis-
solve lipids, does not fix carbohydrates, but is recommended as a fixative
for glycogen. It is a desirable constituent of many fixing fluids because
it does not harden, but it cannot be used alone because of the unfavor-
able shrinkage it produces. Acetic acid frequently accompanies it to
counteract this poor quality.

Potassium Dichromate, K,Cr,O,

This substance, often incorrectly called potassium bichromate (which
would be potassium hydrogen chromate), is a noncoagulant of proteins,
making them more basic in action, but it dissolves nucleoproteins.
Chromosomes, therefore, are poorly shown, if at all. Potassium dichro-
mate leaves tissues soft and in poor condition for paraffin sectioning.
One valuable use, however, is the fixation of mitochondria (as with
Champy fluid); the lipid components are made capable of resisting so-
lution in lipid solvents. After fixation, tissues may be soaked in potas-
sium dichromate to insure that lipids are well preserved.
Potassium dichromate can be mixed with mercuric chloride, picric
acid and osmic acid, but it reacts with formalin and must not be mixed
10 | Fixation (CHAP. 1)

with it until immediately before use. If acetic acid is added, mitochon-

dria are lost, but chromosomes are shown better than when the chro-
mate is used alone. It usually is necessary to wash out the dichromate
with water in order to prevent the formation of an insoluble precipi-
tate of Cr.O3; this will form if the tissue is carried directly to alcohol.

Prichloraceie, Acid, CGl.COOM

This acid never is used alone and is similar to acetic acid in action.
It swells tissues and has a slight decalcifying property. As was noted
under acetic acid, washing should be done in alcoholic solutions.

“Indifferent Salts”
Baker (1958) applied this term to a group of chemicals (sodium sul-
fate, sodium chloride, and others) the action of which is not clearly
understood. Zenker’s and Helly’s solutions often have sodium sulfate
added. Sometimes sodium chloride is added to formalin or mercuric
chloride fixatives, particularly when marine forms are to be fixed.

References: Baker (1945, 1958).

Maceration (a prefixation process)

There are occasions when tissues are extremely dense and cannot be
manipulated while in fresh condition. It may be desirable to separate
the individual fibers of a muscle or nerve, and this is simplified by mac-
eration. The fluids used are not fixing solutions, so maceration usually
must be followed by some form of fixation. The following are various
suggestions for macerating fluids. (Hale, 1958)
1. 30% alcohol, 24 hours or longer (3-4 days).
2. formalin, 1 part in 10% salt (NaCl) solution, 100 parts: 24 hours or
longer.
3. 10% sodium chloride: 24 hours or longer.
4. chromic acid, 0.2% aqueous: 24 hours.
5. nitric acid, 20% aqueous: 24 hours.
6. boric acid, saturated solution in saline (sea water for marine forms),
plus 2 drops Lugol’s iodine solution (page 410), each 25 ml.: 2—3 days.
7. potassium hydroxide, 33% aqueous. Good for isolation of smooth
and striated muscle. After 1-114 hours, tease apart with needles.
Fixing the Tissue 1]

8. Maceration by enzymes, good for connective tissue, reticulum. (Gali-

gher, 1934)
Place frozen sections in:
Ppalicheatin Sicoumy eos. a. ee he edicts 5.0 gm.
sodium. bicarbonate... . . .s.4ss culne ance su 10.0 gm
NSPE rater: cakes ittAlit &..<-a, soda brent ees a bya tard 100.0 ml.

wash thoroughly and stain.

Fixing the ‘Tissue

First consideration in the choice of fixative should le in the purpose

to be served in preparing the tissue for future use. Is a routine all-pur-
pose fixative adequate or must some special part of a cell be preserved?
For example: an aqueous fixing fluid will dissolve out glycogen, and an
alcoholic one can remove lipids. Thought should be given to the rate
of penetration of the fluid and the density of the tissue to be fixed. Ob-
viously an extremely dense tissue might not fix well in a fixative which
penetrates slowly and poorly. With fixatives of poor penetration, the
size of the pieces must be trimmed to a minimum. In all cases, pieces
never should be any larger than is absolutely necessary; the smaller the
bulk, the more perfect the fixation.
The hardening effect of fixatives should be considered. An excessively
hardening fixative might lead to difficulties with liver and muscle. Maybe
some other fixative could be used with less hardening effect. If there is
any doubt concerning future needs for a tissue, place it in formalin;
this can be followed by post-fixation treatments.
Use a large volume of fixing fluid at least ten times the bulk of the
tissue if possible. Remove the tissues from the animal and place them
in the fixative as rapidly as is feasible, thereby reducing post-mortem
changes to a minimum. In most cases, do not attempt to fix the entire
organ; it will be too large to allow rapid and complete penetration of
the fixative. This is particularly true of an organ covered by a tough
membrane. An ideal piece would be | to 2 cm. in size; or place a larger
piece in the fixative for 15 to 30 minutes, then trim it to smaller size
and return it to the fixative. This sometimes is necessary when tissue
is very soft or easily crushed. Trim the piece with a new razor blade or
sharp scalpel. This will cause less damage than the squeezing action of
scissors. Do not crush or tear the tissue while removing it; such material
is worthless. Never allow tissue to become dry before placing it in the
12 | Fixation (CHAP. 1)

solution. Keep it moist with normal saline and wash off accumulated
blood with normal saline. Blood retards the penetration of a fixative.
Gently shake the solution and contained tissues several times to make
certain that the fluid can reach all surfaces and that pieces are not
sticking to the bottom or sides of the container. A chunk of glass wool
may be laid in the bottle to aid in keeping the tissue free of the bottom.
Thin pieces of tissue that show muscular contraction or that may
turn inside out (tissues of the gastrointestinal tract are particularly
likely to do this) may be placed on thick filter paper, outside wall against
the paper, and dropped in the fixative. Tiny, easily lost specimens, bi-
opsies, bone marrow, etc., may be wrapped in lens paper or coffee filter
paper (page 228).
The length of time required for complete fixation depends on the
rate of penetration and action of the fixative. Most coagulant fixatives
produce complete fixation as fast as they can penetrate the tissue. But
some fixatives, such as formalin, exhibit progressive improvement in
fixing action after the tissue has become completely penetrated. Pro-
longed action in this case improves the condition of the tissue and
rarely is harmful. Occasionally some type of post-fixation treatment as
noted on page 23 is advisable.

WashingoO the Tissue

After fixation is accomplished, the excess of many fixatives must be

washed out of the tissue to prevent interference with subsequent proc-
esses. Often washing is done with running water; sometimes the tissue
may be carried directly to 50% alcohol or higher. Some technicians
maintain, for instance, that Bouin’s tissue washed in water may lose
some of the soluble picrates and that this tissue should be transferred
from the fixative directly to 50% alcohol. When a freezing step is
planned, formalin-fixed tissue may be washed a brief moment in water,
but in rush problems it can be taken from the fixing solution directly
to the freezing microtome. The presence of alcohol will prevent freez-
ing action and its use must be avoided, or it must be thoroughly washed
out before attempting to freeze the tissue.
When tissue has been fixed with a mercuric chloride solution addi-
tional treatment is necessary. After washing in 50% alcohol, transfer
the tissue to 70% alcohol containing enough iodine-alcohol (saturated
solution of iodine in 70% alcohol) to give the solution a deep brown
Fixing Solutions and their Uses 13

color. Leave the tissue in this solution from 5 to 8 hours, but not longer.
If during this time the solution loses color, add some more iodine-alco-
hol. The iodine removes some, but probably not all, of the excess mer-
curic chloride precipitate left in the tissue. Later, when staining the
sections, iodine-alcohol or Lugol’s solution (page 410) must be included
in the staining series. This should eliminate remaining crystals which
otherwise will persist as dark clumps and needles in the finished slides.
After washing, the tissue may be stored for several weeks or months
in 70% alcohol, but it is always safer to dehydrate and embed as soon
as possible. Storage in alcohol for long periods of time (a year or longer)
tends to reduce the stainability of tissues. This is also true after long
immersion in chromate and decalcifying solutions, or if traces of acid
or iodine are present.
Do not use corked bottles for fixing or for alcoholic solutions; extrac-
tives from corks can be injurious to tissues.

Fixing Solutions and their Uses

Routine Fixatives and Fixing Procedures for
General Microanatomy
BOUIN’S FIXATIVE: 24 hours or longer—several weeks cause no damage.
picric acid, saturated AQUEOUS 2.2.2 a+ s+ aac oer 75.0 ml.
formalin 25.0 ml.
Se ee ne: . [ere 5.0 ml.

Because of acetic acid content do not use for cytoplasmic inclusions.

Large vacuoles often form in tissues. Wash in 50% alcohol. The yellow
color must disappear before staining sections. It usually is removed in
the alcohol series, but if not, treat slides in 70% alcohol plus a few
drops of saturated lithium carbonate until the color is extracted.

BOUIN-DUBOSCQ (Alcoholic Bouin’s) FIXATIVE (Pantin, 1946): 1-3 days.

BO" netay alcohol). 3. a. gies. <5 RE IS 150.0 ml.
POM Maeda), Se, akon ait 2 S'n Ge 2, «3S Ree gg 8 60.0 ml.
PIAGIAlGACELIC, AGIGk esSs @< lS abe ccc sd» Med $3 15.0 ml.
pienc acid crystals? ai u.. Moga. 2. ee 1.0 gm.

Prepare just before using. This is better than Bouin’s for objects dif-
ficult to penetrate. Go directly to 95% alcohol.
14 Fixation (cHAP. 1)

BUFFERED FORMALIN: may remain indefinitely, progressive action.

HOSA. forma Wes Oe PC oe mls 2. 000.0 mir
sodium acid phosphate, NaH,PO,-H,0 ....... 4.0 gm.
anhydrous disodium phosphate, Na,gHPO, .... 6.5 gm.

Wash in water.

FORMALIN (Baker, 1944): may remain indefinitely, progressive action.

caletumchiorideraey at). ih Eee. fee vee AF. Lie 1.0 gm.
cadmmumerchilonide) $29))2-9°4
aE ThE WE tule 1.0 gm.
forma bin (RENE UE. Pon EE aE) STL See 10.0 ml.
distilledtwaceral who: ACE eR RE, 100.0 ml.

Wash in water.

FORMALIN (Baker, 1958): may remain indefinitely, progressive action.

Fontan te. ck oe ee Meee 2a eae 10.0 ml.
calcium chloride (anhydrous) 10% aqueous so-
htion.(10'em-./100ml. water) .......2.2 a... 10.0 ml.
dastilledawater |Ratti. y. Be EES. waa ee 80.0 ml.

Wash in water.

HELLY’S FIXATIVE (ZENKER FORMOL): 6-24 hours. If the tissue seems to

harden excessively, follow a maximum of 18 hours in fixativg with
a 12-24 hour chromating in 3% potassium dichromate.
pGtassium ‘dichromate: me... 2. . yee tee 2.0. em.
INCHGUMICUEINOTIGG cera ss. . sie es ae 4.0-5.0 gm.
SOCMUMINS ENE ATE™ \ (ee we eee. segs. «2k ogee 1.0 gm.

(may be omitted, see indifferent salts, page 10)

distilled@watert,: .%)AeNarmee Sek eee 100.0 ml.
formalin (add just before using) ............. 5.0 ml.

Formalin reduces dichromate and should not be left in stock solu-

tions. The above stock, without formalin, can be used for Zenker’s
fixative, page 16. Excellent for bone marrow and _blood-forming
organs, also intercalated discs. Wash in running water overnight,
post-treat for mercuric chloride. Maximow modification: add 10%
of formalin instead of 5%, and sometimes 10% of osmic acid. The
latter should be fixed in the dark.
Fixing Solutions and their Uses

HOLLANDE BOUIN’S FIXATIVE (Romeis, 1948): 8 hours—3 days.

COP PemmaACelatey. Yin enc ss eae et Sree 2:9 2m.
prani@acid crystals!.........-. DA e eer te 4.0 gm.
OMAN, Deseo g ae eO AS 5. 5 oo Miata see, ate!) SROs mal.
GiSCUNCG Walle, Myo c 2.0 . 5s ghee toate ee exe, 1 0G.0) mal.
placial: ACCUIG ACI ei 6 ge ctie sas os Oe aoe I5-amil.

Dissolve copper acetate in water without heat; add picric acid slowly
with stirring. When dissolved, filter, and add formalin and acetic
acid. Keeps indefinitely. Hartz (1947) recommends this for fixation of
calcified areas as in lymph nodes or fat necrosis. It is a good general
fixative. Wash for several hours in 2 or 3 changes of distilled water.

ORTH'S FIXATIVE (Gatenby, 1950 and Galigher, 1934): 12 hours at room

temperature, 3 hours at 37°C.
formalin ........ oe eee ee Ore an st 10.0 ml.
potassium dichromate . ce. a2): 252k Bee ee 2.5 em,
SGOMUIMSUTALE ce. feu. ae Ra Peed nce mR 1.0 gm.
distilled water ...... e eee Tere re. 100.0 ml.

Mix fresh. A good routine fixative, also for glycogen and fat. Wash
in running water overnight.
Lillie’s (1954B) variation:
20%, potassium dichromate .....2...4.09s - 100.0 ml.
formal 4.55.3 okde: eee ere 10.0 ml.

STIEVE’S FIXATIVE (Romeis, 1948): 24 hours.

mercuric chloride, saturated aqueous ......... 76.0 ml.
PO UIMANINA 8os & cece ed pide hoe BAR. 6 5 nl ee, 20:0) mil.
Placialpacene acids... :..% 2Bre cheees Be a a 4.0 ml.

Similar in effect to Susa’s below, but simpler to prepare. Penetrates

rapidly, good for large pieces. ‘Time not critical. Go directly to 70%
alcohol. Post-treat for mercuric chloride.

SUSA’S FIXATIVE (Romets, 1948): 24 hours.

mercuric chloride saturated in 0.6% NaCl .... 50.0 ml.
trichlonaceticiacid |) Ais. PaMi.ae oe oh meh, Pia 2:0 om,
glacial dectcraci@ =.) 2... Nge . sss. a ae 4.0 ml.
POMS RAR ees Re ttady « « Sd ass B® % ~ 200ml,
Gistiiledhwaters. 6... 5.4: toe See). . ate, . Gk. os 30.0 ml.
16 Fixation (CHAP. 1)

Good substitute for Zenker’s if dichromate is not required. Hardens

less. Rapid penetration. Go directly to 70% alcohol. Post-treat for
mercuric chloride.

ZENKER’S FIXATIVE:4—24 hours. If tissue seems to harden excessively,

follow a maximum of 18 hours in Zenker’s with a 12—24 hour chro-
mating in 3% potassium dichromate.
POCASSHUTIN CMeMrOMate song... ee os ieee 2.) 2m.
MMEECHEICSCIMONIGE. 5°. sulei sar sree st Meer ree eget 4.0-5.0 gm.
SOGHUUIMISUMEIEC re 2 2. een) eM ok ae 1.0 gm.

(may be omitted, see indifferent salts, page 10)

CIsGUNle WALK 4.5)..0 hy bee As 2 A nS gore 100.0 ml.
placialcacetic ACIG.«.! .. oo, Bae x SOE eater 5.0 ml.
Excellent general fixative, fairly rapid penetration. Wash in running
water overnight. Post-treat for mercuric chloride. Zenker’s may serve
for Helly’s (page 14) if acetic acid is not added to the stock solution.

Fixatives for Cell Inclusions and Special Technics

s

ALTMANN’S FIXATIVE (Gatenby, 1950): 12 hours.

bo, potassium Cicinomate ...>.. <. >see ae. 10.0 ml.
DP OSRMICEAGIC: . URE... cates nocyCeeaue eee eaeere 10.0 ml.

Good for fat and mitochondria.

Wash in running water overnight.

AMMERMAN’S FIXATIVE (1950): 2 hours.

chromium potassium sulfate (chrome alum) ... 3.0 gm.
FOUEAINEREE SP. Cet? oa See ce. | eee ee 30.0 ml.
placial acne ACid, 14.060 ay...) Acct. cena ae 2.0 ml.
GistMedtwatetal... 6 fee ame) 2%)... Bess haga ee 238.0 ml. \

Good for yolk-rich material and insect larvae. Do not fix longer than
2 hours, or swelling occurs. Does not harden and gives good cytologi-
cal detail. Wash in 70% alcohol or water.

CARNOY FIXATIVE (Gatenby, 1950): 3-6 hours.

Formula A:
Pleven ACE LIC: ACI. | AR...) Wen ee Oa ee 20.0 ml.
absolute-ethyl alcohols. . ii... Jab Says eee oe 60.0 ml.
Fixing Solutions and their Uses 17

Formula B:
plagialkacetiGnacial te. 62). ee. ae. 10.0 ml.
absolucesethyivalconoly. : \\.2 a5 0ee: fale 60.0 ml.
chlovorenmi’.; wean ear. hee 6 & te SeeSee 30.0 ml.

Chloroform is said to make action more rapid. Important fixative for

glycogen and Nissl substance, but dissolves most other cytoplasmic
elements. Wash 2—3 hours in absolute alcohol to remove chloroform,
particularly if embedding in nitrocellulose.

CARNOY-LEBRUN FIXATIVE (Lillie, 1954B): 3—6 hours

absolute, ethy! alcohol .......«+ 5 hs eas: eee 3s 15.0 ml.
glacial ACCUG Addis ic. 2... fs 2. SR. Sees 15,0) mal;
Chlototomite gg 2. 28s danciasty 4 pees 3 1.0 anil
mercunic chloride: crystals. «.. 52... 2 Waeee paren 4.0 gm.
Lee of The Microtomist’s Vade Mecum fame considers this a very fine
fixative. It does not keep; mix fresh. Penetrates rapidly. Wash well in
alcohol to remove chloroform.

CHAMPY FIXATIVE (Gatenby, 1950): 12 hours.

potassum dichromate, 3% aqueous (3 gm./100
Mall Wallen) is 22.03. $cu0h geek creek eee 5 eee (Opal.
chromic acid, 1% aqueous (1 gm./100 ml. wa-
LEDC ee nds mens. no gees. ooh 7.0 ml.
osmic acid, 2% aqueous (2 gm./100 ml. water) . 4.0 ml.
Prepare immediately before use. Good for cytological detail, mito.
chondria, lipids, etc. Penetrates poorly; use only small pieces of tis-
sue. Wash in running water overnight.

DAFANO FIXATIVE (Romeis, 1948): 12-24 hours.

eobalimitrate:. 2228): 6 o<.0.9¢s8r<
.«ss URE ERE et 1.0 gm.
formalin <4. Bicteha Fes d x nes . 45+ Se 15.0 ml.
Guistilllediwater sea aoe. 62 a5: eae ee ne 100.0 ml.

Wash in water. Good for Golgi apparatus.

FLEMMING’S FIXATIVES: 12—24 hours.

A. Strong solution
chromic acid, 1% aqueous (1 gm./100 ml. wa-
erg eee eet Ree cas OE a.<< =Su a: oom Obs tees ee 15.0 ml.
osmic acid, 2% aqueous (2 gm./100 ml. water) . 4.0 ml.
glacial .aceuie aGids oe ois. es. hae «2 1.0 ml.
18 Fixation (CHAP. 1)

Mix just before using. Acts slowly: use small pieces with no blood on
outside. Wash in running water for 24 hours.
B. Modification for cytoplasmic study. High acetic content of A
solution makes it poor for cytoplasm fixation.
chromic acid, 1% aqueous (1 gm./100 ml. wa-
BEND) enc 4s" ae pry A Bs aca sm emu tl cages, XV oy0 15.0 ml.
osmic acid, 2% aqueous (2 gm./100 ml. water) . 0) mil.
placialyaceti@!aai *.ao biel n ries peergeh. Gees Foca 2-3 drops
C. Weak solution
chromic acid, 1% aqueous (1 gm./100 ml. wa-
CEL) See ee ee. ae. . 2: Se amma Ae 25.0" mal:
osmic acid, 2% aqueous (2 gm./100 ml. water) . 5.0 ml.
acetic acid, 1% aqueous (1 ml./99 ml. water)... 10.0 ml.
dismilledivater.. ae... See... ok Bee *2oe 60.0 ml.

Flemming’s solutions are good for mitotic figures (not suitable for
general work), but they penetrate poorly, harden excessively, blacken
material, and interfere with action of many stains. The weak solution
is a fine fixative for minute and delicate objects. For dense tissues use
the strong solution. Iron hematoxylin is excellent following these
fixatives.

FORMOL-ALCOHOL (Tellyesniczky) (Lillie, 1954B): 1-24 hours

We eeuvina cohol: © 2am. . «0M spy ces. Sake 100.0 ml.
fORMmaNIN RE © sous AMM, . GUM. cial .. «Ga oe) Grin
glacralaectic acid). Ameer. ame. . 2 SPS.Lae 5.0 ml.
Good for insects and crustacea. Widely used by Botanists. ‘Transfer
to 85% alcohol.

GENDRE’S FLUID (Lillie, 1954B): 1-4 hours, 25°C.

95% ethyl alcoho] saturated with picric acid ... 80.0 ml.
forma lini Se Seer: 0c. eso Byte te 15.0 ml.
glacial @cetiGwcid: .. 2 wae...
.«) Sieh. oe 5: Oomal:

Good for glycogen. Wash in several changes of 80% alcohol.

GILSON’S FIXATIVE (Gatenby, 1950): 24 hours; may be left several days.

HiChiGhacia, CONCEMtrAkeGr eee Ae
ee. oo ee 15.0 ml.
Blamialeacetic aciG):.. 27) Rr ae ae site eres, ele 4.0 ml.
MEKCUTIC CHlOTINE Crystals ¥en 2 ewe ae a eee 20.0 gm.
o0eethyl alcohol |... kos. Gm ae at oie weer ienete 100.0 ml.
cdistilled@wateri..: sae: fe Se ee eee ee 880.0 ml.
Fixing Solutions and their Uses 19

Good for invertebrate material. Does not give a good histological pic-
ture; cytoplasm is badly shrunken. Wash in 50% alcohol. Post-treat
for mercuric chloride. Good for beginners, easy to work with.

JOHNSON’S FIXATIVE (Gatenby, 1950): 12 hours.

potassium dichromate, 2.59% aqueous (2.5 gm./
Orin: Walel\: Aeebes. ry ci edat lores seS 70.0 ml.
osmic acid, 2% aqueous (2 gm./100 ml. water) 10.0 ml.
platinum chloride, 1% aqueous (1 gm./100 ml.
WLC) cetG acl icc. ay 5,2 Hore S00 eee aN as 15.0 ml.
PlacialACEHO.ACIC: ws coe 6. cco agers MMe ot 5.0 ml.

Contracts spongy protoplasm less than Flemming’s. Wash in water.

Hermann’s modification: 12-16 hours.

platinum chloride, 1% aqueous (1 gm./100 ml.
WALT ees peters, += Soa ben he an ce pe 15.0 ml.
PlaGITL ACCHIC ACIG! ads). Heng dins.« = 50> eee 1.0 ml.
osmic acid, 2% aqueous (2 gm./100 ml. water) 2.04.0 ml.

Better protoplasm than with chromic mixtures. Good nuclear stain-

ing, but not of plasma. Some shrinkage of chromatin. Without acetic
it is good for mitochondria.

KOLMER’S FIXATIVE: 24 hours.

potassium dichromate, 5% aqueous (5 gm./100
mil, water) 0.020. sacs bese dees. ee ee 20.0 ml.
HOG? fOrmialig. so ecns os le 6 SRA vee eee 20.0 ml.
Placialvaretic acids. 2 2 ee 5.0 ml
trichloracetic acid, 50°% aqueous (50 gm./100 ml.
WAREU), amy ee fee ae = eee aes + ago eerrr >:0 imal:
uranyl acetate, 10% aqueous (10 gm./100 ml.
WALCO) pia dy 204 2 ode ed ya «bees 5.0 ml.

Good for entire eye (Wall, 1938) or nerve tissue, due to presence of
uranium salts. Wash in running water.

LAVDOWSKY FLUID (Swigart et al., 1960): 12-24 hours.

Gistiled! water ee 2. oo ee bsg wes ce ee eee oe 80.0 ml.
0577, Netian) alcOMOl ow. deg 2 Fo oe é Se ER AG 10.0 ml.
chromic acid, 2% aqueous (2 gm./100 ml. water) — 10.0 ml.
PlacialiaceuiG.acid, 5 82.5 Shans dae be oes Pees 0.5 ml.

Good for glycogen. Transfer to 80% alcohol.

20 Fixation (cHaP. 1)

NAVASHIN’S FIXATIVE (Randolph, 1935): 24 hours.

Solution A:
ChiOMIC ACI se7 VE teehee hes eA. ee 1.0 gm.
migetal acetic*aeidye yi) 2). 4:4: Yea cies 10.0 ml.
Gistilled waters. 04 \inesisis: pe eet ee 90.0 ml.

Solution B:
fornma lin’ 7: fabs Aten bk. arr ey ea 40.0 ml.
distilled” watering 4) Peas. 2. See ts oe 60.0 ml.

Mix equal parts of A and B just before using. At end of six hours
change to a new solution for another 18 hours. Useful for preserving
cellular detail in plant materials; as good as Flemming’s on root tips,
and less erratic. Transfer to 75% alcohol.

PERENYI'S FIXATIVE (Galigher, 1934): 12-24 hours.

chromic acid, 1% aqueous (1 gm./100 ml. water) 15.0 ml.
nitric acid, 10% aqueous (10 ml./ 90 ml. water) 40.0 ml.
gaveecthyl alcohol Av ett ee.. ee 30.0 ml.
GusticGiwater 1.) ieee. .2 eee 15.0ani:

Good for eyes. Always when fixing the entire eye make a small hole
near the ciliary body so the fluids of both chambers can exchange with
the fixing fluid. For the best fixing results, inject a little of the fixative
into the chambers. Decalcifies small deposits of calcium; good fixative
for calcified arteries and glands. Trichromes stain poorly; hematox-
ylin is satisfactory. Wash in 50% or 70% alcohol.

REGAUD (Kopsch) FIXATIVE (Romeis, 1948): 4-24 hours.

potassium dichromate, 3% aqueous (3 gm./100
LOL AES): eS oe, : 5 oem eee eS a 40.0 ml.
formalin. 610. 20. om, BOO EE ce 10.0 ml.

Mix immediately before use. Recommended for mitochondria and

cytoplasmic granules. Tends to harden. Follow fixation by chromat-
ing several days in 3% potassium dichromate. Renew solution once
every 24 hours. Wash in running water overnight.

ROSSMAN’S FIXATIVE (Lillie, 1954B): 12-24 hours.

absolute alcohol, saturated with picric acid ... 90.0 ml.
formalin... \t4s. 2, see A ee, Sah, See oe ee 10.0 ml.

Good for glycogen. Wash in 95% alcohol.

Fixing Solutions and Their Uses 4|

SANFELICE FIXATIVE (Baker, 1958): 4—6 hours.

chromic acid, 1% aqueous (1 gm./100 ml. water) 80.0 ml.
FORMAN: ae sat cine. o. = alee Raat ee etait aul 40.0 ml.
PlAGIAIMACCE Cra eo cues oe se atin eeeas oe 5.0 ml.

Mix immediately before use. Good for chromosomes and mitotic

spindles. Fix small pieces. Produces less final shrinkage than others
of this type. Wash in running water. 6-12 hours.

SCHAUDINN’S FIXATIVE (Kessel, 1925): 10-20 minutes for smears, 40°C.

mercuric chloride, saturated aqueous ......... 66.0 ml.
G5, peruMdmalcOlol 08s 4 o2.< 4 «oH vie ot 33.0 ml.
LACTALCOCE IG AGI |ct, 5 3.3.5 ong nc ¢:2 a ee eee 5.0-10.0 ml.

Recommended for protozoan fixation, smears on slides, or in bulk.

Not for tissue; produces excessive shrinkage. Transfer directly to
50% or 70% alcohol. Post-treat for mercuric chloride.

SINHA’S FIXATIVE: 4—6 days.

picric acid, saturated in 90% alcohol ......... 75.0 ml.
HOPMAN ar AOS) soe al deh es ee 25.0 ml.
nitric AciC. “concentrated ...:..¢...:.+2
eee 8.0 ml.

Sinha (1953) adds that 59% mercuric chloride may be included; possi-
bly this means 5 grams per 100.0 ml. of above solution. Recom-
mended for insects; softens hard parts, but with no damage to internal
structure. Transfer directly to 95% alcohol.

SMITH’S FIXATIVE (Galigher, 1934): 24 hours.

potassium: dichromate’ 2. Peo. ous 5 eee 5.0 gm.
forma ye se fake Retna: . +d 38 ee 10.0 ml.
distilled water 99 sis the at o's ws th a 87.5 ml.
Slacial Acetic aGias | 24 fen 225 a6 05s ee re 2.0 mal:

Mix immediately before use. Good for yolk-rich material (Laufer,

1949). Wash in running water overnight.

The pH of some fixatives changes after mixing and again after tissue
is added. This may be a factor worth considering under certain condi-
tions—when effect on stainability or silver impregnation is important,
for instance (Freeman et al., 1955).
22 | Fixation (cHap. 1)

Fixation by Perfusion
Perfusion (forceful flooding of tissue) is of advantage only for a tissue
that requires rapid fixation but is not readily accessible for rapid re-
moval. A prime example is the central nervous system. Many organs are
not adequately fixed by this method because the perfusion fluid may be
carried away from rather than to the cells.
Special equipment necessary for perfusion includes (1) a glass cannula
which fits the specific aorta to be used; (2) rubber tubing to connect the
cannula to the (3) perfusion bottle.
When the animal is dead or under deep anesthesia, cut the large ves-
sels in the neck and drain out as much blood as possible. Expose the
pericardium by cutting the costal cartilages and elevating the sternum.
Cut the pericardium and reflect it back to expose the large arteries.
Free part of the aorta from the surrounding tissue and place a mois-
tened ligature behind it. Make a small slit directed posteriorly in the
wall of the aorta and insert the moistened cannula. Bring the two ends
of the thread together and tie the cannula firmly in place. Cut open the
right atrium to permit escape of blood and other fluids.
Precede fixation with a small amount of saline (50-100 ml.). Fill just
the rubber tubing leading from the perfusion bottle to the cannula.
(Separate the saline from the fixative with a clamp near the attachment
of the rubber tubing to the bottle.) Fill the perfusion bottle with fixa-
tive (500-1000 ml. depending on size of animal). The fluid should be
warmed to body temperature. The saline precedes the fixative to wash
out residue blood before it becomes fixed to the vessel walls. If a forma-
lin-dichromate fixative is being used substitute 2.59% potassium dichro-
mate for normal saline.
When ready to start perfusion, with bottle at table level open clamp
on rubber tubing. Gradually raise the bottle to increase pressure of
fluid. Continue to raise the bottle gradually until at a height of 4 to 5
feet enough pressure is exerted to force out most of the blood. After
5 minutes, open the abdomen and examine the organ to be perfused.
If the surface vessels are still filled with blood and the organ has not
begun to take on the color of the perfusing solution, it is possible that
the perfusion has failed. But sometimes stubborn cases may require
10 to 30 minutes to perfuse. When blood color is absent, perfusion is
complete.
Observe these suggestions: (1) the cannula used should be as large
as possible to permit as rapid a flow as possible. ‘This aids in washing
Post-fixation Treatments 23

out blood ahead of the fixative. (2) If the head alone is to be fixed, clamp
off the thoracic duct, and if the brain and spinal cord are to be fixed,
clamp off intestinal vessels. ‘The fixative is then directed toward brain
and spinal cord. (3) When the perfusion bottle is being filled, allow
some of the fluid to flow through the rubber tubing and cannula to
release air bubbles. This can also be done with the saline. Air bubbles
will block the perfusion. (4) Do not allow the injection pressure to ex-
ceed the blood pressure; artifacts will result.
If only a small piece of an organ is to be fixed, a modified and easier
perfusion may be undertaken. Inject the organ with a hypodermic syr-
inge of fixative. This usually will be found adequate. Immediately after
injection, cut out a small piece of tissue close to the injection and im-
merse in the same type of fixative.
Lillie (1954B) lists two disadvantages of perfusion: the blood content
of the vessels is lost and perfusion cannot be used if post-mortem clot-
ting is present. But he does favor perfusion as the outstanding method
for brain fixation, saying that immersion of the whole brain without
perfusion “can only be condemned.” If whole brain perfusion is not
possible he suggests the following as the preferred method of fixation
for topographic study.
1. Cut a single transverse section anterior to oculomotor roots and interior
margin of anterior colliculi, separating the cerebrum from midbrain and
hindbrain.
2. Make a series of transverse sections through the brain stem and cerebel-
lum (5-10 mm. intervals) leaving part of meninges uncut to keep slices in
position.
9 Separate two cerebral hemispheres by a sagittal section. On sagittal surface
identify points through which sections can be cut to agree with standard
frontal sections. Make cuts perpendicular to sagittal surface. Cut rest of
brain at 10 mm. intervals.
4. Fix ina large quantity of solution.

References: Bensley and Bensley (1938); Cowdry (1952); and Lillie

(1954B). See also Koenig et al. (1945) and Eayrs (1950).

Post-fixation Treatments

Chromatization

2.5-3%, aqueous potassium dichromate (2.5-3 gm./100 ml. water):

overnight for small gross specimens (1—2cm.), 2—3 days for larger
24 Fixation (CHAP. 1)

ones; 1—2 hours for sections on slides before staining (may be left
overnight).
Wash thoroughly in running water, overnight for large gross tissues;
15-30 minutes for slides.
Improves preservation and staining, particularly of mitochondria.

Deformalization
The removal of bound formalin frequently is necessary in silver im-
pregnation methods, such as Ramon y Cajal, del Rio-Hortega methods,
and the Feulgen technique.

Lhotka and Ferreira’s (1950) Method

1. Wash tissue blocks in distilled water: 15 minutes.
2. Transfer through 2 changes of chloral hydrate, 20% aqueous (20
em./100 ml. water): 24 hours each.
3. Wash in distilled water: 15 minutes.
4. Proceed to any method. With silver stains, time of impregnation
needs to be lengthened: Ramon y Cajal to 2 weeks; del Rio-Hor-
tega to 24 hours.

Krajian and Gradwohl’s (1952) Method, on Slides

1. Place in ammonia water (40 drops ammonia in 100.0 ml. water):
1 hour.
2. Wash, running water: | hour.
3. Fix in special fixative if necessary: 1 hour.
Wash, running water: | hour.
or Proceed to stain.

Removal of Mercuric Chloride Crystals

Iodine method: see page 12.
Gonzalez method (1959A). Gonzalez suggests the following if necessary
to avoid mordant action of iodine. After fixation, wash for a short
time, or immerse directly in cellosole (ethylene-glycol-monoethyl-
ether, Carbide and Carbon Chemical Co.): 24-48 hours, 3 changes.
Follow with toluene: 1-2 hours; infiltrate and embed.

Decalcification

Calcium deposits may be so heavily concentrated that they interfere

with future sectioning and result in torn sections and nicks on the knife
Decalcification or

edge. If deposits are sparse, overnight soaking in water will soften them
sufficiently for sectioning. This is undertaken when the tissue is blocked
and ready for sectioning (page 55). Heavy deposits may be removed by
any of several methods. Opinions are varied as to preferred method,
but do not leave tissues in any of the fluids longer than necessary. If any
doubt arises concerning completion of decalcification, check for calcium
by the following method.
To 5 ml. of the solution containing the tissue, add 1 ml. of 5% so-
dium or ammonium oxalate. Allow to stand 5 minutes. If a precipitate
forms, decalcification is not complete. A clear solution indicates it is
complete. Sticking needles in the tissue to check hardness is sloppy
technic which can damage the cells.

Acid Reagents

After using an acid for decalcification, transfer directly to 70% alcohol

to prevent swelling in the tissue and impaired staining reactions: 3—4
hours or overnight.
a. (forimic saci 2-20
A ee 5.0-25.0 ml.
fOuMa ee ot oh we ee oe ee 5.0 ml.
distilled water to make’ .....7.:2...., 202% 100.0 ml.

With 5 ml. formic acid content, 2—5 days. If increased to 25 ml. less
time is required, but with some loss of cellular detail.
b. formic acid, 50% aqueous (50 ml./50 ml. water) 50.0 ml.
sodium citrate, 159% aqueous (15 gm./100 ml.
WOTEK) 1 © occa nah og aq oh ees 2s ade odd 6 cee 50.0 ml.

GX, TOUMMALITD yor ees oe Se cokcdo ce dad nde oes ee 10.0 ml.
nitric acid, concentrated .................... 5.0 ml.
Gistilled water . 2.2.2. 25. 2 as24.6 4 -as dean < 85.0 ml.

If acidity is a problem for staining, treat with 2% aqueous lithium

carbonate solution, or 5% sodium sulfate: 6-12 hours, then into 70%
alcohol.
d. Citrate-citric acid buffer, pH 4.5 (controlled hy-
drogen ion concentration) (Culling, 1957)
citric acid monohydrate, 7°% aqueous (7 gm./
NOOR -Wwaten ities, = 45 eae ss gas ee = 5.0 ml.
ammonium citrate, anhydrous, 7.459% aqueous
(-ao.em./ LOOM. Water): a. 2.32%.
25g4 eles « 95.0 mil,
zinc sulfate, 1% aqueous (1 gm./100 ml. water) 0.2 ml.
CHIOTOTOMIIE ea. 6 acre be cal ee os ie eS il few drops
26 Fixation (CHAP. 1)

Calcium ions are insoluble at pH 4.5, so buffer solutions may be used.

Slower than other methods, but no perceptible tissue damage.
e. Kristensen’s (1948) fluid is highly recommended.
eNmrornvic acid \(see-page 408) 2 once a: 50.0 ml.
IN sodium formate

(pH 2.2) 24 hours. Wash in running water: 24 hours.

Pros and Cons in Use of Acid Reagents

Schajowicz and Cabrini (1955) observe that strong acids, such as nitric
acid, do alter histochemical behavior of bone and cartilage, and must
be used with care. Formic acid and citrate do not have this disadvantage
if used for only a few days.
Case (1953) added 1% phloroglucinol to formic acid for improved
cell detail and preservation, and for staining qualities. Culling (1957)
disagrees and maintains that staining is poor after phloroglucinol.
Morris and Benton (1956) found 1-2M hydrochloric acid the most
rapid decalcifier (approximately 3 hours). He found that this produced
adequate staining reactions if slides were mordanted in 5% aqueous
ammonium chloride for 30 minutes before staining.

Ion-exchange-resin Method of Decalcification

Dotti et al. (1951) theorized that replacing sodium citrate (formic acid-
citrate mixture) by a cation exchange resin might result in speedier de-
calcification. The liberated calcium could be removed more rapidly
from the solution—requiring about half the time necessary for formic
acid-citrate decalcification. They recommend 40% of resin in formic
acid. If speed is not essential, they recommend 10% of resin. The resin
is Win 3000, ammonium salt of sulfonated resin (Winthrop Stearns Inc.,
NY).

Electrolysis Method of Decalcification

The principle of electrolysis is based on the theory that there is an at-
traction of Ca ion to a negative electrode. The bone is suspended by a
platinum wire, becoming the anode, and a second platinum wire forms
the cathode. ‘Then by electrolysis the calcium ions are freed from the
Decalcification 27

bone." Case (1953) considered the electrolysis method to be inferior to

the use of either nitric or formic acid. Clayden (1952) and Lillie et al.
(1951) demonstrated that an increase in temperature accomplished the
same thing. Probably the rise in temperature was the principal reason
for speed of decalcification. Culling (1957) agreed that heat speeds up
decalcification but causes severe swelling and is not to be recommended.
Lucas (1952) found no evidence that the passage of a current by itself
accelerated decalcification. Experiments by Lillie et al. (1951) indicate
that in respect to temperature there is another story. Decalcification at
room temperature or even cooler produces the best results for staining.
The present author finds nothing to be recommended about the elec-
trolysis method.

Chelating Agents for Decalcification

These agents during decalcification offer the advantage of maintaining
good fixation and sharp staining. They are organic compounds that have
the power of binding certain metals, such as calcium and iron. Versene
(Dow Chemical Company) or Sequestrene (Geigey Chemical Company),
the disodium salt of ethylene diamine tetracetic acid (EDTA), is the
most commonly used agent. The method does have two disadvantages;
the tissue tends to harden and decalcification is slow.

Hilleman and Lee (1953)

200 ml. of a 5.5% solution of either Versene or Sequestrene in 10%
formalin, for pieces 40 « 10 & 10 mm. It may require up to 3 weeks.
Renew the solution at the end of each week. Transfer directly to 70%
alcohol.

Vacek and Plackova (1959)

0.5M solution of EDTA at pH 8.2-8.5 yields better results in silver
methods than does decalcification with acids.

Schajowicz and Cabrini (1955)

Versene is here considered the better of the two solutions. The so-
lution should be adjusted to a pH 7.0 with NaOH and HCl. Hema-
toxylin and eosin stains as usual, but glycogen is lost, and alkaline
phosphatase has to be reactivated after chelating agents.
‘Bone Decalcifier, Portable, Chicago Apparatus Co. Catalog #28-712.
28 Fixation (CHAP. 1)

Decalcification Combined with Other Processes

FIXATION-DECALCIFICATION

1. Lillie (1954B) recommends 1-2 days:

picric acid, 1-2%, aqueous (1-2 gm./100 ml.
WCET) “AE, AEE ON aT Uy TES ane ae LTE 85.0 ml.
Pormialins’ Pe: ee oi. PE Ee eee eh ae, 10.0 ml.
formic acid, 90-95% aqueous (90-95 ml./10—5
mil: Swatery PSR) CAEN 8 EE 2 RRR Sree aA td 88 5.0 ml.
Extract some of the yellow: 2—3 days in 70-80% alcohol.
2. Lillie (1954B) also recommends:
Add 5%, ot 90°, formic acid to Zenker's fixative:
3. McNamara et al. (1940):
MiehCuUnTC- CHIONIGEe 4.5.5 ..e ewe ee 10.0 gm.
Gistitled Water... ic. cok ceeee uc fy} Pane Pee 300.0 ml.
Dissolve with heat. Cool.
taichilomicetic: Acid js eee ce ee ee aa 30.0 gm.
GistmlleGesvaley 0. Swale eee eRe ee 100.0 ml.
Dissolve and then add:
NUCKIiG acid, concentrated -6 ook ae ad eee 5.0 ml.
Go Ueeudyl alcohols es. ea hoe oe ea ay Casa 50.0 ml.
formalin). 2: See See are a ee 40.0 ml.
Mix the two solutions. Change daily until bone is soft. If more
than 7 days are required, nuclear staining is impaired. Running
water: 24 hours; dehydrate and embed.
4. Perenyv’s fluid, also a fixative, page 20.
Good for small deposits. Little hardening effect. Excellent cytolog-
ical detail. Good for calcified arteries, and glands, thyroid. 12-24
hours. Wash in 50-70% alcohol.
5. Schmidt (1956) uses the following for 24-48 hours, pH 7-9:
4%, formalin (4 ml./96 ml. water) plus | gm.
sodium acetate 100.0 ml.
disodium: Versenate 22 4 ener ee. cee 10.0 gm.

No washing necessary. Dehydrate and embed as usual.

Decalcification 29

DECALCIFICATION-DEHYDRATION
Jenkin’s fluid (Culling, 1957):
aDsoluteretuyltalconoll ... ~..:.dWienyess, ce hia pee 73:0 mi:
AIStCMEMRWALEE 2a. Batic + 3 x sed yn eulaged ates ae 10.0 ml.
GhlOrolonmin aaa st ties fo 4c ak alee ee Se 10.0 ml.
SlagialaCCtiCiaClG: clits 55 bch owe wae Sade de 3.0 ml.
hydrochloric acid, concentrated . 724... 2..9:3 2. 4.0 ml.

The swelling action of the acid is counteracted by the inclusion of

alcohol. Large amounts of solution should be used, 40—50 times bulk
of tissue. After decalcification, transfer to absolute alcohol for several
changes, then clear and embed.

The major portion of this book will be devoted to sectioning methods

for preparing tissue for staining, because of complexity and quantity of
such methods. Brief mention, however, should be given to other means
of examining tissues.
Exceedingly thin membranes can be examined directly by mounting
in glycerol or other aqueous media. Considerable detail can be observed
with reduced light or under the phase microscope. Sometimes a bit of
stain can be added to sharpen or differentiate certain elements. More
permanent preparations can be secured with fixation as discussed in
the preceding pages.
“Touch” preparations are made by pressing the cut surface of fresh
tissue against a dry slide. Cells adhere to the surface and can be exam-
ined unstained, or the slide may be immediately immersed in a fixative
and then stained.
Occasionally, free-hand sections of relatively tough tissue are cut for
examination.
Smears are one of the commonest devices for simple slide prepara-
tion: blood and bone marrow (page 219), Papanicolaou (page 357),
fecal (page 386), and chromosomes (squash preparations, a modification
of smears, page 367).
“Cell blocks’—concentrated clusters of individual cells or grouped
cells—are described in detail on page 38.
 Chapter 2

Dehydration

Preparation for Embedding

Tissues fixed in aqueous solutions will maintain a high water con-
tent, a condition that can be a hindrance to later processing. Except in
special cases (freezing method, water-soluble waxes, and special cell con-
tents), the tissue must be dehydrated (water removed) before certain
steps in this processing can be successful.
Tissues, during fixing and washing, lack an ideal consistency for sec-
tioning—cutting thin slices of a few microns in thickness. They may be
soft, or may contain a lumen or hollow spaces and are easily deformed
by sectioning. If the cells were pierced by a knife, their fluid content
could be released and this would allow the cells to collapse. ‘To pre-
clude these problems, the fluids in the tissue are replaced by a medium
which hardens to a firm, easily sectioned material. The cells are filled
intracellularly and enclosed extracellularly with the medium and are
thereby protected during physical handling. The most universally used
media for this purpose are paraffin and nitrocellulose or some variation
thereof. Other media, less frequently used, are gelatin and water-soluble
waxes.
Various conditions determine the choice of medium. Paraffin is suit-
able for most histological and embryological purposes when thin sec-
tions (1-15 ») are required. Thin sections also can be prepared with
nitrocellulose. Serial sections, however, are made more easily with the
former than with the latter; also paraflin preparation requires a far

30
Preparation for Embedding on

shorter time. Impregnating with nitrocellulose has distinct advantages

when it is desirable to avoid heat and when a tissue becomes hard too
readily, or is too large for the paraffin technic. In nitrocellulose, shrink-
age is reduced to a minimum, whereas in paraffin it can amount to as
much as 8-20% of the original size. Gelatin can be used for extremely
friable tissue in the freezing technic, and water soluble waxes are used
when alcohols, hydrocarbons and the like must be avoided. ;
Before embedding in either paraffin or nitrocellulose, all water must
be removed from the tissue. This dehydration usually is achieved in a
series of gradually increasing percentages of alcohol in water. Gradual
changing through 30, 50, 70, 80, 95% and absolute alcohol is said to
reduce some of the shrinkage occurring in the tissue. In cases where
time does not permit such a series, the 30% and 80% steps, and even
the 50% change, may be eliminated without great damage to the tissue.
The time required for each step depends on the size of the object—14,
to 2 hours, maybe 3 hours in extreme cases. A second change of absolute
alcohol should be included to insure complete removal of water. But
tissue should remain in absolute alcohol only long enough to remove
all traces of water; a total of 2-3 hours should be ample, even for large
pieces. Too long an exposure to 95% and absolute alcohol tends to
harden the material, making it difficult to section.
There are other agents which are just as successful dehydrants as
ethyl alcohol (ethanol). The ideal dehydrating fluid would be one that
can mix in all proportions with water, ethyl alcohol, xylene and paraffin.
Two such solutions are dioxane (page 39) and tertiary butyl alcohol
(butanol). Absolute butyl alcohol is miscible with paraffin, and after
infiltration with warm (50°C) butyl-alcohol-paraffin mixture, infiltra-
tion with pure paraffin can follow. If zsobutyl alcohol is used, there may
be some difficulty with impregnation, probably due to the limited mis-
cibility of this alcohol with paraffin and water. In both cases—tertiary
and isobutyl alcohol—there is more shrinkage than with other alcohols.
Isopropyl (99%) alcohol (isopropanol) is an excellent substitute for
ethyl alcohol, and is sufficiently water free for use as absolute alcohol.
Actually isopropyl alcohol has less shrinkage and hardening effect than
ethyl alcohol; it is cheaper and free of internal revenue restrictions. One
disadvantage must be remembered, isopropyl alcohol cannot be used
prior to nitrocellulose embedding, since nitrocellulose is practically
insoluble in it. Also dyes are not soluble in it, so it cannot be used for
staining solutions.
For the preparation of dilutions of ethyl alcohol, it is customary to
use 95% alcohol and dilute it with distilled water in the following
32 . Dehydration (cHAP. 2)
manner: if a 70% solution is required, measure 70 parts of 95% alco-
hol and add 25 parts of distilled water to make 95 parts of 70% dilution.
In other words, into a 100 ml. graduated cylinder pour 95% alcohol to
the 70 ml. mark, and then add distilled water up to the 95 ml. mark.
Absolute alcohol is not accurately 100%, but may contain as much
as | or 2% water. If the water content is no higher than this, the abso-
lute alcohol is considered 100% for practical purposes in microtech-
nic. If it is necessary to make certain that the water content is no
more than 2%, add a few ml. of the alcohol to a few ml. of toluene or
xylene. If a turbidity persists, there is more than 2% water present. But
if a clear mixture remains, the alcohol is satisfactory as an absolute
grade.
Dilutions of isopropyl alcohol (99%) can be handled as a 100% solu-
tion; that is, for 70% use 30 ml. water to 70 ml. of alcohol, etc.
If distilled water is not provided in the laboratory building, a Barn-
stead Still! can provide a sufficient amount of water for an average
microtechnic laboratory. The stills are available in sizes of 14 up to
10 gallons of water produced per hour. A special model will produce
30 gallons per hour.

Special Treatment for Small, Colorless ‘Tissues

Often a tissue is small and lacking in color, and seems to disappear
into the opaqueness of the paraffin. An easy answer to this problem is to
add some eosin to the last change of 95% alcohol; the tissue can then be
seen more readily and oriented with greater facility. This, however,
cannot be done if isopropyl alcohol is being used, since stains are not
soluble in it. The eosin will not interfere with future staining; it is lost
in the hydration series following deparaffinization.
1 Barnstead Still and Sterlizer Company, 49 Lanesville Terrace, Boston 13, Massachusetts.
 Chapter 3

Clearing, Infiltrating,
and Embedding for
the Paraffin Method

Clearing

In most technics that require dehydration and infiltration, an inter-

mediary step is necessary to hurdle the transition between the two. Be-
cause the alcohol used for dehydration will not dissolve or mix with
paraffin (exception, tertiary butyl alcohol as noted previously) some
fluid miscible with both alcohol and paraffin must be used before infil-
tration can take place.
The hydrocarbons benzene, toluene and xylene are reagents com-
monly used for this purpose, but if the tissue contains considerable
cartilage, or is fibrous or muscular, thus tending to harden readily,
sometimes it 1s wise to avoid these solutions. Xylene, formerly one of
the most widely used reagents, is the worst offender of the three in this
connection, and in most cases of clearing for infiltration it should be
abandoned in favor of one of the other two. Benzene presents fewer
hardening problems than either of the others, but because of its low
boiling point (80°C) sometimes is difficult for beginners to use. If a

3
34 | Paraffin Method (cuap. 3)

student is too slow while making his transfers from benzene into paraf-
fin, the benzene may evaporate out of the tissue and leave air pockets.
These will not infiltrate with paraffin. Of the three reagents toluene
probably is the safest to use; it does not harden as excessively as xylene,
and it has a higher boiling point (111°C) than benzene, thus eliminat-
ing some of the hazards of evaporation. (Boiling point of xylene, 142°C.)
In conclusion, benzene produces less shrinkage than either toluene or
xylene.
If tissue hardening does present a serious problem, then one of the
clearing oils can be used. Cedarwood oil is well known and is relatively
safe for the beginner, but overnight usually is required for complete
replacement of the alcohol in the tissue. Also, as is true of all oils, every
trace of oil must be removed during infiltration. Sometimes this condi-
tion is difficult to judge, since the oil may have a boiling point in the
200s and be slow moving out of the tissue. The action may be improved
by mixing oil with an equal amount of toluene. The cedarwood oil
method is an expensive one involving the use of a costly oil as well as
absolute alcohol. In this case the latter fluid may not be eliminated.
Chloroform is used in many laboratories, but has outstanding dis-
advantages. It dessicates some tissues, connective tissue in particular, and
has a boiling point of 61°C, making it highly volatile. Aniline can be
used with good results but it too is difficult to remove during infiltra-
tion. A mixture of equal parts of oil of wintergreen (methyl salicylate)
and aniline followed by pure methyl salicylate, and then methyl sali-
cylate-paraffin offers quicker and surer results. Methyl salicylate may be
used alone; also there are bergamot, clove, creosote, terpinol and other
oils. Amyl acetate and cellosolve (ethylene-glycol-monoethyl ether, Car-
bide and Carbon Co.) do not harden excessively; the latter, however, is
highly volatile.
If at any time during the clearing process the clearer (xylene, toluene
or benzene) becomes turbid, water is present and the tissue is not com-
pletely dehydrated. The only remedy is to return the tissue to absolute
alcohol to eliminate the water, then place it again in a fresh supply of
clearer. The solution originally used may contain water. Embedded
tissue containing water can shrink as much as 50%, and it offers difh-
culties in sectioning and mounting sections on slides.
“Clearing”? may seem a strange nomenclature for this intermediary
step, but it happens to be a special property of the reagents mentioned
above. They remove, or clear, opacity from dehydrated tissues, making
them transparent. Blocks of tissue appear to deepen in color; also they
seem almost crystalline, never milky.
Infiltration with Paraffin 35

Infiltration with Paraffin

Paraffin is considered to be either soft or hard. The melting point

of the former lies in either the 50-52°C or 53-55°C ranges; of
the latter in the 56-58°C or 60-68°C ranges. The choice of melt-
ing point depends upon the thickness at which the tissue is to be
sectioned, or upon the type of tissue—hard paraffin for hard tissues and
soft paraffin for soft ones. If relatively thick sections are to be cut, use a
soft paraffin; otherwise the sections will not adhere to each other in a
ribbon. If thinner sections are desired, down to a thickness of 5—7
microns, use a paraffin in the 56—58° grade. For extremely thin sections
of less than 5 microns, sometimes the best results can be obtained with
a hard paraffin of 60—68° melting point. The sections will retain their
shape and size without excessive compression and will ribbon better
than if the paraffin is too soft. In addition, room and temperature con-
ditions can influence the choice of paraffin. Often hot weather will force
the use of a harder paraffin than might be chosen during cool weather.
If it is impractical to stock more than one kind of melting point paraffin,
usually a 56—58°C is the safest choice.
Except in the case of friable tissues, the following step may be
omitted. After the tissue is well cleared by benzene or other clearer,
begin to saturate the solution with fine shavings of paraffin until some
of the paraffin remains undissolved. Leave the tissue in the saturated
clearer for 4—6 hours, or overnight for large pieces. Then with a warm
spatula remove the tissue to melted paraffin already prepared in an
oven.
In normal cases which eliminate the preceding step, tissues are trans-
ferred directly from clearer to paraffin. Keep the oven temperature only
high enough to maintain the paraffin in a melted state, no higher. This
lessens the danger of overheated tissue, which can initiate hardness and
shrinkage. Paraffin standing in a warm oven in a melted condition for
several days or weeks is better for infiltrating and embedding purposes
than freshly melted paraffin. After }—] hour in the first bath the tissue
is removed to a fresh dish of paraffin for a similar length of time. Two
changes of paraffin are sufficient for most normal requirements, but for
some cases of difficult infiltration, such as horny skin or bone, a third
change may be necessary, and the time of infiltration may need to_be
extended to as much as six hours over-all, even overnight. Such/CTises
fortunately are rare.
The use of a vacuum oven for infiltrating will remove air from some
36 . Paraffin Method (cuap. 3)

tissues (lung) and eliminate holes in the final paraffin block. (Weiner,
1957; Luna and Ballou, 1959)

Embedding with Parafhn

As soon as the tissue is thoroughly infiltrated with paraffin, it is ready
to be embedded; the paraffin is allowed to solidify around and within
the tissue. The tissue is placed in a small container or paper box
already filled with melted paraffin and the whole is cooled rapidly in
water. Before transferring the tissue, warm the instruments which
manipulate it. This will prevent congealing of paraffin on metal sur-
faces. Handle the tissue and paraffin as rapidly as possible to prevent
the paraffin from solidifying before the tissue is oriented in it. Orienta-
tion is important. If the tissue is placed in a known position and care-
fully marked with a slip of paper in the hardening paraffin (Fig. 9), it
remains a simple matter to determine the proper surface for sectioning.
If a paper box is used, the orienting mark may be made directly on one
of its flaps.
Each technician eventually adopts his or her own pet embedding
mold or container. A few suggestions: petri dishes, syracuse watch
glasses, shallow stender dishes, test tubes to concentrate solid contents
of tissue or body fluids, and a neat little dish for tiny pieces—perhaps a
miniature syracuse watch glass (watch glasses, U.S. Bureau of Plant
Industry Model, 20 mm. inside diameter, 8 mm. deep, A. H. Thomas
Co., #9850). Lightly coat the insides of glass dishes with glycerol; then
the solidified paraffin block loosens readily. Cast lead L s (Lipshaw
#334, Diamond embedding boxes) when placed on a small flat metal
rectangle can be adjusted to almost any size for embedding, and, being
metal, they cool the paraffin more quickly than glass. Also they break
loose immediately from the paraffin. Lipshaw also has a Pop-out Em-
bedding Mold.
Paper boxes may be fashioned according to Figs. 1, 2, 3. Perhaps the
one advantage of these is that they and any data recorded on them can
be left permanently on blocks which have to be stored. The Lipshaw
Company offers a disposable mold, called the Peel-A-Way Disposable
Embedding Mold. These molds are made of lightweight plastic, avail-
able in five sizes, and are easily broken at the corners so the sides can be
peeled down and the moll pulled away. Perforated tabs to fit can be
purchased. Various devices such as refrigerator trays with their dividers
can be used for embedding a number of large pieces of tissue. Lipshaw
Embedding with Paraffin 37

Figures 1, 2, and 3
Folding a bond paper or
an aluminum foil box for
paraffin embedding.

——

3
offers an embedding combination with 20 or 30 compartments. The
literature and catalogs are full of ideas; the above are just a few of them.
When small amounts of paraffin are ready to be solidified, they can be
cooled immediately in water, preferably at a temperature of 10—-15°C.
Make certain that a solidified scum has formed over each potential block
before dipping it below the surface of the water. Water colder than
10°C causes the block to contract too strongly and it may finally crack.
Furthermore, normal crystals may form in the center, but abnormal
ones in the periphery of the block. The perfect block is one in which the
paraftin crystals are contiguous with each other and the paraffin appears
clear and homogeneous. Paraffin may contain 7-15% air dissolved in it
and will appear clear when that air is distributed evenly through its
mass. But pockets of air produce milky spots, a condition called ‘“‘crystal-
lization.” Either slow hardening of the block in the air, or too rapid
cooling may cause this effect, particularly in the case of large blocks.
38 , Paraffin Method (cnap. 3)

Quick hardening of outer surfaces will trap the air. When these prob-
lems arise, cool the blocks from the bottom, and with a hot instrument
keep the upper surface melted for a short time, thus releasing some of
the air. Then blow across the top until a scum forms, and ease the block
into water. This treatment also prevents excessive shrinkage in the
center of the block and the enclosed tissue. (Dempster, 1944.)
If the paraffin does crystallize, difficulty may be encountered during
sectioning; the only remedy is to return the block to melted paraffin,
allow it remelt and repeat the embedding process. Experienced tech-
nicians soon learn how fast to handle the paraffin and reduce crystalliza-
tion to a Minimum.
Thoroughly hardened paraffin blocks can be stored indefinitely with-
out injury to the tissue, but they must be kept in a cool place where they
cannot soften or melt.

Embedding Cellular Contents of Body Fluids

“Cell blocks” are clusters of individual cells which have been concen-
trated and embedded for sectioning. The process for embedding them
is as follows:

1. Collect the material in centrifuge tubes and add fixative: 1 hour, or over-
night. Agitate occasionally.
2. Concentrate by centrifuging (preferably in small tubes); decant and add
water or alcohol depending on requirement of fixative. Loosen material
at bottom of tube and stir with small glass rod: 5-10 minutes, or longer.
3. Centrifuge and decant. Add next solution and stir thoroughly. Dehydrate
in this manner, and clear: approximately 10-15 minutes for each step
depending on size of particles.
4. Add melted paraffin and place tube upright in a glass or beaker in oven.
Stir slightly with warm instrument to work paraffin to bottom of tube: 30
minutes. Stir a bit once during this period.
5. Cool test tube.
6. Break tube. Place paraffin block in tiny paper box in a small dish of
paraffin in the oven. Leave only long enough for block to begin to soften.
Quickly cool paper box.

Clark (1947) suggest an alternate method:

1. Warm tube carefully in water, slightly warmer than paraffin. As soon as
paraffin against glass melts, tip tube and allow paraffin block to slide out.
Embedding Cellular Contents of Body Fluids 39

2. Mount block in a small hole dug in a square of paraffin and blend two
together with a warm needle or spatula.
Farnsworth (1956) transfers minute pieces with a pipette to a piece of lens
paper lying on a blotter. The latter absorbs the fluid, then the lens paper
bearing the tissue is laid flat on the surface of melted paraffin. She embeds
in depression slides wiped with glycerine.
Arnold (1952) concentrates small organisms in agar and carries through with
the usual processes.

[See also DelVecchio et al. (1959); DeWitt et al. (1957); McCormick

(1959B); Seal (1956); Taft and Arizaga-Cruz (1960).]

Dehydration and Clearing Combinations

Dioxane Method.
Any procedure which shortens the processing time for embedding has
considerable merit and finds favor among technicians. In 1931 an article
appeared concerning a reagent, which dehydrated as well as cleared in a
minimum of steps. Graupner and Weissberger (1931) proposed the use
of dioxane (diethyl dioxide), miscible with water, alcohol, hydrocarbons
and paraffin. It seemed to eliminate some shrinkage and hardening, and
was a relatively inexpensive method because fewer solutions were re-
quired. Dioxane itself, however, is far more costly than alcohol.
Some technicians claim that it has other disadvantages. Conn (1939)
cautioned that dioxane was cumulatively toxic and that it should not
be used by any person with liver or kidney trouble. Navasquez (1935)
reported that dioxane toxicity for animals was relatively low, that there
Was no accumulative effect, and that a tolerance would develop. Such
cases as were noted in man came after a period of heavy exposure to the
vapor. It was acute, not a chronic, poisoning. Fairly et al. (1936) said
there is some evidence that toxic effects are due to oxidation products:
oxalic acid and diglycolic acid, which are considered to be nontoxic to
man. Perhaps it is well to be cautious; use dioxane only in a well-
ventilated room and away from the nose. Avoid unnecessary soaking of
hands in it. Keep dioxane containers tightly closed at all times.
Because of its low tolerance to water, carelessness in the use of
dioxane can lead to trouble. If the tissue shrinks (it may shrink as much
as 40-50%) during infiltration with paraffin, water is present. The
author has seen this happen frequently for beginning students. Stowell
(1941) reported 35% shrinkage with dioxane and further condemned it,
saying that some brands already contain at least 10% water and other
impurities even before use. Cloudiness appears at 1% water content.
40 j Paraffin Method (cuap. 3)

Miller’s Schedule for Dioxane (1937)

1. Move directly from fixative into dioxane 3 parts, distilled water 1
part: 3-1 hour.
Dioxane: | hour:
Fresh dioxane: 2 hours.
aeParaffin | part, dioxane | part (in paraffin oven): 2—4 hours.
at
Paraffin: overnight.
SsEmbed.

Many technicians consider it advisable to place anhydrous calcium

chloride in the bottom of the dioxane container during dehydration as
an aid in removal of all water, thereby reducing the possibility of carry-
ing water into the paraffin. Mossman (1937) observed that dioxane reacts
with CaCl» and makes it swell as though water is present. He suggested
that CaO (calcium oxide) is better, but feels that this step is unnecessary.
If a fixative containing potassium dichromate is used, the tissues must
be washed thoroughly before using dioxane; otherwise the dichromate
will crystallize.

Tetrahydrofuran (THF) for Dehydration

Faust (1958, 1959) recommends THF for dehydration for several
reasons. It mixes with water in all proportions, also with paraffin de-
pending upon temperature, becoming increasingly miscible as the
temperature rises. It is miscible with nearly all solvents and can be used
as a solvent for mounting media. Most dyes are not soluble in THF, but
iodine, mercuric chloride, acetic acid and picric acid are soluble in it.
THF has a low boiling point (65°C) so it evaporates rapidly and must
be kept tightly closed at all times. It has to be regenerated occasionally
to remove accumulated peroxides and water.

Haust’s Method, Following Fixation

THF 1 part, water | part: 2 hours.
THF, 3 changes: 2 hours each.
THF | part, paraffin 1 part, 53-54°C: 2 hours.
Paraffin: 2 hours.
Of
B®
Or
NO Embed.
Tetrahydrofuran, highest quality, can be obtained from Eastman
Kodak Co., and Fisher Scientific Co.
The Fisher Company in The Laboratory, Clinical Edition (28, #C4,
1960), suggests that instead of repurifying THF over metallic sodium, a
Timing Schedule for Paraffin Method 4]

simpler and safer way involves passing the THF through a column of
activated alumina. ‘

Timing Schedule for Parafhin Method

Size 10 + mm.—smaller pieces will require less time, larger pieces
more time.

Schedule Using Ethyl Alcohol

1. A general fixative: overnight or 24 hours. (Bouin’s, Susa’s, Stieve’s,
Formalin, Zenker’s')
2. Wash in water, running if possible: 6-8 hours or overnight. Excep-
tions: Bouin’s, Susa’s and Stieve’s fixed tissue can be transferred
directly to 50 or 70% alcohol without washing.
3. ‘Transfer to 50% alcohol: | hour.
4. Transfer to 70% alcohol: 1 hour.
(See special iodine treatment for mercuric chloride fixatives, page
12)
Transfer to 95% ethyl alcohol: | to 1$ hours.
Transfer to absolute ethyl alcohol #1: } to 1 hour.
Or Transfer
“ID to absolute ethyl alcohol #2: 3 to | hour.
8. Transfer to toluene #1: $ to 1 hour.
9. Transfer to toluene #2: $} to | hour.
10. ‘Transfer to melted paraffin #1: § to | hour.
11. Transfer to melted paraffin #2: 3 to 1 hour.
12. Embed.

Alternate Schedule Using Isopropyl Alcohol

(steps I through 4 the same as above)

5. Transfer to isopropyl alcohol #1: $ to 1 hour.

6. ‘Transfer to isopropyl alcohol #2: } to 1 hour.
7. ‘Transfer to toluene #1: 4 to 1 hour.
8. Transfer to toluene #2: 4}to 1 hour.
9. ‘Transfer to melted paraffin #1: $ to 1 hour.
‘If tissue fixed in Zenker’s tends to harden, do not leave it overnight in the fixative;
transfer to 3-5% aqueous potassium dichromate for overnight, then proceed as usual on
schedule.
42 | Paraffin Method (cuar. 3)

10. Transfer to melted paraffin #2: $ to hour.

ll. Embedk
If the tissue is well hardened by the fixative, it is not necessary to
dehydrate the tissue through a number of graduated steps, such as 50,
60, 70, 80, 95% and absolute alcohol. Even the elimination of the 50°,
and 60% steps is possible when time presents a problem; also the use of
isopropyl alcohol materially shortens the schedule.
In passing tissues from one fluid to another, use the decantation
method. This avoids excessive manipulation with forceps and reduces
injury to the tissue. After pouring off a solution, drain the tissue bottle
briefly against a paper towel or cleansing tissue to pull off as much as
possible of the discarded solution. This reduces contamination of the
new solution. Since 95% alcohol, absolute alcohol, clearers and melted
paraffin all contribute to the hardening of the tissue, avoid leaving it
in any of these fluids for longer than the maximum time (preferably
leave only for the minimum period), and never overnight. Effective
infiltration takes place only when the paraffin is actually melted. Partly
melted, “mushy’’ paraffin penetrates poorly, if at all.

Automatic Tissue Processors

Most large laboratories now handle the foregoing processes by ma-
chine; the changes are controlled with a timing device, and the tissues
are shifted automatically through a series of beakers or other type of
container. The timing device can be set on a schedule to handle the
changes during the night in order that the tissues will be ready for
embedding when the technician arrives at the laboratory in the morn-
ing. Small metal or plastic receptacles with snap-on lids hold the tissues
and labels, and are deposited in a basket which clips into the bottom of
the lid of the instrument. When the time arrives for removal of the
tissues to a new solution, the lid rises and rotates to lower the basket
into the next container. The two final beakers are thermostatically con-
trolled paraffin baths. The technician can set the timing device for any
interval desired—15 minutes, 30 minutes, | or more hours, etc.—over
a period of 24 hours. The newest models have a clock which can control
the instrument over a week end or several days. (The above description
applies to the Autotechnicon of Technicon Company.)
There are several tissue processors on the market; in addition to the
Autotechnicon, there is a Lipshaw model, and the Tisswematon of the
Fisher Scientific Company. Complete operational directions are sup-
plied with all models.
 Chapter 4

Microtomes and
Microtome Knives

Microtomes

The first instrument for cutting sections was made by Cummings in

1770. It was a hand model that held the specimen in a cylinder and
raised it for sectioning with a screw. In 1835, Pritchard adapted the
instrument to a table model by fastening it to a table with a clamp and
cutting across the section with a two-handled knife. These instruments
were called cutting machines until Chevalier introduced the name
microtome in 1839. Sliding cutting machines were developed in 1798,
rotary microtomes in 1883 and 1886, and the Spencer Lens Company
manufactured the first clinical microtome in 1901. The large Spencer
Rotary with increased precision became available in 1910. (Richards,
JOF9.)

1. The rotary microtome for paraffin sections; the most widely used method.
2. The sliding microtome for nitrocellulose (celloidin) sections; not always
the most practical method, slow and expensive, but often unexcelled for
hard and large objects such as eyes, bone and cartilage, also in cases when
shrinkage must be kept to a minimum.
3. The clinical (freezing) microtome for unembedded tissues; quick, cheap,

43
44 Microtomes and Microtome Knives (CHAP. 4)

and required for some processes when certain cell components must be
retained, such as fat and enzymes, also for immediate diagnosis.
4. The ultra-thin sectioning microtome for sections thinner than | micron,
electron microscopy. Only for special technics and not commonly found in
laboratories.
5. The base sledge microtome for exceedingly large tissues (brains) and hard
blocks of tissues. Only for special technics and not commonly found in
laboratories.

Microtomes should always be kept well oiled to prevent parts from

wearing unnecessarily or sticking. Either of the latter faults can cause
imperfect sectioning—sections of variable thickness. Obtain advice
from an expert concerning the parts to be kept oiled, and consult the
booklet accompanying the instrument. The best oil recommended for
this purpose is Pike Oil, manufactured by Behr-Manning, Troy, N.Y., a
Division of Norton Co., Abrasives.

Microtome Knives

There are three familiar types of microtome knives:

1. The plane-edge for frozen sections and paraffin ribbons.
2. The biconcave used sometimes for paraffin work.
3. The plane-concave for celloidin, sometimes for paraffin.
Because knives seem to demand hours of attention, they often become
the technician’s nightmare, and the task of keeping them in optimum
condition presents problems.
Theoretically, a perfect cutting edge is the juncture of two plane
smooth surfaces meeting at as small an angle as is feasible—ideally 14°
as suggested by Dr. Lorimer Rutty (Krajian and Gradwohl, 1952, page
28). These cutting surfaces are called the cutting facets. The cutting
edge of a very sharp knife, when examined by reflected light under 100
magnifications, appears as a fine discontinuous line. It may vary slightly
in width, but it should show only a slight reflection, a narrow, straight
bright line. At a higher magnification of about 500 times, the edge will
have a finely serrated appearance. The fineness or coarseness of these
serrations depends on the degree of success in sharpening the knife. The
facets are determined by the “back” which is slipped on the knife during
sharpening to raise it just enough to form these facets (Fig. 4). Every
knife must have its own back. Never interchange backs. ‘The back must
Microtome Knives 45

Figure 4. Microtome knife bevel as controlled by knife back.

fit tight enough so that it does not move about on the knife. When it
shows considerable wear, buy a new one, because as the back wears the
cutting facets are widening. Do not ram the knife into the back; hold
the knife in one hand and the back in the other and work it on gradually
with a rocking motion.
The importance of taking good care of a knife cannot be overem-
phasized. Clean it after use; some materials like blood and water, when
left on the knife edge, corrode it. Clean with xylene on soft cleansing
tissue and finally wipe it dry. Do not strike the edge with hard objects
(section lifter, dissecting needle); the edge can be damaged or dented.

Sharpening Knives
The glass plate has had the longest and most successful use as a sharpen-
ing instrument. In 1857 von Mohl used a thick glass plate; a rotating
one was developed by von Apathy in 1913, and a vertical one by Long
in 1927. Leather strops were advocated in 1922, and a couple of years
later carborundum hones, followed by finer grained hones or stropping
on canvas or leather. Many argue against a strop, saying that a knife
can be honed sharp enough for good sectioning. If, however, there are
fine serrations, gentle stropping frequently can remove them and im-
prove the cutting ability of the knife.
If a glass plate is used, it should be at least 34; of an inch thick,
approximately 14 inches long, and an inch or two wider than a micro-
tome knife. Levigated alumina (approximately 1%) added to a neutral
soap solution is excellent for sharpening; the polishing should be done
46 Microtomes and Microtome Knives (CHAP. 4)

Figure 5. First set of strokes for microtome knife honing.

with Diamantine—a small amount. A final stropping will finish off fine
serrations (Uber, 1936).
Arthur H. Thomas manufactures a knife sharpener, the Fanz model,
using a revolving glass disc over which the knife is swept back and forth.
Some pointers on the use and care of hones and strops may be of
help to beginners. Never apply oil to hones, only water and neutral soap
solution or Lava soap. Never allow the hone to dry while in use; always
keep it wet with water, or water plus soap. Two types of hones can be
used, a coarse one for fast grinding of the knife edge when the nicks are
deep, and a polishing hone to remove serrations and small amounts of
metal left by the preliminary honing. A yellow Belgian hone is one of
the best.
Two patterns are followed, using the same number of strokes per
pattern. In Fig. 5 follow course #1, moving from bottom right to top
Microtome Knives 47

Figure 6. Second set of strokes for microtome knife honing.

left, roll knife over on its back, and return over same path in opposite
direction (course #2) moving from top left to bottom right—3 or 4
times. Move knife toward left into position in Fig. 6 and follow course
#3, moving from bottom left to top right, and return from top right
to bottom left (course #4)—same number of times as above. Repeat
these strokes on hone until deep nicks and scratches are eliminated.
When all nicks and deep serrations have been removed (check under
dissecting scope), the knife is ready to be stropped, a final polishing
action. Do not use a sagging hammock-like strop unless it can be held
tightly flat, otherwise it rounds the knife edge. The best type is
mounted on a felt pad on a hardwood block. This allows a bit of
cushion, but on a solid surface. With a soft cloth keep the strop surface
clean and free of dust. Rubbing the leather with the hand improves
the texture. Do not use mineral oil on it. If the leather becomes dry,
work in Neetsfoot oil, working over small areas at a time, not the entire
strop. Buff with soft towelling at once; do not allow the oil to sit on it
and soak in or develop a gummy mess.
48 Microtomes and Microtome Knives (CHAP. 4)

\i|
lj

Figure 7. First set of strokes for microtome knife stropping.

Lipshaw offers several block strop combinations; the revolving one is

especially handy.
First use a honing strop, usually made of pigskin with a fine abrasive
embedded in it. Then follow with the finishing strop, a fine-grain horse-
hide. The two patterns for stropping are similar to those of honing, but
with the cutting edge moving away from the strop. (Figs. 7, 8) Repeat
these strokes on the honing strop until fine abrasions caused by the hone
have been smoothed away, then polish with a few strokes on the finish-
ing strop. Only a dozen strokes on the latter should be necessary if the
preliminary honing and stropping have been properly done. While
honing and stropping, use both hands, left one on the back and adjacent
knife surface, and the right hand on the handle. Press on lightly, guid-
Microtome Knives 49

Figure 8. Second set of strokes for microtome knife stropping.

ing with both hands and with uniform pressure from both. Always use
the same pressure on the forward strokes as on the return ones. Never
hurry honing or stropping; use at least one full second for each stroke.
Mechanical knife sharpeners are on the market; almost every scien-
tific supply company has at least one. One of the best, an automatic
sharpener, is now in production by the American Optical Company.
Most technicians develop a fancy for one type in preference to others;
the author, therefore, considers it expedient not to offer any recommen-
dation.
If the cost of microtome knives and their maintenance is prohibitive
for a class of beginning students, the Spencer Razor Blade Holder can
be used to advantage. A student can use a razor blade once and throw it
away if the edge has become dull, or sharpen it up a bit on an old knife
strop.
50 Microtomes and Microtome Knives (CHAP. 4)

The life of the sharp edge of a good knife can be prolonged if the
technician has another knife for the preliminary trimming of paraffin
blocks. After undesirable parts of the tissue block have been trimmed
away, the good knife can be substituted for the old one, and the re-
quired sections collected.
 Chapter 5

Paraftin Method

Sectioning
The embedded blocks (Fig. 9) are trimmed into squares or rectangles,
depending on the shape of the tissue, with two edges parallel (Fig. 10).
The two short or side edges need not be parallel, sometimes with ad-
vantage as will be indicated later. Wooden blocks or metal object discs,
such as are sold by supply houses (Lipshaw #800), are covered with a
layer of paraffin. (Suggestion: Keep a 1-inch brush in a beaker of melted
paraffin in the paraffin oven.) Then a heated instrument (spatula or

Figure 9. Untrimmed paraffin tissue Figure 10. Trimmed _ paraffin

block. tissue blocks.

ml
52 Paraffin Method (cuar. 5)

slide) is held between the paraffin on the object disc and the under
surface of the tissue block (Fig. 11). When both surfaces are melted, the
instrument is withdrawn and the tissue block pressed firmly into the
paraffin on the object disc.
After cooling, the block is
ready to be sectioned (Fig.
12).
Set the Rotary micro-
tome for section thickness
(5 or 6 microns, etc.) . Raise
the tissue carrier and place
in it the object disc with
its mounted tissue block.
Tighten the clamp of the
tissue carrier onto the stem
of the object disc. If using
a wooden block, allow it to
extend about + inch be-
Figure 11. Mounting paraffin tissue block yond the metal clamps; this
on an object disc. will prevent the paraffin
block from breaking loose
from the wooden block when the clamp is tightened.
Insert the microtome knife and tighten its clamps. The knife must
be held in the clamps at a proper angle for optimal sectioning, produc-
ing a minimal amount of compression, and allowing the sections to
adhere to each other. Many suggestions can be made concerning angle
determination, but in most instances the technician finds the answer
after a process of trial and error. The cutting facets are small as deter-
mined by the back. When placed in the microtome, the knife must be
tilted just enough so the cutting facet next to the block and the surface
of the block clear each other. If the tilt is not sufficient (Fig. 13), the
surface of the block is pressed down with the wedging effect of the facet
and no section results. At the next stroke of the knife, this compression
increases, and finally a thick section is cut, a composite of the com-
pressed sections. Too great a tilt of the knife makes it scrape through
like a chisel, rather than cut through the tissue (Fig. 14).
Turn the feed screw handle (left side of instrument), moving the
feed mechanism backward or forward until the face of the tissue block
barely touches the cutting edge of the knife. With an old knife start
trimming into the block until the desired area is reached. Change to a
good knife and readjust tissue carrier if necessary. Never touch the edge
Sectioning 53

of the knife with anything hard. Fragments of paraffin can be flicked

off with a camel hair brush, or removed with a finger tip. Scratches
appearing in sections often can be remedied by rubbing the knife edge
with a finger tip, up-
wards. Also try this
motion on the back of
the knife; it will re-
move bits of para-
fhn which can cause
scratches. Cleansing
tissue dipped in xy-
lene also is helpful.
(Warning: discard the
first section after
cleaning the knife; it
probably is a_ thick
one.) When all sec-
tioning is completed,
xylene must be used
Figure 12. Tissue biocks mounted on an object
to clean the knife;
disc or wooden block and ready for sectioning.
leave no corrosive ma-
terial on its edge.
Section with an easy rhythm; never rush, or variable thickness of
sections is likely to occur. As the sections move down on the knife they
form a “ribbon,” each section adhering to the one that precedes it as
well as the one that follows it. During the cutting of the sections, a bit
of heat is generated, enough to soften the paraffin and cause the indi-

Figure 13. 9 Insufficient Figure 14. Excessive paraffin knife

paraffin knife tilt. tilt.
54 | Paraffin Method (cuap. 5)

vidual sections to stick to each other. As the ribbon forms, hold it away
from the knife with a camel hair brush and ease it forward so that the
sections do not remain on the knife. This is advisable to prevent the
sections from bunching and piling on each other. They can stack high
enough to topple over the edge and get caught between the tissue carrier
and the next stroke of the knife. The parallel edges of the paraffin block
must be cut clean and parallel. If the edges have not been trinimed, but
remain as the original sides formed by the mold, a ribbon will not form.
In dry weather static frequently becomes a problem during section-
ing. The sections stick to the knife or to parts of the microtome. They
break apart and stay bunched up instead of lying flat. The friction of
the knife as it crosses the paraffin block forms static electricity on its
surface. An inexpensive little instrument, the Reco Neutra-Stat,’ may
be used to relieve this situation. An alpha radiating static eliminator
strip in a slot on the head of the instrument irradiates the air with
harmless alpha particles. These ionize the air and discharge static
charges from the block surface.
The ribbons formed during sectioning can be mounted directly on
slides, floated on a water bath, or laid in order in a box. The latter is
particularly convenient when the slides cannot be made immediately, or
when numerous sections have to be cut, serial sections for instance.
Hosiery boxes make handy containers, and if painted black inside with
India ink, provide an excellent dark background for the sections. If the
shorter edges of the block were not trimmed parallel, a serrated edge
along the ribbon indicates the exact position of each individual section,
minimizing the danger of cutting through one. The sections can be
stored in boxes until all required slides have been mounted. Valuable
sectioning time is conserved by this means.

Difficulties Encountered While Sectioning and

Suggested Remedies?
1. Ribbons are crooked.
a. Wedge-shaped sections caused by poor trimming; sides of paraffin
block are not parallel, or not parallel to edge of knife.
b. Part of knife edge may be dull; try another part of it.
Uneven hardness of the paraffin; one side may be softer than the
other, or contain areas of crystallization. Reimbed.
' #61-579 Reco Neutra-Stat, Model M, for microtomes. E. Machlett and Son, 220 E. 23rd
Street, New York 10, N.Y., or from Lipshaw Manufacturing Company.
* Modified after Richards, 1949.
Sectioning 55

2. Sections fail to form ribbons, usually due to hardness of paraffin.

a. Use softer paraffin (lower melting point).
b. Blow on knife to warm it or dip it in warm water.
c. Cut thinner sections.
d. Place table lamp near knife and block to warm it.
e. Resharpen knife.
f. Lessen tilt of knife, and clean edge.
g. Dip block in softer paraffin and retrim so a layer of this paraffin
surrounds original block.
©9 Sections are wrinkled or compressed.
a. Resharpen knife; a dull knife compresses badly.
b. Paraffin too soft; reimbed in harder paraffin.
. Cool block and knife.
. Increase tilt of knife.
ro
QS . Clean edge of knife with finger or xylene, remove any paraffin
collected there.
f. Tissue is not completely infiltrated,* or too much crystallization
is present.
g. Soak tissue block in water,+ $-1 hour or overnight if necessary.
4. Ribbons are split or scratched longitudinally.
a. Nick in knife; move to another part of edge or resharpen it.
b. Knife dirty or gritty along edge.
c. Dirt or hard particles in tissue or in parafhn, crystals from fixing
solution not adequately removed. Filter paraffin, decalcify or
desilicify tissue.
d. Decrease tilt of knife.
e. Tissue too hard; soak in water.t
¢
aon Tissue crumbles or falls out of paraffin.
a. Poor infiltration;* reimbed.
b. Not completely dehydrated.
c. Not completely dealcoholized.
Too long in paraffin bath or too hot while there; soak in water.
e. Clearing fluid made tissue too brittle: soak in water.
* Most conditions of poor infiltration are due to traces of water or alcohol. This will
have to be corrected by first removing such paraffin as is present. Soak the block in toluene
for 2 or 3 hours (or more); change twice, then place in absolute alcohol for 1 or more
hours. This should remove all traces of water. Clear again in toluene (check against milky
appearance); re-infiltrate and embed.
+ When soaking in water is recommended, the cut face of the tissue is exposed to tap
water for 14-1 hour, or overnight for stubborn cases and when time is of no concern. The
author has found this treatment completely satisfactory, but some technicians advocate the
addition of glycerol (1 part to 9 parts water) or 60% ethyl alcohol (Lendrum, 1944). The
fluid works in through the cut tissue surface and softens tough parts. Exception: Do not
soak nervous system tissue at any time and lymph nodes and fatty tissue only briefly.
 Paraffin Method (cuap. 5)

Sections cling to block instead of knife.

a. Knife too dull or dirty.
b. Increase tilt of knife.
c. Paraffin too soft or room too warm. Try harder paraffin or cool
block.
d. Infiltrating paraffin too hot, or too long exposure to solutions
which harden; soak in water.t
~I Tissue makes scratching noise while sectioning.
a. Tissue too hard; paraffin too hot or too long exposure to solu-
tions which harden; soak in water.}
b. Crystals in tissue; fixing reagents not removed sufficiently by
washing; calcium or silicon deposits present. (See pages 24 and
312s)
. Knife rings as it passes over tissue.
a. Knife tilted too much or too little.
b. Tissue too hard and springs back edge of knife; soak in water.t
c. Knife blade too thin; try a heavier one.
. Sections curl, fly about or stick to things; this is due to electrification
from friction during cutting, especially in weather of low humidity.
a. Increase humidity in room by boiling water in open pan.
b. Ground microtome to water pipe.
c. Postpone sectioning until weather is more humid. Early morn-
ing sectioning often is best.
d. See page 54.
10. Sections are skipped or vary in thickness.
a. Microtome in need of adjustment or new parts.
b. Tighten all parts, including knife holder and object holder
clamp.
c. Large or tough blocks of tissue may spring the knife; soak in
water.f
d. Knife tilt too great or too little.

Mounting

Slides and Cover Glasses

Most microscopic materials (sections, whole mounts, smears, touch

preparations) are mounted on class slides and are covered with micro-
scopic cover glasses. A noncorrosive quality should be purchased to
insure a nonreacting surface. The slides may be obtained in the regular
3 x 1 inch size or in 3 X 2 inch for larger tissue sections. The brands
Mounting 57

labeled “Cleaned, ready for use,” save many technician hours devoted
to cleaning the uncleaned varieties. In addition, they permit a uniform
smear for blood smears and the like. .
Cover glasses are manufactured in circles, squares and rectangles, in
thickness ranging from #0 to #3. Thickness #0 is used rarely, when
an oil immersion objective rides unusually close to the slide. This size
is so thin that it fractures easily. Thickness #1 (about 0.15 mm.) usu-
ally is adequate for oil immersion work. Thickness #2 (about 0.20 mm.)
can be used for whole mounts or when oil immersion is unnecessary.
Thickness +3 (about 0.30-0.35 mm.) is occasionally used when a
tougher glass is required and for large whole mounts. Circles and
squares are purchasable in sizes up to { inch in diameter. Circles are
designed for mounts when ringing the cover is necessary. Whole mounts
in glycerol jelly or any other volatile mountant must be sealed against
evaporation. A ringing table can be used to make the supporting ring
for the cover glass and then the final seal around the edge of the cover
glass. The rectangles come in 22 and 24 mm. widths, and in 30, 40, 50
and 60 mm. lengths. Cover glasses will mount more efficiently if they
are cleaned in absolute alcohol and wiped dry. Fewer bubbles cling to
the glass when it is pressed into place over the mounting medium.

Mounting Technics

If paraffin sections are to be stained uniformly, the embedding solution

is removed, but the sections could lose their unity if they are not affixed
to the slides. Also mounting on slides permits handling of many sections
simultaneously, as is desirable for serial sections.
Before sections are mounted, a glass marking pencil should be used
to make an identifying mark on one end of the slide—a tissue number,
student locker number, research project number or a similar identifica-
tion to prevent confusion with other slides. In addition, this enables the
worker to distinguish on which side of the slides the sections are mounted
and thus prevent failure during the staining process. Types of permanent
glass marking pencils include diamond points (a real commercial dia-
mond mounted in a handle) and s/eel pencils with tungsten-carbide tips
(Fisher Scientific Company #13-378). A vibro-engraver or vibro-tool
can be used for engraved marking on the glass. Wax china-marking
pencils make only temporary inscriptions which dissolve in the staining
solutions. Some inks are reasonably satisfactory; one of the best is Gold
Seal Laboratory Ink (#A-1408, Clay-Adams, Inc., N.Y.). Stafford (1960)
58 | Paraffin Method (cuap. 5)

uses a waterproof pencil (Venus Unique Blue Waterproof Pencil

+1206, American Pencil Co.) on slides with frosted ends, but this may
be removable in xylene or alcohol.
‘The customary means of affixing sections to slides is attachment with
egg albumen and water. (Gelatine and blood serum also can be used,
page 414.) Absolutely clean slides are essential to insure adherence of
the sections throughout any staining procedure. With one finger smear
a thin film of albumen fixative (adhesive, page 412) on the slide, and
with a second finger wipe off excess albumen. This should keep the film
thin enough. Thick albumen picks up stain, makes a messy looking
slide, and can obstruct a clear image of sharp uncluttered tissue ele-
ments. If there is a continued tendency to get too much albumen on
the slide, try a “floating solution”; dilute a couple of drops of albumen
fixative with about 10 ml. of distilled water and float the sections on
the solution on the slide (also see Schleicher, 1951). Lillie (1954B) sug-
gests that since water is used to float the sections on a thin coat of fixa-
tive and since the fixative constituents are water soluble, it is doubtful
that enough albumen remains on the slide to actually be an adhesive
agent. He considers it probable that the albumen acts as a surface ten-
sion depressant and aids in closer attraction of sections to slide.
If a water bath is used for spreading the sections, have the tempera-
ture of the bath about 5°C below the melting point of the paraffin
being used. When removing the sections from the microtome knife or
from the box, stretch them as flat as possible as they are placed on the
water surface. After they have warmed a bit, they can then be pulled
more nearly to their original size with a couple of dissecting needles.
With a hot needle or spatula separate the required sections from the
rest of the ribbon. Dip an albumenized slide under them and with
a needle hold them against the slide while removing it from the bath.
Drain off excess water and dry the slide on a warming table or in an
oven. Sections collected (glossy side down) in a box can be separated with
a spatula or razor and removed to an albumenized slide. Add enough
distilled water to spread well beyond the edge of the sections. Place the
slide on a slide warmer*® to correct any compression acquired during
sectioning and until the sections are stretched flat. Dense tissues will
compress less than the paraffin surrounding them and often will develop
folds because the paraffin does not expand sufficiently on the warm plate
to permit the tissues to flatten. Pieces with a lumen will invariably dem-
onstrate this fault. One of the simplest ways to correct this difficulty is
to break away the paraffin from the outside edge of the tissue. Do this
* One of the best is Fisher’s Slide Warmer, Catalog #12-594-5.
Mounting 59

Figure 15. Compressed or folded paraffin sections.

when the ribbon is floating on the water on the slide, but not while it
is warm, or the paraffin will stick to the dissecting needles. Have the
slide cool and split away the paraffin with care so that the tissue is not
injured (Figs. 15, 16, and 17). A rather violent method for straightening

Figure 16. Breaking paraffin with dissecting needles to permit ribbon ex-
pansion without folds.

stubborn ribbons is the addition of some acetone or alcohol to the water.

Slides that do not take the water smoothly may be dirty slides, but it
is more likely that the albumen was omitted. Excessive heat for flatten-
ing may cause shrinkage of cell structures and tearing and displacement

GLEE L;
Yea) DA NPN

Figure 17. Expanded and flattened paraffin sections,

60 Paraffin Method (cnap. 5)

of tissue parts. A safe temperature is approximately 5—10°C lower than

the melting point of the paraffin. When the sections are spread smooth,
the excess water may be drained off; this is facilitated by touching a
piece of cleansing tissue or filter paper to the edge of the slide and
around the sections. Permit slides to dry overnight on the warm plate
or in an oven of comparable temperature. If they must be stained on
the same day as they are mounted, dry them in one of the mechanical
hot air dryers (Technicon or Lipshaw Companies).
Certain tissues frequently develop cracks through the sections while
drying—an aggravation common to spleen, liver, lymph nodes and
nervous tissue. To prevent this, as soon as the sections are spread, drain
and blot them free of as much water as possible and dry them at a
higher temperature (60—65°) or in the mechanical hot-air dryer.
Properly mounted sections will have a smooth, almost clear appear-
ance. If they have a creamy, opaque texture, and when examined from
the undersurface reflect light, air is caught between the glass and sec-
tions. Such tissues will float off in aqueous solutions. The cause usually
lies either in poorly cleaned slides or in drying at too low a temperature
or not long enough. If, when mounting, an excess of bubbles appear
under the sections, use distilled water which has been boiled for some
time to release trapped air. Poorly infiltrated sections and nervous tis-
sue which has been soaked in water usually fail to adhere; these can
possibly be saved by the following method.
Because of their tendency to loosen from slides, certain types of tis-
sues may require special treatment. These tissues include brittle sec-
tions, tissues containing a large amount of yolk or osmic acid, and
sections which will be subjected to strong acids or alkalis during stain-
ing (as well as the poorly infiltrated sections and nervous tissue men-
tioned above). After drying, and when ready to stain, dissolve out the
paraffin with toluene or xylene. Place the preparations for 1-2 minutes
in absolute ethyl alchohol, followed by 1-2 minutes in a dilute solution
(about 0.5%) of nitrocellulose in ether-absolute alcohol (50:50). Drain
off excess nitrocellulose, but do not allow it to completely dry; wave
slide in air a second and place it in 70% or 80% alcohol. Continue with
the planned staining method.

Serial Sections

With a few exceptions, the usual paraffin method is used for serial sec-
tions, and in most cases, every section is mounted and in correct order.
While sectioning, the ribbons are arranged in a cardboard box from
Mounting 61

left to right and top to bottom (like a printed page). When the sectioning
is finished, the sections are removed to slides in the above order. Serially
number each slide with a glass marking pencil (do not use a grease pen-
cil and have it wash off in staining solutions). ‘This system affords simple
sorting of slides when all processing is complete.
 Chapter §

Freezing Method

The freezing technic tor preparing specimens is unsurpassed in two

ways: (1) it is rapid—essential for diagnosis in hospitals; and (2) it can
be used for the preparation of sections containing such elements as fats
enzymes and some radioscopes that would be lost in alcohol, paraffin
solvents or by heat. One disadvantage is that some distortion may be
caused by the freezing and cutting.
Freezing methods formerly used ether, ethyl chloride or other vola-
tile liquids. Present methods employ compressed carbon dioxide gas
blown into a chamber. There the gas expands, cooling the chamber,
and freezing the tissue mounted on it. For this procedure most labora-
tories use the clinical microtome with a freezing attachment. An attach-
ment also can be used on sliding microtomes, but not, efficiently, on
rotary models. There are also dry ice holders available, such as the one
manufactured by Fisher Scientific Company—a Dry Ice Freezing Cham-
ber. Dry ice is held inside the chamber and the specimen is frozen sim-
ply by pushing the dry ice up against the metal top holding the speci-
men. Nickerson (1944) fitted the object clamp of a rotary microtome
with a small metal box holding dry ice. A new freezing system using
Freon is on the market—the Histo-Freeze—a portable freezing unit,
12 & 18 inches, with Freon 12 circulating through the unit (manufac-
tured by Scientific Products, Evanston, Illinois). Model 670274 fits
American Optical, 67027B fits Bausch and Lomb, and 67027C fits the
Sartorius freezing microtomes. Lipshaw has a tissue freeze refrigerating
unit, counter or desk height, fitting American Optical, Bausch and
Lomb, Sartorious or Reichert microtomes.

62
Fixation, Blocking, and Sectioning 65

Fixation, Blocking, and Sectioning

Fresh tissues may be used, but it is preferable to fix the material,
usually in formalin, which leaves the tissue in a consistency ideal for the
freezing technic. Formalin is a water solution and requires no washing.
Hartz (1945), however, recommends Bouin’s as better than formalin,
but says that the tissue must be washed for a short time before freezing.
If the fixative contains mercuric chloride, the crystals may be removed
immediately after sectioning with an iodine solution, such as Lugol’s,
which contains no alcohol. If the tissues are extremely friable it may
be advantageous to embed them in gelatine (page 66) or immerse them
in a thick aqueous gum syrup, such as gum arabic.
When it is necessary to section immediately (example: surgical bi-
opsy), and yet fixation is desirable, boil a small piece for a couple of
minutes in 10% formalin. Rinse in water and freeze. Reiner (1953)
adds tissue to undiluted formalin at room temperature, then heats the
solution and tissue to 56-60°C in a water bath for 2 minutes. Agitate
the tissue during the heating. Reiner claims inferior results without
agitation and also says that placing tissues in preheated formalin is dis-
advantageous.

Friedland (1951) Method, Using Agar

1. Boil tissue in formalin.
2. Replace formalin with 2% sterile agar (previously heated to its melt-
ing point) in water. Boil gently 1 minute.
3. Pour off and freeze tissue immediately.
The Agar can be kept ready in Pyrex tubes, and melted quickly over
an open flame.

Lev and Thomas (1955) Method

This method fixes tissue at 80°C and produces excellent results. The
following solution is placed in a baby’s bottle-warmer and comes to a
boil in 1 minute. Add the tissue and allow it to boil for 1 more minute.
formalin hee) dc ccs ee ees a bss 10.0 ml.
Placial aCeMe acids. msc 2. tetas
ds De. Os 1.0 ml.
9o°7,,.ethyl aleonOleiss 22% 22..54 >< 25s 80.0 ml.

The tissue will have to be washed to remove the alcohol before freez-
ing can be attempted, possibly a disadvantage when speed is impera-
tive.
64 | Freezing Method (cuap. 6)

The pieces to be cut should be no more than 2—4 mm. thick for rapid
and uniform freezing. One surface should be flat to provide sufficient
contact with the tissue carrier. If, during freezing, there is a tendency
for the tissue to break loose from the carrier, cut a piece of filter paper
to fit the top of the carrier and place it, wet, under the tissue. This may
help to hold the tissue in place while freezing and sectioning.
Consider the shape and type of tissue when it 1s oriented on the car-
rier. If one side is narrower than the other, place that side, or any
straight side, parallel to the knife. If there is a tough membrane on one
side of the tissue, place that side toward the knife, otherwise the mem-
brane can break away from the rest of the block during sectioning.
Usually the amount of water carried over with the tissue is sufficient
for firm attachment to the carrier. (Use normal saline for fresh tissues.)
Avoid too much water and do not allow it to settle around the tissue
in a wall of ice that can deflect the knife or tear through the tissue dur-
ing sectioning. Uneven or torn sections result. If, however, gum is be-
ing used, it is necessary to build the gum around the tissue while freez-
ing it. In this case the tissue must be completely encased by the medium.
With a finger, gently press the tissue upon the tissue carrier. Slowly
freeze the tissue by turning on CO, for a moment or two, and then turn
it off. A series of successive jets of gas freezes better and wastes less gas
than a continuous stream. When the tissue is frozen fast and the finger
can be removed, move the knife over the block so it too is in the path
of the cooling gas. This aids in uniform freezing of the tissue by deflect-
ing some of the gas onto the top of it, and at the same time cools the
knife.
Good sections depend on the proper temperature in the tissue. Ma-
terial frozen too hard forms white brittle fragments on the knife; if too
soft it forms a mushy mass. In both cases, the sections break up when
placed in water. Difficulties may be avoided by slightly over-freezing
the block and then as it warms and reaches the correct temperature, a
number of sections can be cut in close succession. When these fall on
the tissue carrier, allow several to accumulate and pick up the group
with one sweep of the finger. Or, as soon as the section is cut, remove it
from the knife with a finger tip and place it in a dish of water. Do not
allow water to collect on the knife. Keep both knife and fingers dry.
Some tissues (adipose for one) tend to stick to the finger and will not
shake loose in water. ‘Transfer them to 70 or 80% alcohol.
Occasionally, when there is no time for gelatine embedding, one en-
counters a tissue difficult to section because it tends to fall apart. Bush
Mounting 65

and Hewitt (1952) support the tissue with Kodak Frozen Section Film.
The film is softened in water, excess moisture sponged off, and the film
pressed down on the freshly cut surface of the frozen tissue. The film
freezes and adheres to the tissue while a section is cut; this is carefully
raised as the knife passes under it. The film with the section is laid face
down on a slide. As the section thaws, it will cling to the slide, and a
thin layer of moisture develops by capillarity between it and the film.
When this has dried, the section can be stained. (Also see use of cellu-
lose tape, page 394.)

Mounting

If the sections are collected in a glass dish, place the dish on a black
surface—a table top or a piece of black paper. The sections are more
easily manipulated over the black background and the best ones can be
selected. Dip one end of an albumenized slide into the water. With a
needle or preferably a small round-tipped glass rod, gently bring a sec-
tion against the slide. Hold the section at one corner; by maneuvering
under water creases can be unfolded and the section unrolled as the
slide is drawn out of the solution. If the wrinkles refuse to straighten,
drop the section on 70% alcohol and then remove it to a slide. Drain
off excess water or pull it off with filter paper. Press section in place
with filter paper moistened with 50% alcohol. With a pipette add ab-
solute alcohol directly on top of the section; let stand for approximately
30 seconds. Replace with more absolute alcohol (must be ethyl alcohol).
Drain thoroughly, and flood slide with 1% nitrocellulose (dissolved in
absolute ethyl alcohol-ether, 1:1) for 1 second. Drain off and wave slide
in air until almost dry. Immerse in 70 or 80% alcohol, 5-10 minutes or
longer (can be left here for several hours or days). Cawtion: use an old
solution of alcohol, either one from the staining series, or one which
has been mixed for at least a day. A freshly prepared solution forms bub-
bles between section and slide.
Fresh tissue sections may be removed from normal saline to the slide
and thoroughly drained. Add 95% alcohol to remove the water; let stand
30-60 seconds. Drain off and allow the section to almost dry. Place in
95°, alcohol for 1-2 minutes and proceed to stain.
A gelatine fixative can be used to affix the sections. Spread fixative on
the slides and dry them in an incubator. (They can be stored for some
time in a dust-free box.) Float the sections on the gelatine, drain off
66 | Freezing Method (cuap. 6)

excess water and place the slides in a covered dish over formalin. This
converts the gelatine into an irreversible gel, thereby holding the sec-
tions in place. After 30 minutes over the formalin, wash in running
water, 10 minutes, and proceed to stain.

Gelatine Embedding

Culling (1957) Method

Some tissues tend to fall apart when sectioned, and it is necessary to
embed them in some medium such as gelatine.

After fixation, wash.

Place in 10% gelatine in 1% phenol aqueous, 37°C: 24 hours.
Transfer to 20% gelatine-phenol, 37°C: 12 hours.
Embed in 20% gelatin using a mold as for paraffin.
NO
Go
OreAllow to set, preferably in cold. Trim off excess gelatine close to tis-
use. Leave as little as possible on under surface; the gelatine tends to
inhibit freezing. The tissue block should be small with a minimum
of gelatine between the tissue and the freezing stage.
6. Before freezing, immerse trimmed block in 10% formalin to harden:
24 hours.
7. Section as usual on the freezing microtome.
8. Float sections on cold water and transfer to albumenized slides.
Drain, and warm slightly to coagulate albumen. Place in warm wa-
ter to remove gelatine and proceed to stain.

Pearse (1953) Method

1. Fix in cold formalin (15%) at 4°C if it is desired to preserve en-
zymes: 10-16 hours.
2. Wash, running water: 30 minutes.
3. Infiltrate with gelatine, 37°C: 1 hour.

PERTIC aes... ~ cette be He acd hop cok 15.0 gm.

SIVECTON CM... . . eyes 2d ease Ace te 15.0 ml.
astiiledmcmer .. . .ceeho. oe ore oe ee 70.0 ml.
small crystal of thymol.

4. Cool and harden in formalin (40%), 17-22°C: 1 hour. Wash.

5. Store at 4°C or below until sectioned.

See Albrecht (1956) and Thompson (1957) for staining in quantity.

Rapid Staining Methods 67

Staining
Nitrocellulose-mounted sections are transferred from the 70% alco-
hol to water and then into any desired stain. After staining they prob-
ably will require special care. If after dehydration the sections are
placed in absolute ethyl alcohol and show a tendency to fall off, because
the nitrocellulose dissolves, follow the 95% alcohol with isopropyl alco-
hol, 2 changes, and then xylene. Another method is to follow the 95%
alcohol with carbol-xylol (page 410), several changes, until the sections
look clear. Fogginess or colored droplets on the slides indicate traces of
water. If carbol-xylol is used, rinse it off by dipping the slide for 2-3 sec-
onds in xylene. Carbol-xylol must be removed; any of it left in the sec-
tions will fade stains in a short time, almost overnight. Warning: keep
fingers out of carbol-xylol; it contains carbolic acid. Wash it off immedi-
ately if any spills on the skin and use Lanolin for skin irritation.
Irregular staining usually is caused by an uneven coat of nitrocellu-
lose; this can be prevented by tilting the slide immediately after the
solution is applied. If the nitrocellulose is thicker than 1% uneven
staining may result. A colored background indicates a thick coat of
nitrocellulose. Sometimes irregular staining is due to loosening of parts
of the section.

Rapid Staining Methods

Pinacyanole Method (Proescher, 1933; Humason and Lushbaugh,

1961)

Staining and mounting time, 30—90 seconds

1. Mount frozen section (fresh or fixed) on slide; drain off excess water.
2. Cover section with several drops of stain: 3—5 seconds.
(pinacyanole, 0.5% in either 70% methyl or ethyl alcohol; keep stock
solution in refrigerator.)
Sis). Float section off into tap or distilled water: 5-10 seconds or until free
of excess stain (longer washing will not alter intensity).
4. Remount on clean side, blot excess water from around section, cover
with Aquamount,! glycerine or glycerine jelly, and cover glass.
1 Edward Gurr, London.
68 | Freezing Method. (cHAP.. 6)

An alternate method can be used and is perhaps preferred by some.

1. Place frozen section in a small amount of stain, 5-10 seconds.

. Transfer with glass rod into tap or distilled water to remove excess
ie)

stain, 5-10 seconds or longer.

3. Mount on slide, blot off excess water, add mountant and cover glass.

This method is not recommended for paraffin sections, only for fresh
or frozen ones; differential staining is lost in the former. In order to
preserve cytoplasmic staining, sections cannot be dehydrated; alcohol
decolorizes cytoplasmic structures and only the nucleus will retain ap-
preciable amounts of stain. All mounts are temporary and should be
sealed if preservation for several days is desirable. (The stain solution
can also be used for sex chromatin staining, page 362.)

Results (of Pinacyanole Staining)

chromatin—well-differentiated blue to reddish blue
connective tissue—pink
elastic tissue—dark violet
muscle—violet to purple
plasma cells—red granuloplasm
hemosiderin—orange
hemoglobin, neutrophil and eosinophil granules—unstained
neutral fat—colorless to faint bluish violet
lipoids—bluish violet to purple
amyloid—carmine red

Humphrey’s (1936) Method (Perhaps the Fastest)

1. Place section on a slide and wipe off excess water.

2. Add 1 drop of 0.5% brilliant cresyl blue in saline.
3. Cover with cover glass and examine. Never a permanent mount.

Thionin or Toluidine Blue Method

1. Remove section from water to a solution of either 0.59% thionin or
toluidine blue O in 20% ethyl alcohol plus a few drops of glacial
acetic acid: 30 seconds.
2. Rinse in water and float on slide.
3. Drain off excess water, blot around edges of section, add drop of
glycerine and cover glass. This is not a permanent slide.
Rapid Staining Methods 69

Reiner (1953) Method (Uses Stock Giemsa)

L. Add 1-2 drops stock Giemsa to section on slide: 5—10 seconds.

9
“_-Float section off into dish of acidulated water (3 drops glacial acetic
acid to 500 ml. distilled water). Allow to differentiate until dense
clouds of color cease to stream off: 10-15 seconds.
ee Transfer to water and remount on clean slide.
Remove excess water, mount in glycerine jelly.
 Chapter "7

Nitrocellulose
Method

This form of embedding is often classified as the celloidin technic

and celloidin becomes something of a generic term inclusive of the
various cellulose compounds, such’ as nitrocellulose and soluble gun
cotton or collodion. These are solutions of pyroxylin consisting chiefly
of cellulose tetranitrate. Obviously a purified nonexplosive form of
pyroxylin is necessary and there are several on the market: Parloidin,
Celloidin, and Photoxylin. (An excellent prepared embedding medium
can be purchased from the Randolph Products Co., Carlstadt, N.J.,
“Tissue Embedding Solution #4700’—a solution of 30% low viscosity
nitrocellulose in 35% ether and 35% absolute ethyl alcohol. It is a
special low viscosity nitrocellulose (LVN) allowing more rapid infiltra-
tion with solutions of higher concentration than is possible with many
of the other so-called celloidins. ‘This permits formation of harder
blocks in a shorter time and thinner sections can be cut than with the
latter types of media.
The chief advantages in using the nitrocellulose method are that
larger and harder pieces can be cut in nitrocellulose than in paraffin,
and the consistency of nitrocellulose allows mixed (hard and soft) tis-
sues to be cut. This quality is useful for organs such as eyes, teeth and
bones and their surrounding tissues, and for problems of shrinkage and
the formation of artificial spaces. But a slow dehydration and prolonged
infiltration is preferred in these cases. Also the method is costly.

70
Dehydration and Infiltration 71

Walls (1932) comments that the method offers many advantages:

1. It will handle anything organic.
2. Tt produces no artifacts; no shrinkage or swelling; delicate connections,
intercellular bridges are preserved.
“3. There is no compression of the sections during sectioning as with
paraffin, no disruption of delicate parts.
4. Sectioning is independent of temperature, humidity, and atmospheric
electricity.
5. Tissues are not rendered brittle or hard.
6. Mounting is quicker; there is no need for albumen, heating, or drying.
Ny. Hollow objects section better.
8. Celloidin supports tissue constantly; tissues cannot be crushed or dis-
placed.
9. Many stains perform better in it.
10. Time tolerations are great; there is no Jast minute rush to embed, no
worry about tissues staying too long in solutions or hot paraffin.

In this method toluene cannot be used as a solvent, but there are

many other solvents for nitrocellulose. One of the commonest is a com-
bination of two so-called latent solvents (not in themselves efficient sol-
vents, but possessing excellent solvent qualities when mixed with other
compounds), diethyl ether and ethyl alcohol. The solution is made up
of equal parts of the two, ether and absolute ethyl alcohol.

Dehydration and Infiltration

The tissue is fixed, washed and can be stored in 70% alcohol as usual.
The next step is as follows:
95% ethyl alcohol: 4—24 hours, or longer.
absolute ethyl alcohol, 2 changes: 4—24 hours or longer in each.
=
ho
oo ether-alcohol (equal parts of absolute ethyl alcohol and anhydrous
ether): 4—24 hours or longer.
10% nitrocellulose (dissolved in absolute ethyl alcohol-ether, 1:1):
2 days or longer, in a screw-cap jar to keep evaporation to a nyni-
mum.
5. 33-35% nitrocellulose (in absolute ethyl alcohol-ether): 2 days or
longer, in tight jar.
Nitrocellulose solutions should be stored in the dark; light causes the
solution to deteriorate. Ferreira and Combs (1951) warn that old light-
affected solutions cause fading of nervous tissue blocks.
Te. Nitrocellulose Method (cHav. 7)

Embedding
Slow Method

Place the tissue in a small glass-covered dish (stender dish), and over it
pour 33-35% nitrocellulose until it 1s $ to } inch above the tissue. Over
this pour a thin layer of ether-alcohol (1:1). Cover the dish tightly, and
allow to evaporate slowly until a proper consistency. If bubbles appear
in the medium, evaporation is proceeding too rapidy. A little Vaseline
on the ground-glass edge of the cover will help to seal it more tightly;
in dry, warm weather it may be necessary to enclose the evaporation
dish in another dish or jar to slow down the removal of solvent. The
process should take several days to a week or more, the slower the bet-
ter. Do not allow the medium to become too hard; it should reach the
consistency of hard rubber and should feel dry and not show finger
prints. If a tough film forms quickly on the surface of the solution and
sticks to the side of the dish, carefully loosen the film from the glass to
allow more efficient evaporation.
When the nitrocellulose is properly formed, trim it down to cutting
size, and mount it on a fiber or wooden block. (Figs. 18, 19, 20, 21.)
Around the block, tightly wrap a band of hard bond paper, then secure
the band with string or stick it down with a paper label. (Do not use a
rubber band.) The paper must project high enough above the mount-
ing block to enclose the tissue block. Moisten the mounting block and
paper with ether-alcohol, then place a small amount of 33-35% nitro-
cellulose in the container formed by the two. Roughen the underside
of the tissue block with a needle, and cover it with a drop or two of
ether-alcohol. Press the roughened surface tightly into the nitrocellulose
on the paper-wrapped block. After a few minutes in the air, place the
block in a closely capped jar with a small amount of chloroform; leave
it there 20-30 minutes. Add more chloroform to immerse the entire
block, and if possible allow it to “set” in chloroform overnight. ‘There
are other and more haphazard ways of mounting nitrocellulose blocks,
but the above method is relatively sure.
Romets (1948) claims that if wooden blocks are used they should be
pre-seasoned with nitrocellulose. Cook them in distilled water, then
allow them to dry for several days. Extract with equal parts of 70%
alcohol and glycerine for a day, and wash the blocks in distilled water
until shaking no longer causes a foam. Cover the surface with 8% nitro-
cellulose, dry, and put the blocks in a glass bottle.
Embedding 73

|
ry
He
Hy]
iH
hi
||
HTP
MHI
|

| |
I

20 Zl
Figures 18, 19, 20, and 21. Preparation of a fiber block and nitrocellulose
tissue block for sectioning.

Rapid Method

This method will not form as perfect a block as the former one; the
nitrocellulose is not always uniform and may section unevenly.
Wrap paper around the fiber block as for the slow method. Fill the
cavity with some nitrocellulose from the infiltrating jar and transfer
the tissue into the mass of nitrocellulose. Orient the piece of tissue so
it can be sectioned along the upper surface. Set the fiber block and
nitrocellulose and tissue in a tightly capped jar with a small amount of
chloroform. Leave in this situation until the block is firm, but check
frequently to see that the chloroform does not evaporate completely.
When the nitrocellulose has hardened, add more chloroform to com-
pletely immerse the block and tissue until ready to section.
Mounted or unmounted tissue blocks can be stored in chloroform,
74 | Nitrocellulose Method (cHAp. 7)

70%, 80% or 95% alcohol, but for long storage add a little glycerine
to the alcohol.

Sectioning
Clamp the jaws of the tissue carrier tightly against the lower two-
thirds of the mounting block. If the jaws are clamped against the upper
portion, the resulting compression in the block can loosen the nitro-
cellulose.
If the knife has a concave surface, clamp that surface uppermost in
the knife holder. The knife holder must be adjusted so the knife is
pushed back on the instrument far enough to clear the tissue before the
tissue is elevated in preparation for the next section. An automatic
lever on a small slide (sliding surface) on the back side of the knife
block can be moved until the correct knife position is determined. If
the knife does not clear the tissue before the latter is elevated, the press-
ing of the knife can injure the tissue and frequently alters the thickness
of the next section.
The maximum thickness of a section as controlled by the automatic
feed lever may be only 30 or 40 microns on most microtomes. ‘Thicker
sections however can be cut by moving the hand feed counterclockwise
and then releasing it. The hand feed is a large round knob (American
Optical sliding microtome) mounted at the base of the tissue carrier
and near the micrometer screw. Each movement of the hand feed will
equal the number of microns indicated by the automatic feed.
Keep the surface of the slide for the knife block well oiled with the
oil provided with the microtome (or Pike Oil, Norton Co.). A dry slide
produces a jerky knife movement and rough, irregular sections.
Always maintain a pool of 70% alcohol on the edge of the knife and
the top of the tissue block. Otherwise the sections may shrivel instead of
slicing in a smooth sheet. The following methods are suggested for sec-
tioning.
Method 1. Knife and block are wet with alcohol. Draw the knife
through the block until a section is almost, but not quite, cut to com-
pletion, making certain that the cut has cleared the tissue in the block.
If the cut does not clear the tissue, a striation will appear in the tissue
at the point where the knife stopped or hesitated. The section rolls as
it is cut. While the section is still attached at one corner, unroll it
against the knife, and then finish the cut. Squares of paper cut to a
size a little larger than the section are moistened with 70% alcohol.
 Hot Celloidin Technic 75

Hold one of these against the under surface of the knife and slip the
section off onto the paper. Invert the section and paper in a dish of
70% alcohol and press them down against the bottom of the dish. The
sections can be stacked one on another in this fashion and stored until
they are stained. Caution: use plain white paper, filter paper or paper
towels. Lined paper may stain the nitrocellulose and produce poor
staining.
Method 2. An alternate method is to flood the knife with alcohol and
moisten a camel hair brush. Hold the brush close to the nitrocellulose
block, so the alcohol is held between the block and brush by capillary
action. Do not rest the brush too firmly on the block, lest it be cut by
the knife. Pull the knife across the block; raise the brush slightly, and it
will guide the section onto the knife, smoothly and without rolling.
Remove as above. (Walls, 1932)
Method 3. Walls (1936) developed a dry method which eliminates
the constant soaking with alcohol during sectioning. After the block is
hardened, soak it in cedar oil:chloroform (50:50) for 24 hours. Blot the
block and allow it to stand in the air for 10-20 minutes. ‘Transfer to
cedar oil; this lubricates the block and knife during sectioning. The
sections, as removed from the knife, are placed in the same kind of oil,
then rinsed in 95% alcohol, 80% alcohol, and transferred to water for
staining. This type of oil-soaked block can be sectioned on a rotary
microtome as well as a sliding one.

Hot Celloidin Technic

Koneff (1937) Method

This is a more rapid method and can be used when materials are not
injured by heat. The procedures are handled in screw-cap jars in a par-
affin oven, about 56°C.

70% alcohol, 2 changes: - 21 hour each.

80%, alcohol, 2 changes: al2 hour each.
Tt
AS)
SCL 95% alcohol, 2 changes: $ hour each.
Absolute alcohol, 2 changes: } hour each.
We
or Absolute alcohol-ether (equal parts): 1 hour.
=P)
~~ LOC, mitrocelluloge: 1 hour.
~I 25-33% nitrocellulose: overnight.
ee Embed as for rapid cold method.
76 | Nitrocellulose Method (cuar. 7)

Walls (1932) used bottles with thick lips and corks, and wired on the
corks with a wire wound under the lip and over the cork. High pressure
builds up in the bottle, and together with the high viscosity of the
heated celloidin produces rapid penetration. He maintains that heat is
of no concern in this method; in the paraffin method it is the hot paraffin,
not the heat itself which, if used too long, produces a brittle block.
Do not hurry the cooling process; leave warm bottles on a wooden
table top until they have reached room temperature and they will not
break.

Difficulties in Nitrocellulose Sectioning"

1. Scratches in sections. Caused by:
a. nickin knife:
b. hard material in tissue.
c. position where knife was stopped while unrolling section.
. Tissue soft, mushy, crumbles or falls out. Caused by:
ho

a. imperfect infiltration due either to incomplete dehydration or

too short a time in nitrocellulose. Reinfiltrate.
b. embedding too rapid, as in short method.
3. Sections vary in thickness. Caused by:
a. worn parts in microtome.
b. pressing too hard on knife block.
c. insufficient hardening of nitrocellulose. Too soft and compresses
under knife.
d. tissue block is rising before return stroke of knife has cleared it.
e. insufficient tilting of knife. Shoulder of facets compresses block
instead of cutting it and next section is thick.
f. embedding too rapid, as in short method.

Staining and Mounting

Sections are stained before mounting in this method. Staining may be

handled in syracuse watch glasses or similar flat dish. With forceps or
spatula carry the sections through successive watch glasses and follow
each change by draining the section against a paper towel or filter pa-
per. In this way, contamination can be held to a minimum. If, however,
the solutions do pick up considerable stain, change them as ffequently
as seems necessary, or even more often. Also, if they have remained un-
1 Modified after Richards, 1949.
Mounting Before Staining We

covered for some time it is probable that the alcoholic content has been
reduced by evaporation. Never, at any time during the transferring of
sections, allow them to become dry. They may be carried directly from
the 70% alcohol to the stain if it is an alcoholic one, or first into water,
then into stain, if it is aqueous. (Remember to remove undesirable pig-
ments or crystals before staining.)
When ready for dehydration, transfer through 50%, 70% and into
959, alcohol. Then clear the sections by either of the following meth-
ods.
Method 1. Transfer sections into carbol-xylol (page 410), 2—3 min-
utes. Keep carbol-xylol covered at all times to prevent evaporation of
xylene. Dip the edge of a slide into the carbol-xylol dish and slip the
section in place with a needle or forceps. Drain thoroughly and flush
off excess carbol-xylol with toluene or xylene, 2 or 3 times. Add several
drops of mountant and cover glass. Press cover firmly in place. Several
hours later, after the resin has thickened, press down again. If the cover
glass insists on tilting or lifting, place a small weight on it or clamp it
with a clothes pin overnight. Bertram (1958) describes a weight which
will not stick to the mountant and cover glass.
Method 2. Transfer the sections to absolute alcohol containing 5%
of chloroform to prevent the alcohol from dissolving the nitrocellulose.
Clear with benzene and mount as above, omitting the carbol-xylol.
Do not attempt to dissolve or remove the nitrocellulose from sections;
they will become too difficult to manipulate. When using carbol-xylol,
keep fingers out of the solution. Wash off any that does get on the fingers
and rub lanolin on the skin.
A method for transferring sections to slides, particularly thin sections,
is as follows: slip a piece of cigarette paper under a section and spread
it smoothly on the paper with a brush or needle. Place the paper plus
the section on a slide with the section between the slide and paper. Blot
firmly with a piece of filter paper. Peel off the cigarette paper and rinse
the section with toluene or xylene. Add mountant and cover glass.

Mounting Before Staining

Sections are affixed to slides for staining in this method.

Method 1

a. Albumenize slide.
b. Press section on with filter paper.
78 Nitrocellulose Method (cuar. 7)

c. Pour clove oil over it: 5-20 minutes.

d. 95% alcohol, 3 changes: 5-10 minutes.
e. Absolute alcohol-ether, 2 changes, to dissolve nitrocellulose.
f. 70% alcohol, etc., to stain.

Method 2 (Lewis, 1945)

a. Rub on slide one drop Haupt’s fixative (page 413).

. Transfer section on cigarette paper to slide.
. Blot, and firmly press with filter paper.
d. Roll off cigarette paper rapidly; section must not dry.
. Place immediately in absolute alcohol-ether until nitrocellulose
is dissolved: 10—20 minutes.
Remove to 70% alcohol, etc., to stain.

Method 3

. Float sections onto slide and blot firmly with filter paper.
. Dip slide into 0.5% nitrocellulose for few seconds; drain and wipe
back of slide clean.
Harden film in chloroform: 5—10 minutes.
1959 10%, alcohol; iete; to/stain:

Method 4 (Culling, 1957)

. Transfer section to 95% alcohol: 1-2 minutes.

. Float on slide, drain for few minutes, blot lightly to flatten section,
but do not dry.
. Pour ether vapor over sections—only vapor, not liquid. This par-
tially dissolves nitrocellulose and causes it to adhere to slide.
d. Place slide in 80% alcohol: 5 minutes, to harden nitrocellulose.
. Running water: 10 minutes; stain.

Method 5—Serial Sections

. Arrange sections on knife from left to right.

. Lay cigarette paper on sections and saturate with 70% alcohol.
c. With quick sweeping movement, pull paper off knife and carry to
slide. Smooth with brush.
. Place filter paper on top and press flat, until much of alcohol is
absorbed.
Mounting Before Staining (i)

Jerk off paper and cover with clove oil: 3—5 minutes.
ThSubmerge in 95% alcohol.
os 70% alcohol, etc., to stain.

Method 6—Serial Sections (Williams, 1957)

a. Coat clean slides by dipping in warm solution of 1% gelatine.

Allow to drain and dry in vertical position.
Lay sections on blotting paper moistened with 70% alcohol. Keep
covered to prevent drying.
Pick up sections, one at a time, dip in 70% alcohol and arrange in
order on coated slides.
a. Blot sections with dry filter paper, flattening and pressing them
into contact with gelatine.
Place in coplin jar containing 2—3 cc. formalin. Do not allow fluid
to come in contact with sections, it may cause them to float off
slide.
Allow slides to remain tightly covered, 2-3 hours, room tempera-
ture.
70% alcohol, etc., to stain.

Method 7—Serial Sections (Wetmore, 1932)

a. Place on slide in order cut. Keep slides moist with 95% alcohol.
b. Blot off excess alcohol with filter paper.
C. Paint in spaces between sections with 2-4% nitrocellulose and
allow to dry. Thin sections require 2%, thicker ones 4%. Keep as
little as possible from getting on sections or margins may curl.
d. Place slides and sections in 95% alcohol plus a little chloroform to
harden, approximately 30 minutes.
With scalpel or section lifter, remove sheet of sections from slide
and store in 95% alcohol until ready to stain.
The author hesitates to recommend any of the above methods over
others. Adopt the one that cooperates the most effectively.
 Chapter §§

Specialized Embedding
‘Technics

Water-Soluble Wax Embedding and Sectioning

The so-called carbowax compounds and water-soluble polyethylene
glycol waxes, a series of polymers (compounds composed of the same
kind of atoms in the same percentage composition but in different
numbers and therefore having different molecular weights), each of
which is designated by its average molecular weight. These waxes are
used when it is necessary to go directly from fixative or water to the
embedding medium; no alcohols or clearing agents are required.
The following compounds are manufactured by the Carbide and
Carbon Chemicals Company: Compound 4000 is hard and dry, in crys-
talline flakes; Compound 1540 is not so firm, and will liquefy within a
week; Compound 1000 comes in slippery lumps and liquefies within
24 hours; Compound 1500 is a blend of equal parts of polyethylene
glycol 300 (a fluid) and wax 1540.
Other carbowax compounds are the polyglycols E9000, E6000,
E4000, E2000, and E1000 manufactured by Dow Chemical Company,
and HEM (Harleco Embedding Media) by Hartman-Leddon Company.
Wade (1952) finds #000 too hard for his use and suggests a mixture of
1:9 or 2:8 of 1540 and 4000 depending upon weather, and recommends
the latter except in the hottest and most humid climate.
Fixation: any fixative, but after a potassium dichromate fixative wash
for 12 hours before embedding.

80
Water-Soluble Wax Embedding and Sectioning 8]

Infiltration and Embedding: carbowax I—3 hours, 50—56°C; agitate

occasionally.
Prepare the carbowax ahead of time by placing the mixture in a
beaker and allowing it to melt in an oven. If it is melted rapidly over a
flame, a block formed from it will not cut well. The mixture will
keep for an indefinite time in the oven.
Embed by placing the tissue in a second batch of carbowax in a small
container or paper box in the refrigerator until it is hard, approximately
30 minutes. Blocks solidified at room temperature will not section as
well as those made in the refrigerator. Do not chill blocks in water—it
dissolves carbowax. When completely hard, the blocks may be removed
from the refrigerator and will turn opaque as they warm to room tem-
perature. This is of no disadvantage, but keep them in polyethylene or
cellophane bags or containers if storing them for some time. They must
not pick up water, even from the atmosphere.
Sectioning: A cool dry room is recommended. The edges of the block
must be parallel in order to obtain ribbons. Wade (1952) suggests that
if the sections do not adhere, if they break up on handling or do not
ribbon at all, try exposing the block to air for a day or two. For immedi-
ate use, try painting a 25°% solution of beeswax in chloroform on the
upper and lower surfaces of the block. Make the layer uniform and
allow it to become dull dry. Such surfaces should help the sections ad-
here to each other. Plain water may do as well in some Cases.
Hale (1952) found that variable and high humidity produces erratic
results, because with the absorption of water on the surfaces of the
block sectioning becomes impossible. He found that at 24—25°C sections
ceased to ribbon at 40% humidity; at 17-18°C humidity had no effect.
He, therefore, concluded that, as the temperature drops, greater hu-
midity is permissible. At higher temperatures, a higher percentage of
hard wax may be used, but care must be taken that the block does not
become too brittle for good sectioning.
Mounting Sections: Water mounting dissolves carbowax, and will
result in distorted sections and surface tension problems when trying to
affix sections to slides. Various solutions for this problem follow.

Blank and McCarthy (1950) Method

potassitmrdichromidte: 2-5... 5.4. 6. BeBe = 0.2 gm.
Relate. wet See hee a ya 3k Ske Oe 0.2 gm.
dusted water a tiviss. 2. ws ‘sas mes so eee ee 1000.0 ml.

Boil for 5 minutes. Filter.

82 Specialized Embedding Technics (CHAP. 8)

Sections while floating on this solution are picked up on a slide and

allowed to dry.

Wade (1952) Method

Wade prefers to albumenize slides with a thin coat of Mayer's egg
albumen (page 412). Dry overnight or three hours or longer in an oven.
Floating ribbons intact and without wrinkles still remains a problem.
He suggests that the addition of a wetting agent, Turgitol 7 (0.005% in
distilled water), will reduce surface tension effects. Add 10% of 1540
Carbowax to reduce shrinkage while sections are drying.

Giovacchini (1958) Method

Giovacchini thinly smears slides with the following solution: Dissolve

15 grams of gelatine in 55.0 ml. distilled water by heating. Add 50.0 ml.
glycerine and 0.5 gram phenol. Place sections on slides and place on
warming plate at 58-60°C for 15 minutes. Transfer to drying oven,
58°C, for 24 hours. Ready for staining.

Jones et al. (1959) Method

Jones and his coworkers float their sections on:
cienliylene glycol 4 .iep eee cn atk Mi See 100.0 ml.
ommanen bia Pi! 3 0k Se ene eas 1 od SE 7.0 ml
Gam lowe 1)... <.i. SE
eh eee ates saree, 1.0 ml.
GlistilediGwater |) ..JL). (Vier pee ayn ee) eos ye 400.0 ml.

In this method more spreading results from increasing the propor-

tion of water. If the tissues overexpand, add a small amount of Zephiram
chloride concentrate (5-10 drops to 500.0 ml. flotation solution); this
reduces surface tension and prevents air bubbles from being trapped
between tissue and solution surface. Mount on albumenized slides and
dry.

Pearse (1953) Method

This method combines features of the above two methods. Pearse
smears with Giovacchini’s fluid and mounts the sections with the di-
ethylene glycol mixture.

Zugibe et al. (1958) Method

This method cuts off a section from the ribbon with a razor blade.
One edge of the section adheres to the blade. Touch the loose section
edge against a slide coated with a flotation fluid and draw the rest of
the section onto the slide.
Double Embedding 83

Goland et al. (1954) Method

Goland and coworkers follow carbowax infiltration by:

xylene, 61°C: 10 minutes.

paraffin, 61°C: 30 minutes.
embed.

Chill in refrigerator, do not apply ice directly. ‘This overcomes some

of the disadvantages of carbowax and produces less shrinkage and dis-
tortion than a regular paraffin method. After sectioning, the tissues may
be handled as is customary for paraffin sections.
Either albumen or Giovacchini’s fluid smeared on slides have been
successful methods, but it has been easier to mount sections out of a
water bath (of floating solution, Jones et al., 1959) on to gelatine
smeared slides than by laying the sections directly on floating solution
on slide. Albumen slides mount readily either way. The razor-blade
method is tricky, but works.

Double Embedding
Fragile, small and hard objects often crumble when processed in the
paraffin method, and some difficulties can be eliminated by substituting
a double embedding technic.
1. Fix and wash tissues as usual.
2. Dehydrate in 50%, 70% and 90% ethyl alcohol: 2 hours each.
3. Absolute ethyl alcohol: 2-16 hours.
4. Methyl-benzoate-celloidin solution (see below): 24 hours. Pour off
and replace with fresh solution: 48 hours. If tissue is not clear, repeat
for 72 hours.
5. Pure benzene, 3 changes: 4 hours, 8 hours, and 12 hours.
6. Mixture of equal parts of paraffin and benzene, in embedding oven:
1 hour.
.
~I Parafhn, 2 changes: $ to 6 hours, depending upon thickness and
nature of tissue (3 hours for 5 mm. thickness).
8. Embed and proceed as with ordinary paraffin sections.

Methyl-benzoate-celloidin Solution
Add 1 gram air-dried celloidin flakes to 100.0 ml. methyl benzoate.
Shake well and allow bottle to stand upright for an hour or longer.
84 Specialized Embedding Technics (CHAP. 8)

Invert for an hour. Lay bottle on side for an hour, then turn it upright
again. Repeat this process until solution is completed, probably the fol-
lowing day.

Mounting Sections and Preparing to Stain

If the sections do not flatten well on slide, but instead want to curl and
separate from it, float them on 95% alcohol to soften the celloidin. If
there still is a curling problem, soften them with ether vapor, blot with
filter paper and soak on 0.5-1.0% celloidin or nitrocellulose. (Lillie,
19573.)
If albumen continues to fail as an adhesive agent, try a fresh solution
of Masson’s gelatine fixative (page 412) under the sections and warm
slides on warming plate. As soon as they have spread, remove the slides
and allow them to cool for a few minutes. Drain off excess gelatine solu-
tion, blot down sections with filter paper and place in formalin vapor
overnight (40—50°C).
When ready to stain, place the slides in chloroform before proceeding
to 95% alcohol. The chloroform will remove the paraffin and harden
the celloidin simultaneously.

Briggs (1958) Double Embedding Method

1. Fix, wash, and dehydrate through 70%, 95%, and 2 changes of abso-
lute alcohol: 2 hours each.
Absolute alcohol-ether (50:50): 12-24 hours.
4%, celloidin (or nitrocellulose): 48 hours.
DO
09
H Dip tissue in alcohol-ether to remove excess celloidin from surface.
Harden in chloroform vapor for few minutes. Drop into chloroform
and leave until tissue sinks: 12—24 hours.
Sh Infiltrate, 2 changes paraffin: 12—24 hours each.
6. Embed.
Briggs suggests that during staining, add 4 chloroform by volume
to all hydrat:ng and dehydrating solutions above 70%, alcohol to pre-
vent removal of celloidin.

Banhy and Clark (1949) Double Embedding Method

This is similar to the Briggs method with the following modifications:
For step 3:2 changes nitrocellulose, 3% and 6%.
To follow step 4: benzene, overnight.
Polyester Embedding 85

Dioxane in Double Embedding

1. Fix, decalcify if necessary, and wash.

2. Dioxane: 2 hours.
3. Dioxane-nitrocellulose: 3 days.

GION CHE Meee es AS, Gs en ee 70.0 ml.

Zp TMC
OCE NUN OSES oeidies aces che BE ee an eS Ss 30.0 ml.

4. Dioxane: ? hours.
5. Dioxane-parafhin (50:50): 2 hours.
6. Paraffin, 3 changes: 20 minutes, 30 minutes, | hour.
7. Embed, and section immediately.
At UCLA, this method was used successfully on hard fish with scales
intact. (Also see Brown, 1948)

Polyester Embedding
Polyester resins are plastics (polymers) formed by the esterification
reaction (forming a compound from an alcohol and an acid by removal
of water) between polyhydric alcohols and polybasic acids. ‘These
resins exhibit a wide range of properties and are utilized in a diversity
of commercial and scientific endeavors. The alcohols most commonly
used are ethylene, propylene, 1,3 and 2,3 butylene and dipropylene
glycols. Unsaturated dibasic acids may be maleic anhydride or fumaric
acid. Saturated dibasic acids may be phthalic, adipic and azelaic acid,
and chlorinated acids or anhydrides.
A polyester as used here is an unsaturated polyester base resin dis-
solved in a polymerizable monomer, is suitably diluted with the mono-
mer and supplied as a viscous fluid. ‘The monomer (smallest unit enter-
ing into the formation of high molecular weight polymers) can be
styrene, vinyl toluene, diallyl phthalate, methyl methacrylate, triallyl
cyanurate or several other vinyl monomers.
To promote polymerization at the time of use a catalyst must be
added to modify the velocity of the reaction without becoming a part of
the product. The curing, as this is called, is sometimes accomplished
with elevated temperature and sometimes by the use of an “accelerator”
or “promoter” in addition to the catalyst. The speed of cure depends
on the amount of catalyst and accelerator used and varies with different
resins.
86 Specialized Embedding Technics (cHaAP. 8)

References: Manufacturing Chemists’ Association (1957) and Simonds

et al. (1949).

Kuhn and Lutz (1958) Method

Advantages of the method. Polyester embedding supports hard struc-
tures adjacent to soft ones, is faster than the nitrocellulose method and
can be sectioned on a rotary microtome. Size of blocks should be 5-16
mm. in diameter and can be sectioned at 5 to 50 microns. ‘The method
uses a nonrigid plasticized resin prepared by Natcol Laboratories (Route
2, Box 575, Redlands, California), known as C.M.E. Tissue Support
Resin (soft, medium and firm), plus a catalyst (#1) and a promoter
(£2) also supplied by the company.

PROCEDURE

Dehydrate tissue in ethyl or isopropyl alcohol.

Remove alcohol in anhydrous ether: 2—4 hours.
NO
oo
= (a) ‘Transfer soft specimens directly to resin with one drop of catalyst
(#1) added for each 30 ml. of resin. Keep specimen submerged.
Exhaust the ether from the specimen in a vacuum chamber using
reduced atmospheric pressure (10-15 in. Hg); 1—4 hours.
(b) For hard or dense tissues prepare 10—20 ml. of a mixture of the
resin and styrene monomer (1:1), add one drop of the catalyst (#1).
Submerge tissue. Place in vacuum chamber with reduced pressure:
1-4 hours. In stubborn cases, alternate the reduced pressure with
positive pressure (20 lb/sq. in.) at 15 to 20 minute intervals. Follow
this treatment with undiluted resin as in 3a: 1 hour, preferably in
vacuum.
4. For embedding, use gelatine capsules #000 and veterinary capsules
+10 for larger specimens.
5. Remove tissue from resin and allow excess to drain off.
6. Warm 30 ml. of resin in a beaker set in a water bath at 65°C. Have
a stirring rod warming in the resin. Add three drops of catalyst (41)
and four drops of promoter (#2) for a reasonably fast curing solu-
tion. If a thinned preparation (as in step 3b) was used, add four
drops of catalyst, as well as of promoter, to the embedding resin. Stir
rapidly until all mixing lines have disappeared.
7. When resin is thoroughly mixed, fill capsules about half full. Place
tissue in position and complete filling. If the tissue floats push it back
into place until the resin cures enough to prevent floating. If this
takes longer than 5 minutes, warm capsules with a lamp, but only
until the resin has reached curing stage (obviously begins to thicken).
Polyester Embedding 87

Allow to harden at room temperature: 12—24 hours. Peel off capsule

or dissolve it in warm water. If the resin cures too rapidly or too
slowly, the catalyst and promoter may be decreased or increased by
one drop of each.

SECTIONING

If the block is made long enough, the block itself can be clamped
directly in the tissue carrier. Slow cutting usually produces the best
sections. Ueckert (1960) recommends the sledge microtome as best for
polyester sectioning, but most laboratories do not possess this type of
microtome and will have to resort to the use of a rotary type. Sometimes
breathing on the surface of the block facilitates sectioning. If the sec-
tions curl, the knife is dull or the knife angle is incorrect.

STAINING

Place the sections after cutting in distilled water. Stain them un-
mounted, because mounted sections trap the stain under the plastic
which is never removed.
Kuhn and Lutz recommend safranin and fast green dissolved in
alcohol. Ueckert has used hematoxylin-eosin, iron hematoxylin-Van
Gieson, and silver methods. Follow the staining by floating the sections
on absolute alcohol to remove any excess stain. Blot and mount in
Permount or any similar mountant (page 416). Do not use toluene; it
causes the plastic to swell and form wrinkles.
Kuhn and Lutz have used this method for whole decalcified snake
heads, chick embryos, leg joints and undecalcified mouse paw and tail;
they suggest the possibility of its use for plant material.
 Chapter Q

The Microscope

The Compound Microscope

The ordinary laboratory microscope uses brightfield illumination
with direct light furnished by a substage condenser and mirror. The
image of the specimen appears on a bright, well-lit field.
Diagrams designating all the parts of a microscope are provided by
the manufacturers and will not be included here; we will, however, give
a brief listing of the important parts and pertinent information con-
cerning their use.
The lenses of a microscope are the oculars (eyepieces) at the top and
the objectives at the bottom of the body tube. The objectives magnify
the specimen a definite amount, forming an image which is again mag-
nified by the ocular eyepiece. The lenses of a good microscope should
not only magnify, but also should improve the visible detail. This is
the resolving power and is a function of (1) the wave length of the light
used, (2) the lowest refractive index (refractive index is a measurement
of the refraction or bending of light rays as they pass through from one
medium to another and at an oblique angle) between the objective and
the substage condenser (page 90), and (3) the greatest angle between
two rays of light entering the front lens of the objective. Blue light
increases resolving power over white light, ultraviolet increases it even
more, but cannot be seen and results must be photographed.
Oculars are manufactured in a variety of powers of magnification

88
The Compound Microscope 89

(engraved on them); 5X and 10X are the customary equipment for

student and laboratory microscopes—the former for low power over
large areas, the latter for general work. Most of these microscopes are
equipped with three objectives: 50 to 32 mm. (low power); 16 mm.
(middle or intermediate power); and 8 to 4 mm. (high power). All are
engraved with their magnifying power. An additional objective will be
included on the four-objective nosepiece and can be used interchange-
ably with the low power (or any other power) on the three-objective
nosepiece. As one shifts from low power to higher powers, the magnifica-
tion increases but the size and depth of the examining field decreases;
also, the clearance of the objective above the specimen (working dis-
tance) decreases, and more light is required. The working distance is
the distance between the front of the objective and the top of the cover
glass when the specimen is in focus.
Achromatic objectives (producing an image essentially free of color
fringes) are found on most laboratory microscopes. These objectives
are corrected chromatically for the light of two wavelengths, the C
(red) and F (blue) lines of the spectrum and spherically for the light of
one color, usually in the yellow greens. Achromats give a good image
in the central portion of the field but not as sharp an image at the edges
as can be obtained with apochromatic objectives. The latter are essential
for critical microscopy and color photography because they are cor-
rected for three colors, including the G (violet) lines of the spectrum,
thereby reducing the amount of color fringes. Lenses, if not corrected
in this manner, exhibit chromatic aberration, and color fringes will
confuse the border of the specimen image. For best results apochromatic
objectives should be accompanied by a corrected condenser of a nu-
merical aperture at least equal to that of the objective aperture and
compensating ocular eyepieces. A correction collar on the apochromatic
objectives can be adjusted to compensate for the thickness of the cover
glass on the slide. The collar must be used and can be adjusted with one
hand while the fine adjustment is manipulated with the other hand.
The body tube supporting the lenses may have a fixed length or it
may be equipped with an adjustable drawtube. A scale on the latter is
used to determine the length of the tube for the various lenses used in
it—usually 160 mm. for American-made objectives. This becomes more
critical with higher power objectives than with lower power ones for
improving sharpness of detail under a thick cover glass.
At the bottom of the microscope is located a mirror to direct light
through the diaphragm and into the condenser. Usually there are two
mirrored surfaces: one plane (flat) to direct the light, reflecting a
90 The Microscope (cHapr. 9)

moderate amount of light with parallel rays but no change in form; the
other concave to converge the light rays to form a cone, concentrating a
large amount of light. The latter replaces a condenser and can be used
for lower magnifications, but should not be used when a condenser is
present and not for high magnification, when the plane mirror should
be used.
Immediately below the microscope stage is the substage condenser,
whose lenses serve to converge the parallel beam of light from the
mirror so a cone of light passes through the aperture of the stage. This
cone of light is focused on the specimen under examination and then
is extended to fill the back lens of the objective. Without a condenser
the back lens of high-power objectives can not be filled with the proper
amount of light. By opening or closing the iris diaphragm mounted
below the condenser, the diameter of the light entering the condenser
can be controlled. A two-lens Abbe condenser is found on most
laboratory and student microscopes. For critical research microscopy,
a corrected condenser with a centering mount should be used. It should
be carefully centered to the objective and immersion oil should be
used between it and the slide for finest detail of the specimen.
The image seen in the compound microscope is inverted, it is upside
down and turned right side to left. The movement of the slide will be
reversed. Most images depend for clarity on color in the specimen or
color added to it and/or differences in refractive indices in different
parts of the specimen and the medium in which it is mounted.
There are innumerable types and sources of light used in laboratories.
If a table lamp is used, such as is found in many student laboratories,
use a daylight bulb in it. Good, simple types of microscope lamps are
equipped with blue ground-glass filters. Place the lamp 10 to 15 inches
in front of the mirror and with all of the light directed below the stage
of the microscope. The surface of the bulb or of the filter is focused on
the specimen by way of the substage condenser. If the lamp is the small
substage type of lamp, it may be used either in front of the mirror or,
with the mirror removed, is placed under the condenser. A more critical
illumination (Kohler method) can be obtained with better kinds of
lamps provided with condensing lens and a coil or ribbon filament.
Controlled illumination is lighting by a cone of rays whose proportions
are regulated by a stop at the illuminator and by the iris diaphragm of
the condenser. ‘The aperture of the objective is completely used and
none of the refracted light should fall on the lens mounting or draw
tube.
The Operation of a Microscope sik

The Operation of a Microscope

Without a Substage Condenser

. Place slide on stage and center the field to be examined over opening
in stage.
ho Turn low-power objective over specimen a short distance above it—
one-half inch will do.
Adjust concave mirror, moving it back and forth, until light is as
uniform as possible through the hole and onto the specimen.
Raise the body tube by coarse adjustment until the specimen is in
focus. Check mirror, adjusting for better light.
Or Higher powers may be swung into position and light adjusted if
necessary for greater magnification.

With Substage Condenser and Lamp without Lenses

. Place slide on stage as above. Adjust objective, plane mirror and

body tube for focus as above.
Remove ocular eyepiece and while looking down the tube open iris
diaphragm of the substage condenser until it coincides with the
margin of the back lens of the objective, just filling it with light, no
more. Focus the condenser up and down until the light is uniform
through the objective.
. Replace ocular eyepiece. Make similar adjustments for all objectives.
To adjust both parts of a binocular microscope, close the left eye and
focus with the fine adjustment until the image is sharp for the right eye.
Leave the fine adjustment alone and focus for the left eye with the
focusing adjustment on the tube outside the ocular eyepiece. Between
the two eyepieces is an adjustment for changing the interpupillary dis-
tance between the eyes. Turn the adjustment slowly until the right and
left images blend and a single field is seen with both eyes.

Critical Illumination (Kohler method) Using a Lamp with

Condensing Lenses and an Iris Diaphragm

Lenses and an Iris Diaphragm

le Place a slide with fine-detailed specimen in position on microscope

stage. Center the condenser in relation to the stage aperture.
oF The Microscope (CHAP. 9)

2. Place the lamp so the diaphragm is about 10 inches from the micro-
scope mirror and line it up with the microscope so the light filament
centers on the plane surface of the mirror. Insert a neutral filter.
3. Using a 10X ocular and 16 mm. objective, adjust the mirror until
the light passes through the substage condenser to the slide. Focus
microscope on the specimen and center the light.
4. Rack up the substage condenser until it almost touches the slide and
close the substage diaphragm. Close the lamp diaphragm about half-
way and adjust the lamp condenser until an image of the filament
is focused on the closed substage condenser diaphragm. This can be
observed in the microscope mirror or with a small hand mirror.
Partially open the substage iris diaphragm.
Or. Focus with the substage condenser until a sharp image of the lamp
diaphragm coincides with the specimen on the slide. With the mirror
center the diaphragm image.
6. Take out the ocular eyepiece, and, looking down the tube, observe
the back lens of the objective. Open substage diaphragm until its
image edge almost disappears in the back lens, but remains just
visible. ‘The back lens should be filled with light—full cone illumi-
nation. Because the light source is imaged on the diaphragm of the
substage condenser, the light source is in focus at the back focal plane
of the objective and the filament of the lamp can be seen.
7. Replace the ocular eyepiece. If the tube length has to be adjusted,
do so (usually 160 mm. for American objectives).
Certain precautions are imperative for good illumination and bear
repetition. The back lens should always be filled with even light and
the aperture of the substage diaphragm should be closed to a minimum
to furnish as wide a cone of light as the specimen will take. Closing
down the diaphragm to reduce light intensity and thereby increase
contrast in transparent objects is poor technique. Light intensity should
be controlled by the kind of bulb, use of filters, or a rheostat or variable
transformer inserted in the lamp system.
Unless you have had previous experience with a certain microscope,
when changing from one objective to another of higher power (high dry
or oil-immersion) do not take it for granted that the microscope is
parfocal (focal planes of all objectives lie in same position on the tube).
The higher power may crush a valuable specimen. Proceed as follows:
examine slide with naked eye for approximate part to be examined.
Center the area over the stage aperture. Check with low power, then
middle power for the particular area of interest, then change to high
power, but raise the tube } to $ inch at least before revolving the nose-
The Operation of a Microscope 93

piece. Then, while watching the bottom of the objective from the side,
lower the tube until the objective almost touches the cover glass. Look
into the microscope and rack up the tube until the specimen is in focus.
Remember, when an objective of a different numerical aperture is used,
the condenser diaphragm must be adjusted (step 6 above). The numeri-
cal aperture (N.A.) number is used to compare the resolving power of
the lenses; the higher the numerical aperture, the greater the resolving
power and the condenser diaphragm will have to be opened wider.

Use of Oil-immersion Objective

The working distance with this objective is short and great care must
be taken to prevent crushing a cover glass. Thin cover glasses must be
used or the specimen mounted on the cover glass itself (page 385).
Greater illumination is required than with the dry objectives and a
wider cone of light is needed. The space between the objective and the
cover glass, also frequently between the slide and the condenser, must
be filled with a suitable medium of a refractive index and dispersion
like that of the glass of the lenses. This medium is provided by immer-
sion oil (cedarwood oil, crown oil).
1. Using a dry objective locate and center the area to be examined.
2. Raise microscope tube, swing oil-immersion objective into position.
3. Place a drop of immersion oil on the cover glass over the area to be ex-
amined. Watching (from the side) the lower edge of the objective, lower
it slowly until it makes contact with the oil.
4. Observing through the microscope, very slowly lower the objective with
the coarse adjustment until the specimen is in focus. Make more critical
focus with the fine adjustment.
5. Remove ocular eyepiece and adjust the light on the back lens of the objec-
tive; the condenser diaphragm will have to be opened.

After use, clean oil-immersion objective; never leave oil on it. Wipe it
with clean lens paper, then with a little xylene or chloroform on lens paper,
finally drying it with clean dry lens paper. Do not leave xylene or chloroform
on the lens.

Other Hints for Efficient Microscopy

Always keep oil surfaces clean, free from dust and grease. ‘This includes
the lamp condenser, filters, mirror, lenses of the substage condenser,
lenses of objectives, slides, cover glasses, binocular prisms, lens of
of The Microscope (CHAP. 9)

ocular eyepieces. Use a good grade of lens paper and a fine camel hair
brush.
If glasses are worn, protect them from scratching by mounting a
rubber band around the top of the ocular. (Fingers of old rubber gloves
can be cut to fit the oculars.)
If, instead of a mechanical stage, clips are present on a fixed stage,
place one clip over one end of the slide. Then with two fingers of one
hand controlling the loose end of the slide, move the slide for examin-
ing, while manipulating the fine adjustment with the other hand.
Individual preferences will determine which hand to use for which
motion. With a mechanical stage the right hand has to control the knobs
and the left hand the fine adjustment.
If it is convenient to have a pointer in the eyepiece, unscrew and
remove the top lens of the ocular eyepiece. Inside, about halfway down
its tube is a circular shelf. Place a small drop of Permount (or other
quick-drying mountant) on this shelf and, with small forceps, place an
eyelash in this drop. Arrange the eyelash to lie flat and project a little
short of center of the hole formed by the circular shelf. Screw lens back
in place.
When carrying a microscope, carry it upright, preferably with both
hands. Never tilt it; the ocular eyepiece can fall out. If using a micro-
scope for checking staining effects, or any wet mounts, place a thin piece
of glass on the stage. A lantern-slide cover glass (34 x 4 inch) is usually
available and is an ideal size for most microscope stages. Clean it after
use to prevent contamination of future slides or material.

Measuring Devices Used on a Microscope

Reading a Vernier

Most mechanical stages are equipped with a vernier—a device for the
purpose of relocating the same spot previously examined on a slide.
Two numerical scales run side by side, a long one constructed on a
millimeter scale and a short one constructed on a scale of nine milli-
meters divided into ten equal divisions. When the area of reference on
the slide is centered in the objective, read the vernier. Check the point
of coincidence of the zero of the short scale against the long scale. Read
the lower whole figure on the latter, to the left of the zero on the short
scale. This determines the whole number, the number to the left of
the decimal point in the final reading (21. of the long scale in Fig. 22).
Exception: If the zero of the short scale coincides exactly with a whole
Measuring Devices Used on a Microscope 95

e) 10 20 30 40

Figure 22. A vernier scale.

number on the long scale, then the number is recorded as a whole

number with no decimals following it. The decimal is determined by
the point of coincidence of the line of the short scale which perfectly
coincides with any line on the long scale (.5 of the short scale in Fig.
22). The reading, therefore, in Fig. 22 will be 21.5.

Measuring Objects with an Ocular Micrometer

‘The measurement of slide specimens usually is done with a micrometer

disc placed in the ocular, but first the disc’s actual value with respect to
the magnification at which it is being used has to be calibrated against
a stage micrometer. Stage micrometers usually have a 2 mm. scale
divided into .01 mm. divisions, or a 0.2 inch scale divided into .001 inch
divisions. The ocular micrometer has a 5 mm. scale divided into 50 (0.1
mm.) divisions or 100 (.05 mm.) divisions.
Unscrew the top lens of the ocular, place the ocular micrometer on
the circular shelf inside and replace the lens. Focus on the stage mi-
crometer, moving it until the zero line of both micrometers coincide.
Then a definite distance on the stage micrometer will be made to
correspond to a certain number of divisions on the ocular micrometer.
The rest is simple; for example:
The ocular micrometer has 100 divisions.

Suppose that 30 divisions (each measuring .01 mm.) of the stage micrometer
equal the 100 ocular micrometer divisions. Then 30 x .01 mm. = .30 mm.,
the length of the 100 divisions on the ocular micrometer.

Next, .30 + 100 = .003 mm. (3.0 «), the value of each single division.

Therefore, when the ocular micrometer (each division measuring 3.0 ;) is

focused on a specimen, and if the specimen requires 9 divisions to meet its
length, multiply 9 by 3.0 » to equal 27.0 » (.027 mm.), the total length of the
specimen.
96 The Microscope (CHAP. 9)

Obviously every combination of oculars and objectives, also different

tube lengths, must each be calibrated in this fashion.

Specialized Microscopy
The image of a biological specimen can be formed and used in
several ways. The most common form, known in all laboratories, is
the use of color images, either natural color in the specimen or differ-
ential color applied to it. But, with the exception of a few vital stains,
the latter method cannot be used on live material. Most living material
is transparent to light; that is, the light wave passes through it with very
little loss of intensity, so other means for examining it must be em-
ployed.

Dark Field Microscopy

In dark field, the objects themselves turn the light into the microscope
by reflecting it or scattering it, and the object appears luminous on a
dark background. No light from an outside source reaches the eye. A
stop in the substage condenser blocks out the central part of the solid
cone of light formed in the condenser, and only oblique rays in a hollow
cone of light, striking from the sides, illuminate the object.
The simplest form of dark field can be developed with a black central
patch stop inserted in the carrier under the condenser. Dark field
elements in a threaded mount can be used in place of the upper lens of
an Abbe condenser. This is practical with all objectives if the numeri-
cal aperture does not exceed 0.85 and immersion is used with the slide.
Dark field can be modified with colored stops and outer rings below
the substage condenser, instead of black stops, to produce optical stain-
ing. The central stop determines the background color and the outer
ring the color of the object, giving optical coloring to unstained objects
and helping to reveal detail and structure.
Dark field and optical staining can be useful for examining: 1. bac-
teria, yeasts, and molds; 2. body fluids, plant or animal; 3. colloids; 4.
living organisms in water; 5. foods, fibers, and pigments; 6. insects and
scales; 7. crystals; and 8. bone, plant, and rock sections.
It is a common means of studying the results of microincineration,
the investigation of minerals present in different parts of tissues. Paraffin
sections of tissue fixed in a solution of formalin and alcohol are
mounted on slides and placed in an electric furnace. The temperature
Specialized Microscopy 97
—

is then slowly raised until all organic matter is burned away and only
the mineral skeleton of the tissue is left. This appears white under
dark-field observation.

Phase and Interference Microscopy

Although most living material is transparent to visible light, different
components of tissue do alter to a different extent the phase of light
waves passing through them. That is, the light velocity is altered,
advanced or retarded and its vibration is said to be changed in phase.
When two waves come together and are in phase, brightness increases,
but if out of phase and of equal amplitude, interference occurs and the
eye sees black, not light. Waves of intermediate difference in phase pro-
duce a series of grays. It is the aim of the phase microscope, to change
slight phase differences into amplitude differences and produce a vari-
ation in intensity from light to dark contrast observable in the specimen
under the microscope.
Both phase and interference microscopes follow this principle of
interference phenomenon—combining light waves that are out of phase
with each other to produce combined waves of greater and _ lesser
amplitude. The means of applying the principle is different.

PHASE MICROSCOPY

The cost of phase microscopy is not excessive and the parts can be
adapted to any brightfield microscope. The system requires phase con-
trast objectives (achromatic objectives with fixed-phase plates and a
two-lens Abbe condenser with an iris diaphragm and a revolving ring
below the condenser to carry four annular ring diaphragms (stops that
produce different-sized cones of light). The correct annular ring dia-
phragm must be centered to the phase ring of the correct objective and
match its numerical aperture. The annular diaphragm causes the light
to strike the object in the shape of a hollow cone and gives rise to two
types of waves; one type passes straight through the object (undiffracted),
the others are diffracted into a different course. Different components
in the tissue diffract differently producing the differences in intensity.
Images under phase microscopy exhibit a “halo” as a result of the
diffraction of light at the phase-changing annulus (annular ring). The
light which has passed straight through the specimen is made to inter-
fere with the light diffracted sideways by it. Only the refracting struc-
tures are observed, and these edges and abrupt changes of refractive
index produce the “halo” around the images.
98 The Microscope (cHar. 9)

INTERFERENCE MICROSCOPY

A more sensitive and accurate instrument is the interference micro-

scope, which is better adapted to measuring the refractive indices of a
specimen. In this microscope, the light splitting and recombining is
carried out externally to the specimen. Birefringent plates (doubly
refracting) are cemented to the top lens of the condenser and to the
front lens of the objective. One set of rays passes through the object, the
other set passes through a clear region at one side, and they are then
recombined. Any phase differences between them remain constant and
can interfere to give light or dark. A refractile object in one beam causes
a change in light intensity.

Phase versus Interference

Both operate on the same principle—the interference phenomenon

which changes phase difference into amplitude difference.
PHASE: (1) Light passing through the specimen is made to interfere with
light diffracted sideways by it. (2) Only shows up diffracting structures in the
specimen; produces a “halo.” (3) Apparatus is simple, reasonable in cost, easy
to operate and can be added to any conventional microscope. (4) Can be used
to study living material, cytoplasm, cell inclusions, nucleus, action of physical
and chemical agents on living cells. (5) Adequate for routine examination.
INTERFERENCE: (1) Light splitting and recombining carried out on outside
of specimen and under control of experimenter. (2) No “halo”; variations
in the optical path through the object are easily interpreted. Phase change
can be measured. (3) Apparatus is expensive and complicated. Requires con-
stant checking and adjusting. (4) Can be used on living material to determine
dry mass: for example, changes in mass during cell activity; protein distribu-
tion, both in cytoplasm and nucleus.

Polarizing Microscopy
Closely related to the above types of observation is the use of polarizing
attachments. These may be used on most types of microscopes, but for
continued use a polarizing microscope is preferable. When a ray of
plane-polarized light (vibrating in one plane) falls on the object, it is
split into two rays: one obeys laws of refraction and the other passes
through the object with a different velocity. After emerging from the
object, the two rays are recombined, but because their velocity is
different, they will be out of phase. This phase difference is the quantity
measured in a polarizing microscope.
Specialized Microscopy Y9

A polarizing (prism) in the fork substage instead of a condenser, or a

polaroid disc in the condenser slot polarizes the light so it vibrates in
one plane only. Part of the light (ordinary ray) is reflected to the side of
the prism and does not illuminate the object; the other part (extraor-
dinary ray) continues straight through the prism to emerge as polarized
light. (There obviously is a loss of light, so plenty of it must be used.)
If the polarizer is a prism, it usually can be rotated 360° and the amount
of rotation in the field checked.
In or above the ocular is fitted another polarizing prism, the analyzer,
whose vibration direction is set at 90° to that of the polarizer. ‘The
extraordinary ray from the polarizer becomes the ordinary ray in the
analyzer and is reflected out of the field, unless an anisotropic sub-
stance (doubly refracting when placed between the analyzer and polar-
izer) rotates the plane of polarization and interferes with the path of
light. Such a substance divides the light from the polarizer into two
beams, one of which passes through the analyzer, making the object
appear to glow against a dark background. Isotropic substances (singly
refracting) do not polarize and therefore do not divide the beam of light
and do not glow.

SOME USES OF POLARIZING MICROSCOPY

1. Determines whether an object is isotropic or anisotropic (if it rotates the

plane of polarization).
2. Can be used for differences in physical properties in different directions;
study of mitotic spindles.
3. Can be used on fresh unfixed material.
4. Reveals molecular orientation of structures, chemical constitution, chem-
ical and physical intervention in the cell, pressures or tensions; all can
produce anisotropic effects.
5. Can be used on natural and artificial fibers, cellulose fibrils, lamellar
plasm differentiation, pseudopodia, spindles and asters, nerve fibers,
muscle fibers, chromosomes, chemical and mineral crystals, crystallized
hormones and vitamins, dust counts, starch grains, horn, claw and bone
sections.
6. Can be used for determination of refractive indices.

X-Ray Diffraction
All forms of matter scatter X-rays and form diffraction patterns. From
these patterns information concerning the materials can be ascertained;
crystalline proteins show an X-ray diffraction of great complexity,
100 The Microscope (cHap. 9)

fibrous proteins show less detail. Information can be obtained concern-

ing molecular orientation, particle and molecular size and sometimes
detailed molecular structure. The image recorded can be related to
simple absorption processes and thus be interpreted chemically.
The simplest method with X-rays is to place a tissue section in con-
tact with a fine-grained photographic emulsion and expose it. Magnifi-
cation 1s obtained in an X-ray projection microscope by separating the
sample and film. ‘This requires very fine focusing. One of the principal
disadvantages of the method is that vacuum is required; therefore
material has to be dry, no water can be present. Freeze-dried tissue can
be used.

USES OF X-RAY

Tissues with relatively high X-ray absorption are bone and tissues
impregnated with heavy metals. The distribution of mineral salts in
undecalcified sections of bone can be clearly shown and quantitative
information obtained. Substances containing high percentage of ele-
ments of high atomic numbers can be injected into blood vessels, or the
lymphatic system, and photographed with fine-grain film. Recorded
images can be related to simple absorption processes and then be inter-
preted chemically. By using the oblique incidence of X-rays, it is
possible to determine the thickness of bone tissue or nerves.
The determination of dry weight (mass) of cellular structures can be
made with X-rays, but the interference microscope can also be employed
for mass determination. In the latter case, the material can be alive and
in fluid. Sometimes it is advantageous to compare both methods as
counterchecks on artifacts: is either or are both producing artifacts?—
the interference microscope because of fluid being present, or the X-ray
product because the material is fixed or dried.

Ultraviolet Microscopy

Short waves beyond the visible spectrum have a profound effect upon
living matter and are useful for the physical analysis of such matter.
Some living material under the influence of ultraviolet radiation visibly
radiates, glows and fluoresces. Some does not and remains dark. A
microscope for this type of observation has been designed with optical
glass in the objectives but is equipped with a quartz substage condenser.
A quartz lamp condenser is part of the system to help concentrate the
maximum amount of near violet on the specimen. The object itself
should not fluoresce, but will absorb, partially absorb or transmit the
Specialized Microscopy 101

ultraviolet and thereby reveal structural differences in a photographic

image without the use of stains. The specimen must be mounted in a
nonfluorescent medium: water, glycerol or mineral oil. Glass fluoresces
and will reduce contrast and possibly obscure some detail in the speci-
men, so quartz cover glasses and slide should be used for best results, also
fluorescent-free immersion oil.
Resolution is increased over that of the conventional microscope and
differences in structure in the specimen are enhanced by the ultraviolet
absorption. ‘The technique is not complicated, nor expensive.
But for greater resolution and higher selective absorption, a quariz
ultraviolet microscope is better than the above. It is, however, more
difficult to handle and focus, also more costly. The quartz microscope
is equipped with fused quartz objectives, crystalline quartz eyepieces,
quartz substage condenser, and quartz right angle prism. Quartz slides
and cover glasses should be used. The light source must be ultraviolet.
Some cells are immune to ultraviolet, others are only mildly affected
and can be used with excellent results under this microscope. But in
the case of those that are killed by ultraviolet, formation of artifacts
should be expected. Because of the possibility of damage to these cells,
focusing can be done with visible radiation and then ultraviolet used
only during photography or photoelectric measuring. Tissue cannot
be fixed for ultraviolet—this decreases absorption—but ice solvent or
freeze dry techniques can be used. Ultraviolet photomicrographs are
difficult to interpret at times and require considerable experience.
The ultraviolet microscope, therefore, has become a useful instru-
ment for measuring the selective absorption of cellular components
(measured at specific wavelengths). Its chief uses have been for the
measurement of cellular concentration and localization of nucleic acids,
RNA (ribonucleic acid) and DNA (desoxyribonucleic acid) content, ob-
servations on normal and neoplastic (tumoring) tissue (in the latter the
nuclear RNA content is high). Some substances can interfere with such
observations. Perhaps among the more interesting are the barbiturates,
which absorb heavily and should not be used for sedation or anesthesia
in animals to be prepared for this technic.

Fluorescent Microscopy

An object fluoresces when it absorbs ultraviolet light reflected on it or

transmitted through it and then emits the energy as visible light of a
specific violet, blue, green, yellow, orange, or red color. Secondary flu-
orescence can be induced by the use of fluorochromes (strongly fluores-
102 The Microscope (CHAP. 9)

cent dyes or chemicals) applied to the specimen. Even though many

objects fluoresce naturally, fluorescence can be intensified in them or
induced in others by the use of fluorochromes which are simple to use.
As with all ultraviolet preparations, nonfluorescent solutions, mounting
media and oil immersion must be used. Quartz slides and cover glasses
can be used, but are not essential for the technics included in this
manual.

APPARATUS REQUIRED FOR FLUORESCENT MICROSCOPY

Fluorescent microscopes can be purchased from several of the optical
companies (Reichert, Zeiss-Winkel, American Optical). In addition to
a compound microscope with a focusable condenser (A bbe is satisfac-
tory) minimum equipment includes: a mercury lamp and filter and
yellow check filters for each ocular eyepiece. Although some technicians
feel that a front surfaced aluminized mirror should be used, this is
not essential. A monocular microscope gives a brighter image than a
binocular microscope.

AMERICAN OPTICAL EQUIPMENT

1. A merc-Arc illuminator (lamp housing and optics are separate from

the power supply), complete with glass optics. Osram HBO-200 lamp
and power supply.
i) Multiple filter holder for use with the illuminator.

3. Exciter filter for higher light transmission (between light source

and microscope condenser) .
4. Barrier (check) filter for ocular.

ZEISS SMALL EQUIPMENT

1. Lighting unit: lamp housing, transformer, mercury burner Osram

HBO 74, Schott BG 12 filter, and gives ultraviolet in 350 my—450
myp ranges.
2. Barrier filter (OG 5) for eyepiece.

ZEISS LARGE EQUIPMENT

1. Osram HBO 200 lamp (44 times brighter than above), 2 Schott BG
12 filters.
2. Zeiss OG 4 and OG 5 filters for eyepiece.
Keep all equipment free from grease, which is opaque to ultraviolet
light.
Fluorescent microscopy differs from ultraviolet microscopy in that
the former observes the specimen directly by innate autofluorescence or
Specialized Microscopy 103

by secondary fluorescence induced by fluorochromes, while the latter

records specific absorption of the ultraviolet light by certain structures
in the specimen.

USES FOR FLUOROCHROMES

Granules of islets of Langerhans: Hartroft (1951); mucin: Hicks and

Matthaei (1958); blood vessels and lymphatics: Schlegel (1949); muco-
polysaccharides: Kuyper (1957); bone marrow: Werth (1953); amyloid
and connective tissue: Vassar and Culling (1959); nerve cells: Zeiger
et al. (1951); fat: Metcalf and Patton (1944); nucleic acids, nucleopro-
teins: Armstrong (1956), deBruyn et al. (1953); cancer: Vinegar (1916),
von Bertalanffy and von Bertalanffy (1960), and Umiker and Pickle
(1960); alkaline phosphatase: Burstone (1960). This bibliography will
easily lead to many others.

Electron Microscopy

In the electron microscope magnets focus an illuminating beam of

electrons onto the specimen. The scattering of the electrons by the
specimen forms shadows that can be photographed on film. Discussion
concerning this method is found on page 397.

Microscopy References
Barer (1956, 1959); Beck (1938); Belling (1930); Bennett (1950);
Davis (1958); Dempster (1944A and B); Engstrém (1956, 1959); Gage
(1943); Ham (1957); McClung-Jones (1950); Munz and Charipper
(1943); Needham (1958); Nurnberger (1955); Oster (1955); Popper and
Szanto (1950); Richards (1954); Ruch (1955); Scott (1955); Shillaber
(1944); Vickers (1956) and Walker (1958).
 Chapter 10

Stains and
Staining

Stains and ‘Their Staining Action

Most tissues after processing do not retain sufficient color to make
them and their components visible under a brightfield microscope. It
is therefore expedient to add colors to tissues by staining (coloring,
dying) them with the proper stains (dyes).
Baker (1958) argues logically against the nomenclature “stains and
staining,” saying that they are not accurate terms. For example: “basic
fuchsin ‘stains’ chromatin; silver compounds ‘stain’ nerve fibers and
sudan black ‘stains’ lipids,” yet from a physical and chemical point of
view the three processes differ markedly. He suggests that the terminol-
ogy “colour and colouring agent or colourant” (British spelling) be
applied to methods of imparting color on or in organic material. At
present it does not seem advisable to depart in this direction. Supply
houses still list their products as dyes or stains and specify dye content.
The Biological Stain Commission has not seen fit to change its name.
To the author, therefore, it appears politic to continue to use “dye,”
“stain” and “staining” until such a time as Baker’s suggestion has taken
hold. When a colored substance which is not a dye is formed, as is true
of many histochemical tests and silver impregnation, the appropriate
terminology will be applied.

104
Natural Dyes 105

Natural Dyes

Cochineal and Carmine

These are members of a group of dyes called “natural” stains. Unlike

other natural stains, cochineal and carmine are derived from an animal
SOUTCE a minute insect, the cochineal insect, Coccus cacti, living on
spineless cacti. The dye is present as a purple sap in the females, which
are harvested, dried and pulverized to produce cochineal. This dye by
itself has little affinity for tissue unless iron, aluminum or some other
metal is present. With the salt of one of these metals as a mordant (see
page 107), staining will result. Alum cochineal, a commonly used form
of this dye with mordant, can be an efficient nuclear stain. The dye
carmine, is derived from cochineal by boiling the latter with a salt,
usually alum, to produce a precipitate. This precipitate is insoluble in
water and before it can be used as a stain must be converted into a
soluble compound such as ammoniacal carmine or aceto-carmine, a
process that will be described under Mordants, below.

Hematoxylin

In many respects, hematoxylin can be regarded as most important

among the natural dyes. It was one of the first histological dyes, and
still remains one of the most widely known and used dyes. Hematoxylin
is extracted from the heartwood of logwood trees from South and
Central America and the West Indies. The tree is Hematoxylon cam-
pechianum, one of the legumes (Conn, 1953), similar to acacia or cassia
trees. Ihe crude material is exported as logs, chips or as dried aqueous
extract of the heartwood. This then is extracted with ether in a con-
tinuous extraction apparatus, evaporated to dryness, dissolved in water,
filtered and crystallized out of solution. All of these steps are slow and
difficult to handle and require costly apparatus, thus making hematoxy-
lin one of our most expensive dyes.
In this condition it is not yet a dye, and its color must be allowed to
develop by oxidation into hematein (color acid—no relation to hematin,
the colored constituent of red blood cells). Oxidation may be accom-
plished in either of two ways: “naturally’’—a slow process of exposure
to air for 3 to 6 weeks, as in Heidenhain’s hematoxylin—or artificially
by the use of mercuric oxide, hydrogen peroxide, or other oxidizing
agent—a more rapid process as in Harris’ hematoxylin. Used alone,
hematein is only a weak and diffuse dye with little affinity for tissues.
106 Stains and Staining (cHaAP. 10)

A weak acid will not combine with nuclear elements in sufficient quan-
tity to produce efficient staining. Some form of mordanting (page 107)
is required to form a base from this dye, which will then stain the acidic
nuclear elements. Ihe most commonly used mordants are alum salts of
aluminum, potassium or iron, as already mentioned.
Baker (1958) recommends oxidation with sodium iodate for pre-
paring hematoxylin solutions. Start with a wholly unoxidized he-
matoxylin dye powder, not hematein powder. Solutions started with
the latter tend to lose their strength by flocculation (sedimentation)
of the products of oxidation. The hematoxylin dye when in solution
should be only partly oxidized by the sodium iodate. Use less chemical
than would be required for complete oxidation; about one fourth to
one half of the full amount of oxidizer is adequate. The solution will
then continue to gradually ripen by atmospheric oxygen and thereby
maintain its strength. Such solutions are allowed to ripen slowly, six
weeks or more. (Mayer’s hematoxylin is an example of this type.) They
will produce brilliant staining for many months.
The rate of oxidation is also affected by the solvent. A neutral
aqueous solution forms hematein in a few hours; an acid solution does
this more slowly and an alkaline solution more rapidly. Alcoholic solu-
tions are slow, and the addition of glycerine retards them even more.
Color changes which take place in a stock solution indicate its efficiency.
The changes, with no mordant present, are from water white through
lilac, bright purple, deep purple, red, orange red, orange brown to
brown. At the purple stage, the solution is most vigorous; at the red
stages less so; and at the brown stage it is no longer useful. ‘The lifetime
of alcoholic solutions is five times greater than that of aqueous ones.
(Cole, 1943)
Hematoxylin is an exceedingly powerful dye with various shades of
staining from purples, through blues and into blue-blacks. The iron-
mordanted form is one of the most valuable dyes for mitotic study, and
gives to the chromatin a precise black or blue-black color. This black
color is the result of the presence in hematoxylin of some tannin, and
the latter in combination with iron salts produces a lasting black color.

Other natural dyes are SAFFRON from stigmas of Crocus; INDIGO from
plants of the genus, /ndigofera; BERBERINE from barberry; ORCEIN and
LirMus from the lichens, Lecanora and Rocella; and BRAZILIN from
brazilwood—a redwood tree of the tropics. Orcein, a specific dye for
elastin (present in elastic fibers), is prepared by boiling the plants in
water. The lecanoric acid in them splits to produce orcinol—a resorcinol
Mordanis 107

with a methyl group attached to it. Orcinol with NH; (ammonia) and
atmospheric oxygen forms orcein.

Mordants

Carmine, dissolved in a solution of aluminum sulfate, becomes posi-

tively charged and acts as a highly basic dye. Such a compound, formed
by a dye radicle with the salt or hydroxide of a divalent or trivalent
metal serving to attach it to tissues, is called a lake. The salt used for
this purpose is called a mordant (meaning “‘to bite’). Lakes may be
unstable or insoluble. Usually the tissue is treated first with a solution
of the mordant and then placed in the dye, and the lake forms in the
tissue. The term mordant should not be used for all substances that
increase staining action—only when salts and hydroxides of di- and
trivalent metals are used.
The use of mordants has many advantages. Once the mordant-dye
has combined with the tissue, the dye is relatively permanent, is in-
soluble in neutral solutions, and can be followed by many forms of
staining. Dehydration will not decolorize them. There are three meth-
ods of use:
1. Mordant preceding dye.
2. Mordant and dye together.
3. Mordant following dye (rare).
For carmine and hematoxylin the mordants commonly used are
aluminum, ferric and chromium salts, and alums (potassium alum,
ammonium alum, iron alum and chrome alum). Ferric chloride also
may be used as a mordant for hematoxylin, causing the tissues to stain
more rapidly and more intensely than after iron alum. A 4% solution
of ferric chloride can be used in place of the 4% solution of iron alum,
and Cole (1933) recommends the use of a phosphate-ripened hematoxy-
lin with it.
For long-lived solutions of combined mordant and dye, mordants
with little or no oxidizing action must be used: ammonium alum,
potassium alum, phosphotungstic acid, phosphomolybdic acid and iron
alum-ferrous sulfate. If a long life is not requisite, mordants with vigor-
ous oxidizing action can be used. Since their usefulness is only a matter
of hours, these solutions must be prepared immediately before use:
ferric chloride, ferric acetate and ferric alum. (Cole, 1943).
When using two separate solutions, a mordant of any kind can be
108 Stains and Staining (CHAP. 10)

used if followed by a well-ripened hematoxylin. If the solution is un-

ripened, then the mordant should include a substance of considerable
oxidizing power, a ferric or chromium salt. The value of two solutions
lies in the fact that the dye can be preceded by a salt, which can not be
used in combination with the dye in a single solution. Ferric chloride,
when added to an ammonia-ripened hematoxylin, will throw down a
precipitate of ferric hydroxide. Double mordanting can be profitable.
A mordant followed by a solution of hematoxylin containing a mordant
gives excellent results. The mordants for separate use are: ammonium
or potassium alum, ferric ammonium alum (2-3 drops of HCI increases
contrast), and ferric chloride (HCI increases contrast). They yield the
following colors with hematoxylin: ammonium alum, bright blue
nuclei; potassium alum, lilac or violet; chrome alum, cold gray blue;
iron alum, blue to black. (Cole, 1943)
Relative to the use of metals with hematoxylin, the following list of
tissue elements and the metals effectively used on them may be of prac-
tical interest. (Mallory, 1938)

Nuclei: aluminum, iron, tungsten

Myelin sheaths: chromium, iron, copper
Elastic fibers: iron
Collagen: molybdenum
Fibroglia, myoglia, neuroglia, epithelial fibers: tungsten
Axis cylinders: lead
Mucin: iron
Fibrin: tungsten
Lakes, formed by mordant and dye, can be used progressively, but
when mordant and dye are used separately, regressive staining usually
is more effective. In progressive staining the stain is added to the tissue
until the correct depth of color is reached. In regressive staining, the
sections are overstained and excessive amounts are removed by one of
the following methods.

Method 1: By Use of Excess Mordant

With an excess of free mordant present outside of the tissue, the

mordant-dye complex in the tissue is broken up, and, since the amount
of mordant in the tissue is small in comparison with that in the differ-
entiating fluid, the dye moves out of the tissue into the latter. (If the
tissue is left long enough in the fluid, it is conceivable that most of the
dye could move out of it into the excess mordant and the sections be-
Synthetic Dyes 109

come colorless.) Because the nuclei hold considerably more dye than
the cytoplasm, the dye is lost more completely from the latter, while
some still remains in the former. At the proper point of extraction from
the cytoplasm and when correct intensity is left in the nuclei, the slides
are taken out of the mordant and thoroughly washed, usually in run-
ning water, to remove excess mordant. Remaining traces can cause the
stain to fade in time.

Method 2: By Use of Acids

Acids are effective differentiators for some dyes, but a completely

adequate explanation for their action is not available.

Method 3: By Use of Oxidizers

Oxidizers furnish a third method of regressive staining, and by this

method the dye can be oxidized to a colorless condition. Oxidizers are
slow in action; the parts of the cell which hold only a small amount of
dye will be bleached before those possessing greater quantities of it. A
complete explanation is not available in this case. Picric acid, a com-
monly used chemical in this category, has both a moderate oxidizing
and a weak acidic action.
Not to be confused with mordants are accentuators and accelerators.
Accentuators are substances which, contrary to the action of mordants,
do not become a part of the dye complex or lake. Instead they increase
the selectivity or stainability of the dye (example: phenol in carbol-
fuchsin). Accelerators, as their name implies, accelerate the action—
usually of importance here in silver impregnation (chloral hydrate).

Synthetic Dyes

Natural dyes had no competition until the middle of the nineteenth

century when William Perkin worked out the processes for making
aniline or coal-tar dyes.
Synthetic dyes, like natural ones, can be used either progressively or
regressively. An acid solution often is used to remove excess basic dye;
an alkaline solution is used to remove excess acidic dye. In some cases,
alcohol can act as a differentiator, particularly for basic dyes; but in
general a sharper differentiation is achieved by using an acid.
The real importance of synthetic dyes lies in their use for double
110 Stains and Staining (cHAP. 10)

and triple staining, the use of two or more stains on the same slide. By
placing several different contrasting colors in a tissue, this type of stain-
ing has definite advantages. The dyes, if properly chosen, will stain
histologically; that is, each dye because of a known specificity will elect
to stain only specific parts of the cells. Due to their chemical nature
synthetic dyes make this kind of staining possible. By being synthesized,
their formula can be controlled and the significant part of the dye is
either anionic (acid) or cationic (basic) in action. Actually dye powder
as purchased is a salt, but the salts of the so-called basic dyes give up
OH ions and act as cations, while the acidic dyes give up H+ ions and
act as anions. Therefore an acid dye is the salt of a color acid, usually a
sodium salt; a basic dye is the salt of a color base, usually a chloride.
Basic dyes have an affinity for nuclei, which are basophilic (readily
stained by basic dyes) and acidic dyes have an affinity for the cytoplasm,
which is acidophilic (readily stained by acidic dyes). Also acid and base
dyes may be combined to form “neutral dyes” which give results differ-
ing from those obtained with ordinary double staining using separate
acid and base dyes. The action of neutral dyes is an example of poly-
chroming, a process in which a dye forms other dyes spontaneously—
the basis of the development of modern blood staining (Romanowsky
stains) in which methylene blue and eosin combine in a polychroming
mixture. Through polychroming, a new group of dyes, azures, is pro-
duced for the multiple-staining action which is so desirable in the differ-
entiation of white blood cells.
Polychroming must not be confused with another type of staining,
metachroming, in which certain substances are stained in one color
and others in another color by the same dye. In the case of the dye
thionin, the explanation for this reaction may be as follows:
Thionin stains chromatin blue; it stains mucus, ground substance of
cartilage, and granules of mast cells, red. The dye seems to exist in aqueous
solution in two forms: (1) the normal color, blue; and (2) the metachromatic
color, red. Both forms are always present, but the red is in a polymerized
form of the blue. The blue form is favored by increase of temperature, a
lowering of pH, a decrease of dye concentration, and addition of salts, al-
cohol, or acetone. The red form is favored by a decrease of temperature, a
raising of pH or increase in concentration of dye (Bergeron and Singer,
1958). Also certain substances, sulfuric esters of high molecular weight and
their salts, increase the production of the red form. Mucin, the ground sub-
stance of cartilage, and the granules of mast cells contain substances of this
nature, and therefore take up the metachromatic form.
Nature of Staining Action hit

The majority of dyes do not stain metachromatically, but are ortho-

chromatic in action. This means that their action is direct and predict-
able under normal conditions; if it is a blue dye, it stains blue, if it is a
green one it stains green, and so forth.

Structure of Synthetic Dyes*

The synthetic (coal tar or aniline) dyes are derivatives of benzene, all
built on the benzene ring. When certain chemical groups, called
chromophores, are attached to a benzene derivative, the compound ac-
quires the property of color and is known as a chromogen. A chromogen,
however, still is not a dye. It has no affinity for tissues, will coat them
only mechanically, and can be easily removed by mechanical means.
The compound must also contain a group which gives it the property
of electrolytic dissociation (the formation of cations and anions in solu-
tion). This auxiliary group, known as an auxochrome, furnishes the
required salt-forming properties. As mentioned above, most of the dyes
are sold as salts and are stable powders until put into solution, then
becoming acidic or basic by dissociation.
Nomenclature of stains has no absolute conformity. The color may be
used—orange G, Martius yellow; or perhaps some chemical term—
methyl green and so forth. If the term is followed by a letter or numeral
(Sudan III, IV, ponceau 2R, 4R), one dye is being distinguished from
a related one. B indicates a more bluish color, Y or G a more yellowish
color, WS means water soluble; and A, B, C distinguish among certain
azures.

Nature of Staining Action

Biologists and biochemists argue concerning the nature of staining

action—is it chemical, physical or a combination of both? If chemical,
this can mean that some parts of the cells are acid and others alkaline,
the former tending to combine with cations and the latter with anions.
There is an absorption and diffusion of the dye penetrating the cellular
elements, combining with them and remaining there in a state of more
or less chemical combination. ‘This action can be combined with physi-
cal action, where there is an adsorption of the dye, an attraction of plus
and minus charges for each other and a condensation of the dye on the
surface of the cell parts. Minute particles of the dye are deposited on
*For a complete discussion of dyes, consult Conn, 1953 or Gurr, 1960.
112 Stains and Staining (cHAP. 10)

the surface of the tissue by selective adsorption and then enter into
combination with the tissue. ‘The proteins, nucleic acids, and other
components of the protoplasm proceed along lines of chemical laws by
exchanging ions. The Stearns (1929, 1930) maintain that the confusion
lies in the term adsorption, that it may be either a chemical or a physical
force, and the adsorbent can form ions and then proceed along chemical
lines.
In any case, the staining properties depend on these three factors:
1. strength of dye
2. rate of ionization of tissue proteins and dyes
3. pH value of dye solution and tissue proteins
In addition, staining can be affected by other conditions:
alcoholic or aqueous solution of dye
. low or high temperature during reaction
=
INO
oo simple or multiple combinations of dyes
aie strong or weak concentration of dye in solution

Standardization of Stains

In the early days of tissue staining it was difficult to secure reliable

dyes. The textile dye industry was the sole source of dyes and products
received were often unsatisfactory. Standardization was crudely done as
to color, and this was no standardization as to chemical content. The
impurities were extremely variable in quantity and quality. Griibler in
Germany was the first to try to standardize dyes and he built a highly
specialized business in this field. He did not actually manufacture dyes,
but he bought up batches from other firms and tested them for technical
use. After the beginning of the twentieth century, certain events
changed Griibler’s hold on the business of standardization. Perhaps of
ereatest influence were two world wars, causing Germany to lose its
monopoly in the dye industry. No country could afford to remain de-
pendent on another country for its source of dye if that country was
likely to remain an enemy.
‘The lack of German dyes led to a new form of standardization in the
United States. A body was organized called the Commission on Stand-
ardization of Biological Stains, later to become the present Biological
Stain Commission. The object of this commission is to work with the
manufacturers showing them what the biologists require, testing their
products and permitting approved batches to be put on the market with
Standardization of Stains iS

the stamp of the commission on them. Specifications of the most impor-

tant dyes now have been drawn up by the commission putting these dyes
on a certification basis. The specifications are partly chemical and partly
spectrophotometric and contain detailed statements as to how the dyes
should be tested for their behavior with the results to be expected from
their tests. Batches of dyes approved by the commission bear a special
label furnished by the commission and known as a certification label.
On it is a C.I. number (Colour Index number) indicating the certifica-
tion number of that particular batch. This number means that (1) a
sample of that batch was submitted to the commission for testing and a
portion of it is on file; (2) the sample proved true to type by spectro-
photometric tests; (3) its dye content met specifications and is correctly
indicated on the label; (4) it was tested by experts in the procedure
named on the label and found satisfactory; and (5) no other batch can
be sold under the same certification number. Any description of the
use of the stain should be followed by its C.J. number or a C.C. indi-
cating that it is Commission Certified.”
Unless otherwise specified, dyes can be purchased from most of the
scientific supply houses with chemical outlets: Coleman and Bell Co.,
Hartman-Leddon Co., Fisher Scientific Co., Allied Chemical Corpora-
tion (National Aniline Division), and others.

Sources of foreign stains:

G. Griibler and Company, West Berlin, Germany.
Esbe Laboratory Supplies, 459 Bloor St. W., Toronto, Canada, outlet
for Gurr Stains (Michrome Stains).
Edward Gurr, London, England.
Pfatz and Bauer, Inc., Empire State Building, New York City.
Roboz Surgical Company, Washington, D.C.

References: Baker (1945, 1958); Cole (1933, 1943); Conn (1946, 1948,
1953); Holmes (1929); Singer (1952); and Stearn and Stearn (1929,
1930).
*C.I. numbers used in this text are from the new revised list.
 Chapter |]

Staining
Procedures

Usually certain standard principles apply to the processing of tissue

on slides, but many exceptions and variations occur and will have to be
handled individually.
The sections first must be deparaffinized, because most stains are
applied in either aqueous or alcoholic solutions and would not pene-
trate efficiently through paraffin-infiltrated tissues. ‘The customary sol-
vent for paraffin is xylene. ‘This is followed by the removal of the xylene
with absolute alcohol because stains rarely can be applied successfully
in a xylene medium. After the removal of the xylene, a general rule is
to transfer the slides to a medium comparable to the solvent of the dye
being used. That is, if the dye is a water solution, the slides are hydrated
through a series of decreasing alcoholic and increasing aqueous dilu-
tions, such as 95%, 80%, 70% and 50% alcohol, or the like, and
finally they go into water. If the dye is dissolved in a 50% alcoholic
solution, then the slides are carried only to 50% alcohol before going
into the staining solution.
During the hydration process, undesirable pigments or other ma-
terials (mercuric chloride crystals, formalin pigment, etc.) are removed
and the slides washed well to remove the responsible reagent. Counter-
stains (background color) or other special treatments must be applied
in their proper sequence, to allow each dye or chemical to maintain its
specific effect on the tissue elements. Improper sequence of staining,

114
Staining Procedures Lib

decolorizing, and other solutions can result in a poorly stained slide.

Hematoxylin-eosin staining (page 130) provides a simple example. If
the eosin (counterstain) is applied before the hematoxylin (nuclear)
stain, the former stain will be completely removed during the action of
the latter stain.
If slides are to be transported in quantity, rather than individually,
several types of holders are useful and are on the market. 4.H. Thomas
has one holding 50 slides; Wards has one for six slides. Some are baskets,
others are clips, and fit into special staining dishes. ‘The tissue-processing
machines are equipped with slide carriers to fit the instrument. Phos-
phor-bronze spring wire, 0.05 inch di-
ameter, can be fashioned into coils of
3 inch diameter and cut into any length
to hold anywhere from 3 or 4 slides up
to 15 or 20. (Fig. 23) This combination
of slides and coil can be used in rectangu-
lar staining dishes with the slides resting
on their long edges, or standing upright
in tall stender dishes.
A final processing of slides is necessary
to make permanent preparations for ex-
amination and storage without deteriora-
tion. All alcohol and water must be
extracted (with certain exceptions) and
a medium applied which maintains the
tissues in a clear and transparent condi- Figure 23. A staining coil
tion, does not alter the color or intensity carrying multiple slides.
of the stains, and holds a cover glass in
place. The water is removed through increasing concentration of alco-
hol until absolute alcohol is reached. The final reagent is xylene (or
the like) to remove the alcohol and make the sections lose their opacity
and thereby become clear. Finally a mounting medium (mountant)
is applied and the cover glass lowered into place completely covering
the sections. (The solvent of the mounting medium usually is either
toluene or xylene.)

Mounting a Cover Glass

The tidiest method for mounting a cover glass is this: (1) apply a thin
streak of medium on the cover glass (Fig. 24); (2) turn cover glass over
and rest on edge (shortest side of rectangles) on the slide, to left of
116 Staining Procedures (CHAP. 11)

sections (unless technician is left-handed) (Fig. 25); ease cover glass into
place slowly to allow air to displace from under cover glass; press firmly
from center outwards, to evenly distribute the medium. An alternate
method is to apply mounting medium along one edge of sections on
slide; rest one edge of cover glass adjacent to mounting medium and
lower gradually to ease out air without bubble formation; press gently
in. place.
If, during microscopic examination of stained and mounted slides,
dull black spots replace nuclear detail, the clearing solution partially
evaporated out of the sections before the cover glass was in place. Re-
turn such slides to xylene, dissolve all mounting medium to allow the
air to leave the sections, and remount. If the slides appear dull, almost
milky, instead of crystal clear, water is present. Remove all mounting
medium in xylene and return slides to absolute alcohol, preferably a
fresh solution. Clear and remount.

Mounting Media (mountants)

Formerly natural resins were used as mounting media: Canada bal-
sam, composed of terpenes, carboxylic acid and esters; gum damar,
composed of unsaturated resin acids and a little ester; or gum sandarac,
an unsaturated acid resin. These dried slowly, were variable in compo-
sition and unpredictable in behavior. Some developed acidity and faded
stains, would turn yellow and crack after a few years. One still used,
particularly for blood smears, is Ewparal, refractive index 1.483, a
eutectic (melts at low temperature) mixture of oil of eucalyptus, gum
sandarac, salol, paraldehyde, menthol and camphor. Euparal Vert
(green because of copper salt content) is claimed to intensify hematoxy-
lin stains. Dried smears may be mounted directly in Euparal, but
sections must be carried into 95% alcohol, and then into Euparal
Essence before mounting. (If alcohol is carried into the Euparal, sec-
tions will fade.) Drain off excess Essence and apply Euparal.
The synthetic resins now available have proven superior to natural
resins in most respects. The composition of synthetic resins can be con-
trolled and they are stable and inert. They dissolve readily in xylene or
toluene, do not require long drying, and adhere tightly to glass. They
have the correct refractive indices, are pale in color, and do not yellow
with age.
The most widely used synthetics are the 8-pinene polymers—terpene
resins, such as Permount (Fisher Scientific Company) and Piccolyte
Mounting Media (mountants) 7

Figures 24 and 25. Placing mountant on cover glass, turning cover over, and
lowering it onto slide.
118 Staining Procedures (cHAP. 11)

(General Biological Supply House) with a refractive index of 1.51 to

1.52. Hartman-Laddon Company sells “HRS,” Harleco Synthetic Resin,
index of refraction 1.5202; Will Corporation has a Bioloid Synthetic
Resin, index of refraction 1.5396; Ward’s sells Kleermount; also the
Technicon Company sells one. ‘The resins should have refractive indices
of Je53: to 1.54 orubetter. (Lillie cf aig 993) Where is no feason te
recommend one of the above products over the others; all are equally
efficient. ‘They are soluble in xylene, toluene, aromatic hydrocarbon
solvents, and in chlorinated hydrocarbons such as chloroform, but not
in Dioxane.
Hyrax (Fisher Scientific Company), refractive index 1.71, is a perma-
nent, neutral synthetic naphthalene resin, formulated by Dr. G. Dallas
Hanna for mounting diatoms. It is good for some unstained botanical
and parasitological materials which lose detail in other synthetics be-
cause they become too clear. Hyrax brings out structure of colorless
fibers and spines.
Lillie et al. (1950) recommend Lustron 2020 for good preservation of
Prussian blue mounts, which tend to fade in some resins. Lustron is a
polystyrene manufactured by Monsanto Chemical Corporation and has
a refractive index of 1.59. It is water resistant and soluble in varying
degrees in aromatic and chlorinated hydrocarbons. ‘The above authors
used it in diethylbenzene as solvent. ‘he present author has had excel-
lent results using toluene or xylene without the formation of air bays
as the xylene evaporated. If such trouble is encountered, diethylbenzene
is recommended; Lillie et al. suggest adding 5 ml. dibutylphthalate to
70 ml. xylene and 25 em. polystyrene. This does not delay hardening.
‘There are also the German synthetics, Caedax and Rhenohistol, and
the British ones—Xam, Cristalite and Clearmount. These are similar
in most respects to American products.
Most synthetic mountants are allowed to air dry, but if quick drying
is a must, try this method of Manikas and Umiker (1959): After slides
are covered, place them on metal trays in a drying oven, 160—170°C, 3
minutes. Remove and chill in freezing compartment of refrigerator, 2
minutes. Slides can be cleaned, marked with ink and stored without
dislodging cover glasses.
Aqueous mounting media are indispensible for the preservation of
tissue elements which are soluble in alcohol or hydrocarbons, or are
demonstrated by the use of dyes soluble in these fluids. A number of
media have been proposed using (1) gelatine and gum arabic as solidify-
ing agents with water, (2) sugars and salts for increasing the refractive
Mounting Media (mountants) 119

index, and (3) glycerols and glycol as plasticizing agents. Gum arabic
slowly hardens by drying, gelatine sets by cooling, and the glycerol keeps
them from cracking or overfrying. The addition of phenol, thymol,
merthiolate, or Zephiran prevents mold growth.

Kaiser’s Glycerol Jelly

ACC ig ore, ands BM in:Une o:daca Oui Bea aeeine + 93% 52:0°ml.
SCANS cae stseke, Se =< x 1s Sp eh el 8.0 gm.
CAV ECON oe oc oP ag oes ade hee ees doe 50.0 ml.

Preserve with either (1) carbolic acid (phenol)

0.1 gm.; (2) merthiolate, 0.01 gm.; or (3 9
—
.

Zephiran 0.1 ml.

Allow gelatine to soak for 1-2 hours in water, add glycerol and pre-
servative. Warm for 10-15 minutes (not over 75°C) and stir until
mixture is homogeneous. This keeps well in covered jar in refrigera-
tor. If heated above 75°C, the gelatine may be transformed into meta-
gelatine and will not harden at room temperature.

Apathy’s Gum Syrup

puna arabic: (ACACIA). 8 ost dees a a ep eee 50.0 gm.
SUIETOSO (Came SUP BI) 4. 6 2 ae 62 eee 50.0 gm.
Gistilled) Wate fede ale ts cose die ees CR ee 50.0 ml.
TORII Gos of pee tn See Se oe As ae Bee 1;0° ml.

Dissolve lumps of gum arabic in warm water; add sucrose. When

dissolved, filter and allow to cool. Add formalin. Do not use powdered
form of gum; it makes a milky solution, whereas the lump form pro-
duces a clearer medium. The cover glass does not have to be sealed as
it does with Kaiser’s glycerol jelly above.

Farrant’s Medium
GIStMECGWANeR ny fies Lila ec Wigs ees 1 a 40.0 ml.
Pui araAVlG( ACACIA)! 444540523). 42% chee 40.0 gm.
BIVECTOU arin tene ace £84 5 hkad oS tS aR 20.0 ml.
carbolic acid) (pHeEROl) 2... 05 s..002 02S e ee eee. 0.1 gm.
(or merthiolate or Zephiran)

It is difficult to obtain a good quality of gum arabic to make a clean

and clear solution, so purchasing of the finished product from Amend
Drug and Chemical Company is preferable.
120 Staining Procedures (CHAP. 11)

Gray and Wess’ Medium (PVA) (1952)

PVA‘ (polyvinyl alcohol) 71-24"... 2... Bere Ns. 2.0
gm.
TORRACCUOINE$ (502 cere.c. 2. oe eee ; 7.0
ani:
PINGEROIN. 30 hiss Gee eh os et RO ae coe. 5.0
ml.
lace macid (s+ a5. 2! ee 5.0
ml.
distilled water 202%.) 0,:0° yt ee eee ee ees toe 10.0 ml.

Make a paste of the dry alcohol with acetone. Mix half of water with
elycerol and lactic acid; stir into paste. Add rest of water drop by
drop with stirring. Solution will be cloudy but becomes transparent
as warmed in water bath about 10 minutes.

PVA (polyvinyl alcohol) Mounting Medium

Add 15.0 gm. PVA 71-24 powder (DuPont. See note below) slowly
to 100.0 ml. cold water. Heat in water bath (80°C) with stirring until
the solution becomes as viscous as thick molasses. Filter out undis-
solved lumps through two layers of cheesecloth. Solution will appear
milky, but after standing for several hours, will clear. A thin film can
be spread over stained blood smears when cover glasses are undesira-
ble.
The following are recommended for small whole mounts, insects,
worms, tiny invertebrates and the like.

Berlese Mounting Medium

1. GRAY’S FORMULA (1952).
WADLEIY i y8 2). Acacia ceo ie oe! 10.0 ml
Slacial ACC tC aACiC ysgy seer mete: fs0.326 atte 3.0 ml.
EXE OSE\SVEUPi. fs pee eee ne, © gt os ee 5.0 ml.
SumiparaDic: (acacia) %Vy Wane. Se 8.0 gm.
cul@ral hydrate: 24 see eee en: 2, ela 2 75.0 gm.
Mix water with acid and syrup; dissolve gum in this. Requires week
or more. Stir at intervals. When solution is complete, add chloral
hydrate. This is one of the best media for insects. As was suggested
for Farrant’s medium, it is difficult to obtain good gum.
2. THICK FORMULA.
SSGUMIEG Water... |... new ie ei yee: + ere 40.0 ml.
saturated aqueous solution of chloral hydrate .. 30.0 ml.
guimarabic, (acacia) o4r eet Ae Hee %. 3 ad oe Oa em.
CIV GEEON ie cy. es oR Ie ee 5.0 ml.
1 Obtainable from E. I. DuPont de Nemours and Co., Wilmington, Del.
Mounting Media (mountants) 121

Dissolve gum arabic in water, add chloral hydrate and then glycerol.
Filter:
3. THIN FORMULA.
AIStUNLCO EWALD nccciare.cle os e's «ROO meg we Ske OES 100.0 ml.
gum arabic (acacia) .......... se roe .... 60.0 gm.
PIV CCTON sean & Soko
whoa Ss i a, Love sheer Pete ih 2.oe 40.0 ml.
saturated aqueous solution of chloral hydrate .. 100.0 ml.

Monk’s Karo Medium (1938)

white Karorsyrup™. v2.2... : ues Ce ee Ae urs 5.0 ml.
Certo (fruit pectin) ...... ee ee 5.0 ml.
VEEola ee ee a aS Oa 3.0) aml:
thymol for preservative

This is recommended for mounting a group of small parts which are

easily disarranged. A thin layer is spread on a slide and the parts to
be mounted are removed from glycerol and immediately arranged
in the medium. The mixture begins to set in about 2 minutes, and
holds parts in place. Dry to hardness over heat. Cover glass can be
added with a drop of mixture or with Euparal. If using mixture, use
enough to prevent air pockets from forming when the medium dries.

Lacto-phenol Mounting Medium

melted phenol (carbolic acid) .....:..:.22.%22 3 parts
NCIC ACIAY =F aa athe e oe nes le 3% ee ry. ] part
plycerols 2. . as. Wee es ook eee . 2 parts
distilled water ow Rese yay ae eens ] part

CMC-10 Mounting Medium

This is a nonresinous mounting medium supplied by General Bio-

logical Supply House. It is primarily for small arthropods, such as fleas,
ticks, mites, etc., but cannot be used for stained material. Small speci-
mens containing little air can be mounted directly, but larger ones are
mounted from water or alcohol.

Yetwin’s Mounting Medium for Nematodes and Ova (1944)

10% bacto-gelatine, granular, Difco .......... 150.0 ml.
plycerol’... 5 sie eay 4 « Me ges OY, 1) «eee 50.0 ml.

(chromic alum)! 6. a4 14 29s eee te oes ee 100.0 ml.

phenol canvolic acid), amelted|. .....:
he. 2.4% 2% 1.0 ml.
122 Staining Procedures (CHAP. 11)

Dissolve gelatine in boiling water, add glycerol. After mixing add

chrome alum solution and phenol. Liquefies in 15 minutes at 65°C.
May transfer from glycerol or formalin directly. Hardens to form
permanent mount.

Abopon
This mounting medium is water miscible and can replace glycerol
jelly in several special staining techniques. It can be purchased from the
Glyco Chemicals Company, Williamsport, Pennsylvania, in the form of
lumps or solution. Dissolve the solid form, approximately 50 gm., in
25 ml. distilled water. The fluid form usually is too thick and is diluted
with distilled water.

Diaphane
This medium is available in two forms, colorless (r.i., 1.4777) and
green (r.1., 1.4792). Slides can be mounted directly from absolute, 95%
or 70% alcohol. It and its solvent can be purchased from the Will Cor-
poration.

Fluorescent Mounting Medium (Rodriquez and Deinhardt, 1960)

Elvanol 51-05 (DuPont. See note, p. 120) ...... 20.0 gm.
0.14M Sodium chloride buffered with 0.01M
KH,PO, — Na,HPO,:12H,0, pH 7.2 ......
addygiveerol 2 oes. 2... ie 2 3 ae 104% Sage iCall

Agitate another 16 hours

Remove undissolved particles of Elvanol by centrifuging, 12,000 rpm,
15 minutes. The pH should be between 6 and 7.

Aqueous Mounting Technics

Aqueous mounts—sections or whole mounts—are removed from the
water, placed on a slide and covered with a drop of mounting medium.
The cover glass, held in a horizontal position, is placed directly on the
medium. Do not drop it from a slanted position. By the former method,
the object can be kept centered and not carried to one side. In many
Cases it 1s not necessary to press the cover glass into place; its own weight
is sufficient. ‘he sections or objects are not attached to the slides and
too much pressure may result in disarranged and broken material.
Aqueous Mounting Technics and Ringing Slides 23

The Two-cover Glass Method

The material is mounted out of glycerol into glycerol jelly between
two cover glasses, one of which is smaller than the other. Clean away
excess jelly and air dry overnight. Invert the pair of cover glasses on a
drop of resinous medium, such as Permount or Piccolyte, on a slide.
This will permanently seal the mount, and it can be treated like any
resin mount.

Ringing Slides
If the mountant contains a volatile substance like water and the
slides are to be rendered relatively permanent, the cover glass must be
sealed with a ringing material. Ringing cements are sold by supply
houses, such as General Biological’s ‘“Yurtox Slide Ringing Cement.”
Others easily obtainable are Duco cement, colorless nail polish, gold size
and asphaltum. Lustron 2020 (Monsanto Chemical Corp.) dissolved in
xylene can be used. Orange shellac in alcohol can ring a cover glass and
then be covered with black asphalt varnish. Prepare the ringing shellac
by dissolving flake orange shellac in 95% alcohol. It dissolves slowly, so
keep the bottle in a warm place, and shake it occasionally. By adding 1
drop castor oil to each ounce of liquid, the ring will not dry out com-
pletely. (Needham, 1958)
Conger (1960) recommends Dentists’ Sticky Wax (Kerr Manufactur-
ing Co., Detroit, Mich.). It is solid and slightly tacky at room tempera-
ture, but flows easily when melted. It adheres well and does not leak, but
will crack off cleanly if frozen with dry ice or liquid air. Good for
acetocarmine preparations.
A firm and reasonably permanent ringing cement is cover glass ce-
ment “Kronig” from Riedel-de-Haen AG, Seelze-Hannover, Germany.
At present it is not handled by American importers, but it can be pur-
chased from George T. Gurr, London.
For temporary mounts, melted paraffin can be used to ring a cover
glass, but it is susceptible to temperature damage and will crack away
from the cover. It, therefore, is not recommended for slides subject to
hard usage and the passage of time.
If the cover glasses are round, a turntable rotating on a steel pin or
ball bearing facilitates the ringing operation. (Watson and Sons, Lon-
don) ‘The turntable spins rapidly and, with the ringing cement on a
brush, a neat seal can be made by following concentric guide lines.
 Chapter | 2

Hematoxylin
Staining
SUBSTITUTES AND COUNTERSTAINS

Single Solutions
Delafield’s Hematoxylin (CARLTON, 1947)
Dissolve 4 gm. hematoxylin in 25 ml. absolute ethyl alcohol. Mix
eradually into 400 ml. ammonia alum, Als(SO4)3(NH4)2SO4-24H2O,
saturated aqueous (approximately | part alum to 11 parts distilled wa-
ter). Leave exposed to light in a flask with a cotton plug for 3-5 days.
Filter. Add to the filtrate, 100 ml. glycerine and 100 ml. methyl alcohol.
Ripen for at least 6 weeks. The ripened solution will keep for years in
a stoppered bottle.

Ehrlich’s Hematoxylin (curRR, 1956)

Nem aroxcy lan... 7 (te eemmnmeene eee hehe eee 2.0 gm.
ammonia alum, Al,(SO4)3(NH4)2SO,°:24H.O |. 3.0 gm.
aiceholymethyl or ethyl essa). | eae 100.0 ml.
PUVEEKOM ae.) hn. ee one ee See 100.0 ml.
disediled water ”.:...°o 2. Seenines
=skiers oe oe ae ae 100.0 ml.

eee
Single Solutions 125

Ripens in 6-8 weeks, or may be ripened for immediate use with 2.4
em. sodium iodate.
Add 10 ml. glacial acetic acid. Keeps for years.

Harris’ Hematoxylin (MALLORY, 1944)

Dissolve 1.0 gm. hematoxylin in 10 ml. ethyl alcohol. Dissolve 20
gm. potassium or ammonia alum, Als(SO4)3K2S5O4:24H2O or Als(SOx)3
(NH4)2SO4:24H2O, in 200 ml. water and boil. Add hematoxylin and
boil $ minute. Add 0.5 gm. mercuric oxide. Cool rapidly. Add few
drops of glacial acetic acid to keep away metallic luster and brighten
nuclear structure. Does not keep longer than a month or two.

Mayer’s Hematoxylin (MALLorRy, 1944)

Add 1 gm. hematoxylin to | liter distilled water. Heat gently and
add 2 gm. sodium iodate and 50 gm. potassium alum, Alo(SO4)3;K2SO4-
24H.O. Heat until dissolved and add 1 gm. citric acid and 50 gm.
chloral hydrate. Preferably ripen for 6-8 weeks, but can be used within
1-2 weeks.

Papamiltiades Hematoxylin (1953)

hematoxylin aqueous « 4-15 .40544 aaa 100.0 ml.
aluminum sulfate, 59, aqueous 2... 22.44.05 50.0 ml.
7ime Suliate; OF ages, fd. 4a... ashame 25.0" mil.
potassium iodide, 477, aqueous .... <1...
2: .6s8. 25.0 ml.
glacial acetic acid ... Ae ee cs 8.0 ml.
glycerol .... igejek Basn Ae ts eke 62 eee 25.0 ml.

Ready for immediate use; keeps approximately 2 months.

Phosphotungstic Acid Hematoxylin (LIEB, 1948)

phosphotungstic acid ..... ts \gera’s¢ ak een VOLO erat,
MENGAL OXWANsy 4.42.22, o 25 o ac bus dos Cee 0.05 gm.
FEC, METCUNIC ORNIGE... 6. i 54,22. Mons eee ee 0.025-0.05 gm.
hvdropen peroxide... 4.5.05 .h20s 2s See 2.0: mi:
Gistilled: water i: hs oa 22. .c cm: 4 0-2 oe ee 500.0 ml.

Dissolve hematoxylin in a little water with heat. Dissolve phospho-

tungstic acid in rest of water with heat; add hematoxylin solution.
Bring to boil. Cautiously add mercuric oxide, and remove from
flame. Cool. Add hydrogen peroxide. Ready for use in 5-7 days;
should be brownish red in color.
126 Hematoxylin Staining (CHAP. 12)

Double Solutions
Weigert’s Iron Hematoxylin (MALLORY, 1944)
SOLUTION A.
iron-chloride, KeCl;, 29%, aqueousue 3... .. #0 mi.
distilled (water yn) ads bes @ ea Pes 95.0 ml.
hydrochloric acid (sp.gr. 1.88—1.92, 37-38% HCl) 1.0 ml.
SOLUTION B.

hemiaitoxeylinitt (28s OEY Ns Eee BAe EO 1.0 gm.

95oethyl alcohol & 80. Re Eiger 204 100.0 ml.

Mix equal parts of A and B. This is best prepared each time, but will
keep for 7—8 days once mixed.

Groat’s Variation of Weigert’s Hematoxylin (1949) (a single solution)

distilled Water | 3 2. hss cysede ee ecstacy Sie 50.0 ml.
sulfuric acid (sp.er. 1:84, 949; sbipsO,) 2...5. 0.8 ml.
ferric alum, Fe,(SO4)3(NH4).SO,:24H,O ..... 1.0 gm.
goy,zethyl alcohol (0)... 5. ay th piln tae 50.0 ml.
Hematoxylin: . x2...sc a2 1 eee:
AYse oe 0.5 gm.

Mix in order given at room temperature. Filter. Groat recommends

his solution as better than Weigert’s; it stains well within 10 minutes.

Lillie’s Variation of Weigert’s Hematoxylin (LILLIE AND EARLE, 1939A)

SOLUTION A.

ferric alum, Feo(SO,4)3(NH4)2S0,:24H,O ..... 20.0 gm.

Gistilled: water. \7:1.4343, aeeS ee 200.0 ml.

SOLUTION B.

WeRICORYLI . . 4).5 ee Rea Mian, 6. tn oa 2.0 gm.

apsolmre: methyl alcoholye soem ot. css aden 60.0 ml.
SERVGETOUEE Sc... o's se eee et. <2: eee 60.0 ml.

Janssen’s Hematoxylin (LILLIE AND EARLE, 1939A)

SOLUTION A.
ferric alum, Fe2(SO,4)3(NH,4).SO,:24H,O ..... 15.0 gm.
ferrous;sultate; FeSO, HHO. ee. ie 15.0 gm.
distilled}water: . 1140.78,
RE ER el ie 100.0 ml.
Double Solutions }27

SOLUTION B.

eT atom ylaINs Cerys e eile aca AA aes she e's 1.0 gm.
Ono emaveaicOnol oo) ic ee Pee oa te ae 50.0 ml.
(edPS) Us AeA a Se eae PL ee ae 50.0 ml.

Mix 4 and B in equal amounts. Can be kept unchanged for several

months, but if solution has turned brown, it is no longer usable.
Keeps better than Weigert’s.

Hansen’s Iron Trioxyhaematin (PANTIN, 1946)

SOLUTION A.

ferric alum, Fe (SO4)3(NH4)2SO4:24H,O ..... 10.0 gm.

Ammon Suliate “a5. ..->-45o9 ean oeeee 2 14 ein:
Gis Ed sWALER Ono o.04.cte'..3'.'s-2.<
x ee eee 150.0 ml.
SOLUTION B.

HEMACONVNO ie Os Coase. g. os oa ht Re eee 1.6 gm.

aistilled’ Water <4 ary aeiek4.4 1s sad aeeee ee 79.0 mal.

Dissolve both solutions with gentle heat. Cool. Pour solution B in a

porcelain evaporating dish. Add 4, stirring constantly. Heat slowly
without stirring just to boiling point. Cool rapidly by floating dish
on cold water. A deep violet color turns brown. Filter into stoppered
bottle with little air space above solution. Keeps 6-8 months, but if
it develops a green sheen it is unsatisfactory. Can be used progres-
sively or regressively.

Double Solutions, Never Mixed Before Use

Heidenhain’s Iron Hematoxylin

SOLUTION A.
ferric alum, Fes(SO4)3(NH4)2SO,°24H2O ..... 4.0 gm.
distilled water ....... (cnt ea ees 100.0 ml.

Keep in refrigerator to prevent precipitation on sides of bottle.

SOLUTION B.

MEMALOX VINE op bess WAG ao oa «ala oo Ye Seg eE 10.0 gm.

Sor ecthyl alcOHOl ans .2cans ose, haem: 100.0 ml.
128 Hematoxylin Staining (CHAP. 12)

Let stand until a deep wine-red color; 4—5 months is not too long.
Add 4—5 ml. of this stock solution to 100 ml. distilled water; this
gives a practically aqueous solution and is already ripe. Saturated
aqueous lithium carbonate—3 drops—added to the working solution
improves color.
Never mix 4 and B. A is used as a mordant solution, and precedes B.

Mallory’s Iron Chloride Hematoxylin (1944)

SOW TON WAT:

ironychiloride; BeClo- 32.) aise

oe ee ee 5.0 gm.
aistilllediswatend) 265.5 a6.) she ae eee Ae 100.0 ml.

SOLUTION B.

nemlatoxy li spas ecstacy sa ee cs sus Oe 0.5 gm.

GISGIMER AVateM Se) ce tes) coReees ee 100.0 ml.

Prepare fresh each time. These solutions are never mixed. Rawlins
and Takahashi (1947) say that bubbling air through hematoxylin so-
lutions ripens them more rapidly. American hematoxylin solutions
may ripen in 2—3 weeks. Hance and Green (1959) ripen solutions even
more rapidly by bubbling oxygen from a tank into the bottom of a
container of hematoxylin.

Testing Hematoxylin Solutions

Add several drops of the solution to tap (not distilled) water. If it turns
bluish purple immediately it is still satisfactory, but if 1t changes slowly,
stays reddish or brownish, it has weakened or broken down and should
be discarded.

Substitutes for Hematoxylin Solutions

Gallocyanin
Seal Mey ARNDT» ha) a2". ee RR me fo5 cheso 0.15 gm.
chrome alum, Cr,(SO,4)3;K,SO,:24H,O 5%
AQUEOUS! +, 210) S2TRARIEe
ATES. BS. BOM 100.0 ml.

Boil 2-3 minutes. Filter. Keeps about a week, then deteriorates

slowly. An iron lake may be prepared by substituting 5% aqueous
iron alum for the chrome alum. (Proescher and Arkush, 1928)
Other Acceptable Counterstains 129

Hematein (Hemalum) (KORNHAUSER, 1930)

hemateluee 2 eee o lve ae tle .cnkn eo ad 0.5 gm.
GoU etal aCOWOL..2 ons. xe ss eek aes F414 10.0 ml.

Grind hematein with alcohol in glass mortar;

Add to potassium alum, Als(SO4)3K.SO,-24H.O
DPA MEOUS WS, Lek. »2.2.< 14d cee aad ase, 2A oe 500.0 ml.

Immediately ready for use.

Counterstains for Hematoxylin,

Gallocyanin and Hematein
Eosin
POS MGs 25500). . 5.04 2a cee een ee 1.0 gm.
VOU, cthydnalcOnol s 2... 26s ase ve eee es « 1000.0 ml.
PlAGial@CelC ACI: <5 255555 a4 a4 foe Phere as 5:0" mil.

Dilute with equal volume of 70% alcohol for use and add 2-3 drops
of acetic acid.

Eosin (puTt’s, 1948)

oO icshOlayate Oh [25
2)«||GPa ea ee Pee mS 1.0 gm.
potassium Cichnomate: .. 2.5 iac. 202k ee 0.5 gm.
saturated aqueous picric acid ...... 2222.4 244 10.0 ml.
apsoluberctuml alcohol, . 2... «2,1. 2% baked 10.0 ml.
iste water Wns oan cated baby bee Reem 80.0 ml.
glacial acetic acid (optional) ................. 1 drop

Eosin-Orange G
1% eosin Y, C.I. 45380, in 95% ethyl alcohol . . 10.0 ml.
orange G, C.I. 16230, saturated solution in
95% ethyl alcohol (approximately 0.5 gm. per
LAO)UISseit RE 2 a a ere ar ry6. . 5.0 ml.
Obe7, ctwicalcouol...22 34.2954 28.06 sha eek , 45.0-mi:

Other Acceptable Counterstains

1. Acid fuchsin, C.I. 42685, 5°% aqueous (slightly acidified improves

stain). If overstained, rinse with tap water.
130 Hematoxylin Staining (CHAP. 12)

. Orange G, C.I. 16230, saturated in 95% ethyl alcohol.

. Van Gieson or substitute, page 165.
. Bordeaux red, C.I. 16180, 1% aqueous.
. Biebrich scarlet, C.I. 26905, 1% aqueous, a good counterstain.
HO.
oo
#®
Ot
MD Additional “‘eosins”
a. Eosin Y, C.I. 45380, 0.1-0.5% in 95% ethyl alcohol.
b. Erythrosin B, C.I. 45430, 0.1-0.5% in 95% ethyl alcohol.
c. Phloxine B, C.I. 45410, 0.5% aqueous, plus a few drops of acetic
acid, page 229.
7. Congo red, C.I. 22120, 0.5% aqueous.
8. Light green SF, yellowish, C.I. 42095, 0.2-0.3% in 95% ethyl alco-
hol.
9. Aniline blue W.S., C.I. 42780, pages 147, 149, 150, 160.
10. Fast green FCF, C.1. 42053, similar to light green or aniline blue.

Hematoxylin Staining Procedures

Delafield’s (or Harris) Hematoxylin

Progressive Method
FIXATION: any general fixative or one specific for nuclear detail.
SOLUTIONS:
Hematoxylin, page 124.
Counterstain, page 129.
PROCEDURE: The slides are passed through a ““down”’ series of jars, a
process often termed running down slides to water (or hydration) be-
cause a series of alcohols of decreasing strength is used. (From left to
right, top row of Fig. 26) Never at any time during this procedure
allow the slides to dry.

1. xylene (or toluene) 2-3 minutes or longer.

(2 changes may prove profitable to insure complete removal of
paraffin.)
Zeapsolite alcohol’ :35/.49) sane: 2-3 minutes or longer.
apeoo, ealcohol ......1:/.
9 wae 2-3 minutes or longer.
Ae ealCOUOL 25. oc ae 2-3 minutes or longer.
If mercuric chloride was absent from fixative, skip steps 5 through
7 and proceed into step 8.
Hematoxylin Staining Procedures 151

omar WATER
t/X 4
& (Ss 70% \oe=
& (Ss ALC

ABS ABS 95% 70-75% (<m

(=)

Figure 26. A suggested arrangement of staining jars. An alternate arrange-

ment can include two jars of xylene in the “down series” (left to
right) when many slides are being stained. An absolute alcohol-
xylene in “up series” (right to left) is optional.

5. Lugol’s solution (page 410) 3 minutes.

6. TunnMing water. ...)....... 3 minutes.
7. 5% sodium thiosulfate, Nag
oO) SO A ee a 2-3 minutes.
0) DUMMIMG Water Joa... e. 3—5 minutes.
9. hematoxylin, Delafields’s ... 2—5 minutes, check after 1 minute
for stain intensity. Fresh solutions stain faster than old ones. If
not dark enough, return slides to stain. Rinse off stain in tap wa-
ter before checking under microscope. If slide becomes too dark,
convert to regressive type of stain, page 000.
10s: running Waler 66 eli<s)<<.. 3—5 minutes.
11. Scott’s solution (page 412) . 3 minutes.
12. running water .......... .. 3-5 minutes.
15, Counterstain 3652.2 544% 5 ia 4 1 or, more minutes, depending on
stain used.

Transfer slides through “up” series, termed running up slides (or

dehydration) using a series of alcohols of increasing strength. Steps
14 and 15 can control intensity of counterstain. Carefully watch tim-
ing in these solutions. (From right to left, bottom row, Fig. 26)
14. TOP), AlCONONS Fegee ied tas ace 3 1 or more dips.
Lo; D9 UCONNOl arene aaa o's few dips.
16. absolute alcohol ........... 3 minutes or longer.
I7. absolute alcohol ........ .. 2-3 minutes or longer.
18. absolute alcohol-xylene (1:1)
(6)C16) 01) 2-3 minutes or longer.
132 Hematoxylin Staining (CHAP. 12)

lO gexvilene SAAS): oe ee 2-3 minutes or longer.

ZUR SVIEMERR AI!) AAoh Lie 2-3 minutes or longer.
21. mounting medium; keep sections moist with xylene during this
process. They must not dry. Add cover glass, page 115.
RESULTS:
nuclei—deep blue
cytoplasmic structures—pink, rose, etc., depending on counterstain
COMMENTS:

ily Slides may be left in higher alcohols and xylene longer than sched-
uled (indicated by “‘or longer’), but do not leave them indefinitely
in any solutions weaker than 80 or 95% alcohol. Lower alcohols and
water can loosen the sections from the albumen.
no. Isopropyl alcohol can be substituted for ethyl alcohol in this sched-
ule, but cannot be used as a stain solvent.
. Two to five minutes for Delafield’s (step 9) is an approximate time
only. This may have to be varied according to the tissue used. ‘Tis-
sues can be individualistic, often due to the type of fixative employed
on them, and a certain amount of trial and error may be required
to develop the exact timing schedule.
The cause of some difficulties can be problematical. Poor staining
can result from improper fixation, leaving gross tissues too long in
alcohol or iodine, faulty processing during preparation for embed-
ding, or careless handling of the slides in preparation for staining.
In many instances no amount of trial and error will produce a per-
fect stain. The only correctable faults are those made during de-
paraffinization and hydration of the slides and the slides should be
reversed through the solutions, back to xylene. Change to fresh solu-
tions and try again.
Sometimes too many slides have been taken through the fluids and
they are contaminated. If as many as 20-40 slides are being stained,
use two changes of xylene in step 1 and follow step 20 with a third
change. Prevent further contamination by draining slides properly.
On removing them from a solution, touch the edges against the inner
surface of the staining jar, and then against paper toweling. But do
not be too thorough and let the slides dry. Merely drain off excess
liquid.
Hematoxylin staining must be left in a “blued” condition. The orig-
inal pinkish color of the hematoxylin, like Delafield’s, must be con-
verted to a blue color by alkalinity. In the progressive method this
is accomplished in running water to remove excess dye and start the
Hematoxylin Staining Procedures 133

bluing action. Then use Scott’s solution (step 11), a couple of drops
of ammonia in a final wash, or a weak solution of sodium bicarbon-
ate, to insure the alkaline condition. In the regressive method (be-
low) the same applies, and as a precaution against neutralization
caused by carrying one solution over into the other (step 5), after
several exchanges of slides, add a drop or two of acid and base to
them. If there is any pinkness left in the nuclei, they are not ade-
quately blued.

Regressive Method

PROCEDURE:
1. Deparaffinize and run slides down (hydrate) to water, removing
HeCl, if present.
ho Stain in Delafield’s hematoxylin, until slides are well overstained:
15-20 minutes.
3. Wash in running water: 3—5 minutes.
4. Transfer to 70% alcohol.
5. Destain in 0.5% (app.) HCl (2-3 drops in 60.0 ml. of 70% alcohol)
few seconds. Remove to 70% alcohol with drop of ammonia added
until sections turn blue. Examine under microscope. If nuclei are
too dark, repeat above procedure. When nuclei stand out sharply
blue against colorless background, blue slides thoroughly in alka-_
line solution: 3—5 minutes. “a6 |
6. Transfer to 70% alcohol. SSS
f PY, Pg ;

7. Counterstain. (:—
- one bts Gy pf ry
8. Dehydrate as for progressive staining. Zz i dapkes
9. Clear and mount. o\
\ & NS bac
RESULTS: . .G
x—
“Seuss
fe,
a e . ‘oP
nuclei—deep blue, sharper than in the progressive method ————
other elements—colors of counterstain

Mayer’s Hematoxylin
FIXATION: any general fixative or one specific for nuclear detail.
SOLUTIONS:
Hematoxylin, page 125.
Counterstains, page 129.
PROCEDURE:
1. Deparaffinize and run slides down (hydrate) to water, removing
HegCl. if present.
 Hematoxylin Staining (CHAP. 12)

Stain in Mayer’s hematoxylin: 11 minutes.

Wash in running water: 3 minutes.
Blue in Scott’s solution: 3 minutes.
Wash in running water: 3—5 minutes.
Counterstain.
Dehydrate quickly through 70 and 95% alcohols.
Finish dehydration, absolute alcohol.
Clear and mount.

RESULTS:
nuclei—deep blue
other elements—colors of counterstain

Heidenhain’s Iron Hematoxylin

FIXATION: any general fixative, preferably one containing HgCle.

SOLUTIONS:
Hematoxylin and iron alum, page 127.
Counterstains, page 129.

PROCEDURE:
i Deparaffinize and run slides down (hydrate) to water, removing

2. Mordant in 4% iron alum: 15-30 minutes (or overnight, see com-

ments below).
3. Wash in running water: 5 minutes.
4. Stain in hematoxylin: 15-30 minutes (or overnight).
5. Wash in running water: 5 minutes.
6. Destain in 2% aqueous iron alum or saturated aqueous picric acid
(see comments below) until nuclei stand out sharp and clear against
colorless background. Dip slides in water containing couple of
drops of ammonia before examining them. Warning: too much
ammonia will loosen sections.
7. Wash in running water: 15-30 minutes or longer.
8. Counterstain, if desired.
9. Run slides up through alcohols (dehydrate), clear and mount.

RESULTS:
nuclei—blue black to black
muscle striations—blue-grey
other elements—colors of counterstain, or very light grey to colorless
if no counterstain used
Hematoxylin Staining Procedures 135

COMMENTS:
The shorter staining schedule produces blue-black nuclei and the
overnight staining a truer black. The latter is recommended for
mitochondria. Picric acid usually perfects a more sharply differenti-
ated nucleus than destaining with iron alum. Prolonged destaining
in the latter case can leave a tan or yellow tinge in the cytoplasm.
Yeilow color left in the tissue after picric acid is due to insufficient
washing following the destaining. Striations of muscle and some
protozoan structures are better differentiated by iron alum. After
either method thorough washing is essential or the sections will con-
tinue to destain slowly and fade after a time. (Picric acid destaining,
Tuan, 1930)
The destaining agents act both as an acid and an oxidizer, reacting
in two ways: (1) as an acid they extract stain faster from the cytoplasm
than from the nucleus; and (2) as an oxidizer they bleach the stain
uniformly. If an acid is used alone, the staining of the nuclei can be
favored, and if the oxidizer is used alone, the staining of the cyto-
plasm can be enhanced.
Hutner (1934) recommends for nuclei:
1. Mordant in 4% iron alum: | hour.
2. Stain hematoxylin: | hour.
3. Destain in saturated aqueous picric acid.
4. Wash and blue by adding I—2 drops ammonia to 70% alcohol.
Hutner recommends for cytoplasm:
1. Mordant in iron alum: 30 minutes.
2. Stain in hematoxylin: 30 minutes.
3. Destain in freshly made 95% alcohol 2 parts, Merk’s superoxol
(30% H2Oz) | part.
4. Rinse in | change 70% alcohol, 10 minutes.
For brevity, intermediate steps were omitted in the above. Oxidizer and
acid can be combined to approximate iron alum destaining effects:
0.25% HCl in 95% alcohol, 2 parts; Superoxol, | part.
Wash in 70% alcohol and blue.

Iron Hematoxylin GROAT’s SINGLE SOLUTION (1949)

FIXATION: any general fixative, preferably containing HgCl».
SOLUTIONS:
Hematoxylin, page 126.
Counterstains, page 129,
136 Hematoxylin Staining (CHAP. 12)

PROCEDURE:
1. Deparaffinize and run slides down (hydrate) to water, removing
HegClo.
Stain in Groat’s iron hematoxylin: approximately 10 minutes.
Rinse off excess stain with running water: 10 minutes.
Counterstain, if desired.
Dehydrate, clear, and mount.

RESULTS:
nuclei—black
other elements—color of counterstain

COMMENTS:
The 10-minute timing is approximate: check under the microscope
for depth of color. If the background is too grey it may be decolorized
in:
distilled water a). 52h). sere ee Sy crs. al 50.0 ml.
Ob°F -alcOWOW | cece c.5 2 ks eee = © SA TEER: 50.0 ml.
sulfuric acid (sp.gr. 1.84, 95.5-96.5% H2SOx4) ... 0.18. ml.

Follow by neutralizing in:

distilled swater !..4 0% 30 eRe = as ou ee 50.0 ml
Doe ralCOMOl. 5g. ae «che Rc rh 50.0 ml.
SOdium@M) bicarbonate .....8..-5

Phosphotungstic Acid Hematoxylin (As MODIFIED BY LIEB 1948)

FIXATION: any general fixative, preferably containing HgCly. Lieb sug:
gests if tissue is formalin fixed, mordant sections before staining in
saturated aqueous mercuric chloride, rinse and treat with Lugol’s.

SOLUTIONS:
Hematoxylin, page 125.
Potassium permanganate, 0.5%
potassium permanganate wes. fies iS eee 0.5 gm.
distilled: ‘water: \:\ Sry eee 2 ae Eee 100.0 ml.

Oxalic acid, 2%
Sc MACLG 5 oa.ss,<1 eee Ee os | eee 2.0 gm.
distilled: water’. .'. Uiieshne emery 6.4 6S ae eee 100.0 ml.

Iron alum, 4%, page 127.

Hematoxylin Staining Procedures 137

PROCEDURE:
1. Deparaffinize and run slides down (hydrate) to water, removing
HeCle.
Oxidize in potassium permanganate, freshly prepared: 5 minutes.
Wash in running water: 5 minutes.
Bleach in oxalic acid: 5 minutes.
IND
©9
es
Or Wash in running water: 5 minutes; follow with rinse in distilled
water.
6. Mordant in iron alum: | hour.
7. Wash out excess alum in running water 10 minutes or longer,
rinse in distilled water.
8. Stain in phosphotungstic acid hematoxylin: 2—24 hours.
Usually complete in 2—3 hours, rarely requires overnight.
9. Transfer directly to 95% alcohol: brief rinse.
10. Dehydrate, absolute alcohol; watch that elements stained reddish
brown are not destained.
11. Finish dehydration in isopropyl alcohol (99°%), 2 changes: 3
minutes each.
12. Clear and mount.
RESULTS:
nuclei, mitochondria, centrioles, fibrin, fibroglia, myoglia and neu-
roglia fibrils, and contractile elements of striated muscle—blue
collagen, reticulum, elastin, cartilage and bone—yellowish to brown
red

COMMENTS:
Mullen and McCarter (1941) use a chromium-chloride mordant after
formalin fixation: Deparaffinize and hydrate to water, mordant for 2
hours or longer in
chromium: chloride; CrGly. 2. s..<242s...62
2988: 5.0 gm.
distilled water ......... he os > 37 Sees 1 LOUD inl:
glacial acetic acid ........ ‘or juie.2 feuigh ageens 5.0 ml.

Good for several weeks. After this mordanting, the staining will be as
brilliant as following Zenker fixation.

Weigert’s Iron Hematoxylin. (MALLORY 1944; JANSSEN AND LILLIE)

FIXATION: any general fixative or one specific for nuclei.
SOLUTIONS:
Hematoxylin, page 126.
Counterstains, page 129.
138 Hematoxylin Staining (CHAP. 12)

PROCEDURE:
1. Deparaffinize and hydrate slides to water, remove HeClp.
2. Stain in Weigert’s hematoxylin: 3—5 minutes or longer.
3. Wash, running water: 5 minutes.
4. Counterstain.
5. Dehydrate, clear, and mount.
RESULTS:
nuclei—black
other elements—color of counterstain

COMMENTs: This is used progressively, check results under microscope.

Mallory’s Iron Chloride Hematoxylin (1944)

FIXATION: any general fixative, or one specific for nuclet.
SOLUTIONS:
Ferric chloride, page 128.
Hematoxylin, page 128.
Counterstains, page 129.
Ferric chloride for destaining (0.25%): dilute 5 ml. of the 5% stock
solution (above) with 95 ml. distilled water.
PROCEDURE:
1. Deparaffinize and hydrate slides to water, remove HgCle.
Mordant in ferric chloride: | hour or longer.
. Wash in running water: 5 minutes.
. Stain in hematoxylin: | hour or longer.
. Wash in running water: 3—5 minutes.
PO.
OO
PB
Ot
D Destain in ferric chloride, 0.25%; agitate slides, check under
microscope.
7. Wash thoroughly, running water: 10-15 minutes.
8. Counterstain, if desired.
9. Dehydrate, clear, and mount.
RESULTS:
nuclei—deep blue
other elements—color of counterstain

Hematoxylin Substitute Procedures

Gallocyanin (ROMEIs, 1948)
Gallocyanin is an excellent substitute for hematoxylin and can be
used in most procedures designed for the latter dye. The solution is
Hematoxylin Staining Procedures 139

made in a few minutes and ready for immediate use; also an iron lake
can be prepared. No differentiation is required, and it is better than
hematoxylin for tissues of the central nervous system. It can replace
thionin for ganglia and glia cells, but not for myelin sheaths. It is good
for negri bodies. The preferred fixatives are aceto-formol, Zenker’s
(acetic or formol) or formalin—fixatives which preserve chromophilic
substance.

SOLUTION: page 128.

PROCEDURE:
1. Deparafhinize and hydrate to water.
. Leave in stain overnight, or 3 hours at 56°C.
© . Wash thoroughly and proceed to counterstain.
KO

4. Dehydrate, clear, and mount.

RESULTS:
nuclei—blue

Hematein (Hemalum). (KORNHAUSER, 1930).

FIXATION: any general fixative, preferably containing HeCls.

SOLUTIONS:
Hemalum, page 129.
Counterstains, page 129.

PROCEDURE:
1. Deparaffinize and hydrate slides down to water, remove HegClp.
2. Stain hemalum, progressively: about 5 minutes.
3. Wash in running water: 5—10 minutes.
4. Counterstain.
5. Dehydrate, clear, and mount.

RESULTS:
nuclei—blue
other elements—color of counterstain

Red Nuclear Staining

Darrow Red. (POWERS ET AL., 1960).

FIXATION: any general fixative.

140 Hematoxylin Staining (CHAP. 12)

SOLUTION:
Darrow Red:

DATO WN GEC. hcp tsle od ee ck s 50.0 mg.

OAM elacialvacetic acid (pPH277)\ ewe
a Ae 200.0 ml.

Boil gently: 10 minutes. Cool and filter. Stable for 1 month.

PROCEDURE:
Ie Use frozen sections in water, or deparaflinize paraffin sections and
hydrate to water.
2. Stain in Darrow red: 20—30 minutes.
3. Rinse in distilled water.
4. Differentiate and dehydrate through 50%, 70%, and 95% alcohol,
not so slowly as to remove too much dye from nuclei, but just to
decolorize background.
5. Complete dehydration in n-buty! alcohol; clear, and mount.

RESULTS:
nuclei—red
other elements—depending on other stains applied
COMMENTS:

This is a good nuclear stain for contrast with blue cytoplasmic

stains—following luxol fast blue for example. It cannot be used with
nitrocellulose because the medium stains with Darrow red.

Scarba Red (SLIDDERS ET AL., 1958)

FIXATION: any general fixative.

SOLUTIONS:
Scarba red:

melted phenol (carbohic acid) Pic. ..-...... blake 2.0 gm.

MEUERAIOTEG: <0. ic 2 See ere ES 2 aA 1.0 gm.
Mix thoroughly, allow to cool, dissolve in:
Goeeeetiay lialcohol™ ....<.4 Vesneeee a: Se 15.0 ml.
Add:
Zo eaniline: 1 Water ee eee|=2. Ae 85.0 ml.
slacialvacetic:acid ja.) arenes
2. 2= Se 1.0-3.0 ml.

Mix well and filter. Keeps for at least 6 months.

“Matheson Coleman and Bell.
Hematoxylin Staining Procedures 141

Differentiator:
TOO scthviealicOnoll so 5.23 %ss'oo ame Aeeuee es ane 85.0 ml.
LOMMAANNON PB e Bets bs 4 b cancun Ran wie ee 15.0 ml.
Slacialacche acid: Qoac. ooo ddawee nee cages a 15.0 drops.

PROCEDURE:
1. Deparaffinize and hydrate slides to water (remove HgCly if pres-
ent).
2. Stain in scarba red: 5 minutes.
3. Rinse in distilled water.
4. Transfer to 70% alcohol: 2-3 minutes.
5. Differentiate until nuclei are clear and sharp against colorless
background.
6. Dehydrate, 95% and absolute alcohol.
7. Clear in xylene and mount.
RESULTS:
chromatin and calcium—red
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|

SPECIFIC
STAINING
METHODS
 Chapter 13

Connective
Tissue

Connective tissue, so named because its chief function is to connect

the other tissues of the body, also provides support for these tissues.
Connective tissue is far too complex to discuss at any length in this type
of book, but a few terms will be covered briefly.
Stroma is a term referring to the supporting tissues in an organ; the
parenchyma is composed of the cells that perform the function of the
organ.
Collagenic fibers (collagen) have tensile strength (tendons); they may
be single or aggregate in bundles, and they do not branch.
Reticular fibers are delicate and often connected to and supported by
collagenic fibers. (For silver methods, see page 179.) Reticular fibers,
often forming dense networks, support cells, capillaries, nerve fibers
and other tissue units, and form parts of basement membranes (inter-
cellular substance lying between epithelial membranes and their sup-
porting connective tissue).
Elastic fibers are long and narrow and are not composed of fibrils,
but are homogeneous. They have the ability to stretch and are located
in such places as the walls of blood vessels or in the respiratory system.
Areolar tissue lies between muscles and fasciae and contains both
collagenic and elastic fibers embedded in a ground substance.
The ground substance is an amorphous kind of material that lies
around fibrous intercellular substances; thus the fibers are seldom

145
146 Connective Tissue (CHAP. 13)

found by themselves. In most stains ground substance is uncolored, but

it contains mucopolysaccharides and can be shown by metachromatic
staining (page 280) .
Hyalin(e) is a descriptive noun or adjective meaning glassy. A sub-
stance so described is a translucent, refractile albuminoid. Amyloid
deposits (amorphous material accumulating in intercellular spaces in
certain pathologic conditions) is said to be hyaline in nature. Old
collagen can become hyaline.
Fibrin (page 230) is formed from fibrinogen (a protein in blood
plasma) and as it forms develops a mesh of fibers at the site of cut blood
vessels.
Mast cells are numerous in areolar and other types of connective
tissue. Staining of mast cells has been included with technics for cyto-
plasmic elements (page 276) because of their characteristic granules.
The cytoplasm of mast cells is stuffed with these granules that usually
are stained metachromatically.
Plasma cells are more common in hemopoietic (concerned with blood
formation) tissue than in loose connective tissue and if numerous in the
latter appear to be associated with low-grade inflammation.
Keratin is a horny material formed by the fusing together of the
surface cells of dry epithelial membranes. (Reference: Ham. 1957)

Mallory Staining
Mallory’s Triple Connective Tissue Stain
Innumerable modifications of this method appear from time to time
in the literature, particularly for the use of phosphomolybdic and phos-
photungstic acid. Experiments by Holde and Isler (1958) support the
need for phosphomolybdic acid if specific staining of connective tissue
is desired. The acid diminishes the background and nuclear staining,
leaving only the connective tissue stained with aniline blue. They feel
that the staining property of connective tissue is due to the presence
of the acid on the fibers. Baker (1958) suggests that phosphomolybdic
acid acts as a “colorless acid dye” in the tissue, chiefly on collagen. It
acts as a dye excluder with acid dyes such as acid fuchsin, excluding
them from collagen. Then the aniline blue stains the collagen selectively,
but is excluded from other tissues. Phosphomolybdic acid should not be
termed a mordant, since it also opposes the action of aniline blue. With-
out phosphomolybdic acid present, aniline blue stains more strongly
and with very little selectivity. As to the use of phosphotungstic acid,
Mallory Staining 147

it may be of interest to recall that certain tissue elements react more

specifically to certain metals than to others; collagen to molybdenum,
and fibroglia, myoglia, neuroglia and epithelial fibers to tungsten. Lillie
(1952) writes that phosphotungstic acid intensifies plasma staining and
phosphomeolybdic acid the fiber staining. The acids may be used sepa-
rately or together depending on the final desired effect.
Oxalic acid is often used. Mallory (1944) claims that it makes the
aniline blue stain more rapidly and intensely. Baker (1958) seems to
agree when he suggests that oxalic acid lowers the PH, seeming thereby
to aid the staining of aniline blue, also of orange G.
No polychroming or metachroming takes place in the combination
of dyes; they react orthochromatically in varying intensities of their
own colors. In Mallory’s combination of stains, aniline blue (acidic)
stains connective tissue and cartilage; orange G (acidic) stains the blood
cells, myelin and muscle; acid fuchsin (acidic) stains the rest of the tis-
sue (including the nuclei) in grades of pink and red. This is not, how-
ever, an efficient method for staining nuclei and they will fade in a few
years. Modifications of Mallory’s using basic fuchsin, carmine or azo-
carmine result in more permanent nuclear staining, since the basic
stain reacts more reliably than the acidic one with the nuclei. The
method also may be preceded with hematoxylin staining for a brilliant
permanent nuclear stain. .
The final staining may be followed by an acetic acid wash (0.5-1%)
for 3-5 minutes or longer, producing more transparent sections with-
out altering the color.

Method (pANTIN, 1946)

FIXATION: any general fixative, preferably containing HgClo.
SOLUTIONS:
Mallory I:
Veisle ive okyh cae@) DAS 724¢)—12 Sana a mn reg oP 1.0 gm.
distilled wae tes e oa Sed mic’ 5 cae doe 4 ane 100.0 ml.
Phosphomolybdic acid:
phosphomolybdicacid «2. .:..+++<.2.2¢ eee os 1.0 gm.
cistilled |Water Moy oa t..24 op niece ened oe deena: 100.0 ml.
Mallory II:
aniline: blue, WS,/C.L 42780 ..,... 8 Ser. 0.5 gm.
oranee GWOT. W6230) o c5 nc as ee 2.0 gm.
Gistilled Water a5 Seo: 4 cosas
aim «iu ceteak Le 100.0 ml.
148 Connective Tisswe (CHAP. 13)

PROCEDURE:

1. Deparaffinize and hydrate slides to water; remove HgCle. If HgCl,

is absent from fixative, mordant in saturated aqueous HgCle, plus
5%, glacial acetic acid: 10 minutes. Wash, treat with Lugol’s and
sodium thiosulfate, wash and rinse in distilled water.
Stain in Mallory I: 15 seconds.
Rinse in distilled water: 10 or more seconds, to differentiate reds.
Treat with phosphomolybdic acid: 1-5 minutes.
Rinse briefly in distilled water.
Stain in Mallory II: 2 minutes.
Rinse in distilled water.
Differentiate aniline blue in 90% ethyl alcohol.
ere)
Sa
ee Dehydrate in absolute alcohol, clear and mount.

RESULTS:

nuclei—red
muscle and some cytoplasmic elements—red to orange
nervous system—lilac
collagen—dark blue
mucus, connective tissue and hyaline substance—blue
chitin—red
yolk—yellow to orange
myelin and red blood cells—yellows and orange
dense cellular tissue (liver)—pink with red nuclei
bone matrix—red

COMMENTS:

From distilled water (step 7) the slides can be run through the “up ce ”

series of alcohols, thereby controlling the blue color. Aqueous-alco-

holic solutions differentiate the amount of Mallory II left in the
various parts of the tissue. After the slides have remained in the ab-
solute alcohol for a couple of minutes they should have changed from
a muddy purple to a clear blue and red. An acetic acid rinse, as men-
tioned (page 147), following step 7 contributes to the transparency
of the sections.
For better nuclear detail, Mallory I can be preceded by alum hema-
toxylin staining. (Results: nuclei—blue)
Landrum and McFarlane (1940) recommend addition of celestin
blue. Follow step 1 with:
Mallory Staining 149

(a) celestin blue: 2 minutes

celestinmolueds; Gils 51050) occ
eee he ote 0.5 gm.
LUGOVIM AUTOM ee eek ee a. Ge ae eae 5.0 gm.
aistilledmwater 24 O05 ac.< 2) Wood bees Le 50.0 ml.

3 minutes;
add glycerol ih p.8 0.4 es oo 14.0 ml.

(b) hematoxylin: 2 minutes

(c) running water: 5 minutes
(d) proceed to step 2
Krichesky (1931) proposed the following for solution (Mallory) II.
A. aniline blue, WS, C.I. 42780 ........... eon 2.0 gm.
distilled water... . Aa dh ae
ae ee 100.0 ml.
Be aranve G.C.1 16250) . o. oe ses in dete ees x : 1.0 gm.
distilled water ......................0-- ese 10050 ml
Ge paosphomolybdie aad .........2tekoeae
ach 1.0 gm.
distilled water ......... We pee LOOM smi.

Keep solutions A, B and C in separate bottles because the mixture

deteriorates on standing. When ready to use, mix equal parts of each.

Azan Stain, Mallory Heidenhain’s (MODIFIED AFTER KONEFF, 1938)

FIXATION: Zenker-formol; other general fixatives fair, but following
them is improved by mordanting slides overnight in 3% potassium
dichromate.
SOLUTIONS:
Azocarmine:
avocammmeyG. Cay 500BD: .5.5-<%40 26 4 apie 0.2-1.0 gm.
Gistilled water: 0.6 fee oba + odes ts bee pees 100.0 ml.
glacial acetic acid . ne eer 1.0 ml.

Boil azocarmine in water 5 minutes, cool, filter and add acetic acid.

Aniline alcohol:
aniline ae es ee YT 1.0 ml.
60-0 ethyl alcohol ¢. 560.2040)
ok eee ee 1000.0 ml.
Acid alcohol:
glacial acetic acid gen Se alse 4 tne ee 107 ani,
90-95%, -ethylaleoholl s,.2.. os: ox cle ages een 5 100.0 ml.
150 Connective Tissue (CHAP. 13)

Phosphotungstic acid:
pliesphetungstie acid? «. -.': See emeamee
soe 5.0 gm.
disulled water! ©4342 SR os 100.0 ml.

Aniline blue stain:

antiine. blue. VS. Gal 42780, eee
te oi oe 0.5 gm.
oranee G, Cal. W6230.. aac 5 eens 2.0 gm.
Oxalic ACIS ccc hoes ts Ay team 52. Sot 2.0 gm.
distilled, water (4) parse ot(stn CAP aon. 8 100.0 ml
5% phosphotungstic acid (above) ............ 1.0 ml
Acidulated water:
Placial ACetie, aCiCl soc, sree ye net 1.0 ml.
distilled“ water "77. 2.0. ie eee ee. 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides down to water; remove HeClbs.
2. (aniline alcohol: 45 minutes)!
3. (acid alcohol: 1-2 minutes)
4. Stain in azocarmine, 56°C: | hour. (2 hours) Check temperature
carefully; if too high, azocarmine differentiates poorly.
5. Rinse in distilled water.
6. Differentiate in aniline alcohol. Check under microscope for
brilliant red nuclei and very light red cytoplasm.
7. ‘Treat with acid alcohol: 1-2 minutes.
8. Transfer to phosphotungstic acid: 2-3 hours. (4 hours)
9. Rinse in distilled water.
10. Stain in aniline blue solution: 1-2 hours. (4 hours)
11. Rinse in distilled water.
12. ‘Treat with phosphotungstic acid: 3-5 minutes.
13. Rinse in distilled water.
14. Rinse in acidulated water: 1-2 minutes or longer.
15. Rinse in 70% alcohol: briefly dip.
16. Dehydrate 95% alcohol, several dips in each of 2 changes.
17. Dehydrate, absolute alcohol, clear and mount.

RESULTS:
nuclei—brilliant red
collagen and reticulum—blue
muscle—red and yellow
basophil cytoplasm—light blue
‘For strikingly beautiful results, particularly for pituitary, Konefl’s longer method is
recommended. Changes in timing are indicated in parentheses.
Mallory Staining 15

acidophil cytoplasm—orange-red
chromophobes—colorless or light gray
COMMENTS:
The azan method is recommended particularly for pituitary and pan-
creas but also is outstanding for connective tissue. If a tissue does not
“take” the azocarmine, it may be due to formalin content of the fixa-
tive. To correct this condition, mordant the sections overnight in 3°%
aqueous potassium dichromate.
Romeis (1948) suggests the substitution of acid alizarine blue for
azocarmine; this shortens the time and does not require heat.
PROCEDURE:
1. Stain in acid alizarine blue: 5 minutes.

aluminum :.sultate, Al,(SO,)5.: .. .-nslese esse 10.0 gm.

acia alizarine pluic, G.L. 58610: .... . eae: 0.5 gm.
distilled water ................. 0.00000. ee 100.0 ml.

Bring to boil: 5-10 min. Let cool. Fill to 100 ml. with distilled
water. Filter. Red violet color.
Wash, running water.
NO Treat
©9 with phosphotungstic or phosphomolybdic acid, 5°% aque-
ous: 30 minutes.
4. Wash in distilled water.
5. Proceed to aniline blue stain.
RESULTS:
After phosphotungstic acid, the alizarine color turns red, stays in nu-
clei and muscles, but is removed from collagen.
After phosphomolybdic acid, the nuclei and muscle will be blue.

Mallory-Heidenhain Stain: Rapid One-Step Method (caAson, 1950)

FIXATION: any good general fixative.

SOLUTION:
Staining solution:
aise twaGen 5. tesa. pe aa 6s ke oat meee 200.0 ml.
Dissolve each of below before adding next stain:
phosphotungstic-acid 26) 2.04.10. 2.0secemee ; 1.0 gm.
orange Gy CAMO 2Z50% 205.65 aus 108k ese ete 2.0 gm.
aniline Dine WSC A2780". on. ioe soe Baan 1.0 gm.
acid’ tuchsin, Giule472685. 2 ocho. 54 oo Seas ee he 3.0 gm.

Keeps for several months,

152 Connective Tissue (CHAP. 13)

PROCEDURE:
1. Deparaffinize and hydrate slides to water, remove HgCls.
2. Stain: 5 minutes.
3. Wash in running water: 3—5 seconds.
4. Dehydrate rapidly, clear, and mount.
RESULTS:
collagen—blue
eround substance of cartilage and bones—shades of blue
mucus, amyloid, other hyaline substances—shades of blue
nuclei, fibroglia, myoglia, neuroglia fibrils, axis cylinders, fibrin—red
erythrocytes, myelin—yellow
elastin—pale pink or yellow

Trichrome Staining
Masson Trichrome Stain
Masson (1928), when he developed this method, called it a trichrome
stain, although it included four dyes. Since then the trichrome name
has been applied to many modifications of Masson’s method using many
combinations of different dyes.
FIXATION: any general fixative.
SOLUTIONS:
Iron alum:
femic. ammonimmysullater, Waser cue yt. 2h 4.0 gm.
distilled water, 9nsi eyMeee ks fc SUG 100.0 ml.
Masson A:
aed fuchsin, C:l. 42685 197 aqueous..." 2. 10.0 ml.
ponceau de xylidine, C.J. 16150 1% aqueous .. 90.0 ml.
placialtacetic acide 2700s") Se aeemmmeeeens
te Ss Sener 1.0 ml.
Lillie (1940) suggests alternate stains for ponceau de xylidine: pon-
ceau 2R, nitazine yellow, Biebrich scarlet, azofuchsin 3B, G and 4G,
Bordeaux red, chromotrope 2R, chrysoidin, eosin Y, orange G and
crocein.
Masson B:
phosphomolybdic acid, 1% aqueous (0.5 gm./
Bunce water): ~.t+. Eeygeeeeeemen
e. .1 Oe eee 50.0 ml.
phosphotungstic acid, 19% aqueous (0.5 gm./
50 ml. water)
Trichrome Staining 153

Masson C:
fase oneen POH uC.LyA2009: 5.2 ven teri sane et o4 2.5 gm.
miStilledewaltenr 2 secu. ri can SHS at ote hoe 100.0 ml.
glacialpAacete acid 1.4.4) su. sc ae arts
be eae es 2-5, mil.

Aniline blue can be substituted for fast green. If so, after staining
with Masson C (step 11 below), extract excess stain for 5 minutes with
1% phosphomolybdic acid, aqueous, followed by a quick rinse in
distilled water and then proceed with step 12, below.

Hematoxylin, see page 124.

Acetic acid rinse.

CUA ACEMC CIE 45 5252 aie cope ees | 1.0 ml.

iste MWAteN x a s6 a6 5.esas 2 ae Aa OO - 100.0 ml.

PROCEDURE:
i Deparathnize and hydrate slides to water. Remove HgCls. Mor-
dant formalin-fixed tissue in saturated aqueous HgCls: 5 min-
utes. Wash, running water: 5 minutes. Treat with Lugol’s and
sodium thiosulfate.
no Mordant with iron alum: 5 hour at room temperature or 5 min-
utes at 40—50°C.
Wash in running water: 5 minutes.
SaitStain in hematoxylin (Delafield’s or the like): $ hour, room tem-
perature, or 5 minutes at 40—50°C.
Wash in running water: 5 minutes.
Differentiate in saturated aqueous picric acid.
Wash thoroughly, running water: 10-20 minutes.
Stain in Masson 4: 5 minutes.
Rinse in distilled water.
Treat with Masson B: 10 minutes.
. Stain in Masson C: 2—5 minutes.
. Differentiate in acetic acid rinse: 1-2 minutes.
Dehydrate quickly 70% and 95% alcohol.
. Dehydrate, absolute alcohol, 2 changes: 3 minutes each.
Clear and mount.

RESULTS:
nuclei—blue to blue-black
collagen, mucus—blue or green
cytoplasmic elements, keratin, muscle—reds
154 Connective Tissue (CHAP. 13)

Masson Trichrome Stain, Modified (GuRR, 1956)

FIXATION: any general fixative.
SOLUTIONS:
Iron alum:
FEEHIC AMIIMONLUUN SULLA: <2 2 sceneries poe 4.0 gm.
distilled water |e sea: ces eRe Bs ny ot 100.0 ml.
Hematoxylins, see page 124.
Acid fuchsin:
acid tuchsin; (C.1°42685" . 22) see ee. ke se 1.0 gm.
distilled, watete fs 5-1.)-.oys ee eka 100.0 ml.
Placial -aACeuCGraClG: \...- A> Seeas Oe 1.0 ml.
Ponceau de xylidine:
ponceau de'xylidine, ‘C.l- 1GUbOmae es. ... oan 1.0 ml.
Cistiled swatelie 3457) :.: .v) - eG ae 2 100.0 ml.
placial “acetictacid 2/476 ).) VFR ER) fe), Ee 1.0 ml.
Fast green:
fasteoneenel Gly Cle 42003 ac pee
hs oo «eat 2.0 gm.
csgnlledt watenager Aone. Mune Be he on as 100.0 ml.
SLACTA A CELIC ACIC a oars... , 2 MOR ees x Sulyieuge 2.0 ml
Phosphomolybdic acid:
phosphomolybdiciacid $7. Besa ees. . ee 1.0 gm.
distilled water: .../... 2 AR ee POs Ee 100.0 ml.
Acidic water:
placiall aACetiC -aACIG EERE Nesia)c:«55s -<capers 1.0 ml.
distilled: Water: =a.s%..cis Seer oa. og teeae 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water, remove HgCl,. Mor-
dant formalin-fixed tissue in saturated aqueous HgCls: 5 min-
utes. Wash in running water: 5 minutes. Treat with Lugol’s and
sodium thiosulfate.
. Mordant in iron alum: $ hour.
Wash in running water: 5 minutes.
. Stain in hematoxylin (Delafield’s or like): 3 hour.
Wash, running water: 5 minutes.
. Differentiate in saturated aqueous picric acid.
. Wash thoroughly in running water: 10 minutes or longer.
®NO. Stain in acid fuchsin: 5 minutes.
Or
MD
con
OF
Trichrome Staining 155

9: Rinse in distilled water until excess stain is removed. Check un-

der microscope.
10. Stain in ponceau de xylidine: 1-5 minutes.
IE Rinse in tap water, control under microscope for proper intensity
of acid fuchsin and ponceau de xylidine.
IZ, Differentiate in phosphomolybdic acid: 5 minutes.
lo: No rinse, transfer directly into fast green: 2 minutes.
14; Differentiate fast green in acidic water and dehydrating alcohols.
1b: Dehydrate in absolute alcohol, 2 changes: 3 minutes each.
16. Clear and mount.
RESULTS:
nuclei—deep mauve blue to black
cytoplasmic elements—varying shades of red and mauve
muscle—red
collagen, mucus—green

COMMENTS:
Among the many modifications of Masson’s stain, this is one of the
best, because it seems to offer good control of both red dyes by the
use of two separate solutions. In most cases, the full 5 minutes in the
acid fuchsin is advisable, but time in the ponceau de xylidine is not
quite so critical.

Pollak’s Rapid Method (1944)

FIXATION: any general fixative. Formalin-fixed tissues are improved by
treating slides overnight with saturated aqueous HgCle.
SOLUTIONS:
Hematoxylins, see page 124.
Trichrome stain:
acid fuchsin. Gl. 42089 oe ccc. ct oa sn Ooh 0.5 gm,
poncean' 2h, Gil, T6150 eon oe eee ie 1.0 gm.
light green SF, yellowish C.I. 42095 .......... 0.45 gm.
erangenG, (Ce 16250 ooo eid ss were ek Ce 0.75 gm.
phosphotungpstic acd: o 00 65.6 awn is oe ER 15em.
phosphomolybdic acid.) occ<.a.ssesateeuee hb-em:,
placial AceMC AO 4 he baa cS ses Shae te 3.0 ml.
DOS ethyl alconol Up: (0-.66.. 065 6 64 cos gee 300.0 ml.
Add acetic acid to alcohol; split solution in 4 parts. Dissolve acid
fuchsin and ponceau in one part; light green in the second part;
orange G and phosphotungstic acid in the third part; and phospho-
156 Connective Tissue (CHAP. 13)

molybdic acid in the fourth part. Mix four solutions and filter. Keeps
well.
Acidified water:
placialacetic acid: 003% 25)... aimee3 jsonal os 0.2 ml.
Gistilled, Water p26 > s+, ES oS he ae 100.0 ml.

PROCEDURE:
. Deparaffinize and hydrate slides to water; remove HgClbs.
. Stain in Mayer’s hematoxylin: 10 minutes. (Pollak uses Weigert’s:
10-20 minutes.)
. Wash in running water: 10 minutes.
. Stain in trichrome stain: 7 minutes.
. Rinse briefly in distilled water.
. Differentiate in acidified water: only a few seconds; check under
microscope if necessary.
. Dipa few times in 70% alcohol.
. Dehydrate in 95% alcohol: several seconds in each of two changes.
. Dehydrate, absolute alcohol, clear and mount.

RESULTS:
nuclei—dark blue
muscle, elastin—red
fibrin, calctum—purple
hyalin—pale blue
collagen, mucus—blue green

Gomori (1950B) Method

FIXATION: any general fixative. Formalin-fixed tissues are improved by
treating slides overnight with saturated aqueous HgClo.
SOLUTIONS:
Hematoxylins, see page 124.
‘Trichrome mixture:
eimomotrope 2h. GC. 16570meer- .:.... Sak 0.6 gm.
fast green FCF, C.1. 42053 or light green SF, C.I.
BOO D. .. . 3.kun 5 he Oe ee A. ( O 0.3 gm.
phosphotungstic acid. 4 osyAaeees.. .....2 Mee 0.6-0.7 gm.
platcialacetic acid: «24.0. eeeenme
st). ii. see 1.0 ml.
GuStINed: Wate, ..%> cc.t aa Rays Sach, slocictou age 100.0 ml.

Keeps indefinitely. Aged solutions stain less red and more green than
fresh ones.
Trichrome Staining ] Co~I

Acidic water:
SIACA PACEMCACIGs 22%, «5 sis acing
Ake aay 0.2 ml.
Gistuledawatens co. ek. is sw dead ae cea Pee oe 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
Slightly overstain with any hematoxylin.
Wash in running water: 5 minutes.
Stain in trichrome mixture: 5—20 minutes.
Rinse briefly in distilled water to remove some of excess stain.
Rinse briefly in acidic water.
Rinse in 70% alcohol.
Sos
SeeDehydrate in 95% alcohol, 2 changes; be careful not to remove
too much green.
9. Dehydrate, absolute alcohol, clear and mount.

RESULTS AND COMMENT

There have been a number of attempts to combine the azan and Mas-
son methods in a quicker one. Gomori considered Pollak’s as a disap-
pointingly dull color scheme, lacking real red shades. Almost any
combination of an acid triphenylmethane dye with a sulfonated azo
dye in the presence of phosphomolybdic or phosphotungstic acid will
give good results. The former favors the green and blue shades, the
latter the reds. A short staining time produces more red and _pro-
longed staining more green and blue. Rinsing in tap water weakens
the reds.
If more red is desired—in muscle and epithelial fibers, for ex-
ample—before staining treat with warm Bouin’s 56-60°C: 2—5 min-
utes. Follow by washing until yellow color disappears.
The rinse in acetic acid does not change the colors, but makes them
more delicate and transparent.
Lillie (1954B) says that Gomori’s trichrome gives a rather diffuse
and incomplete picture of the more finely fibrillar stroma, and the
reticulum of the liver and spleen are difficult to discern.

The author has had good results with most of the trichrome meth-
ods and can only recommend that the selection of a routine stain will
have to be made by each individual. All of the short trichrome meth-
ods offer clearer and sharper returns on thin (5-7 microns) sections
than on thicker ones. For the latter, Mallory’s, Masson, or Azan meth-
ods should be used.
158 Connective Tissue (cHAP. 13)

Churg and Prado (1956) Method

FIXATION: mercuric chloride type excellent. Improve formalin fixation
by treating slides overnight in saturated aqueous HgClb.
SOLUTIONS:
Crystal violet stain:
erystal violet, (Gal 42 DOD) og ce e330 0.1 gm.
distilled awatetrigc 52 ec es Se OUR, os 100.0 ml.
glacial acetic Meta! irs... 2 Ca SER PIE olen as ot 0.2 ml
Acidic water:
glacial taceucracid 7A: Saree). -Wedloxn tt 0.2 ml.
distilled: watera3rmd \iencisee.
tee hey. Pe?! 100.0 ml.
Phosphomolybdic acid:
phosphomolybdic acid) --. tacos 2... ==: 1.0 gm.
CIStINlEQWalee: m2 Sart= =, eee ts. cos axes 100.0 ml.
Chromotrope-aniline blue stain:
chromotrope! Zit; Gil 6570 Ber ees. os 2 shies: 1.0 gm.
anitinesblue .VWS;y Cle 477 SONG ety. guys: 0.25 gm.
HCl, 0.02N (1.7 ml. conc. HC] in 998.3 ml. dis-
tilledrwater)ps<dey re. sheers fee. ia dae 50.0 ml.

Dissolve aniline blue in acid with gentle heat. Cool and add chromo
trope. Keeps well for months.
PROCEDURE:
1. Deparaffinize and hydrate slides to water. Remove HgCly.
. Stain in crystal violet: 2-3 minutes.
. Differentiate in acidic water: 3-45 seconds.
. Rinse well in distilled water.
. Treat with phosphomolybdic acid: 3-45 seconds.
. Rinse in distilled water.
. Stain in chromotrope-aniline blue: 2 minutes.
PO
OO
#
Or
MD . Rinse in distilled water.
CONTI

9. Dehydrate quickly through 70% and 2 changes 95% alcohol.

10. Dehydrate, absolute alcohol, clear and mount.

RESULTS:
Formalin-fixed tissue without HgCls:
nuclei—purplish
cytoplasm—pale blue to pink
Trichrome Staining 159

muscle—red
collagen, basement membrane—brilliant blue
fibrin, serum protein—red
red blood cells, keratin—bright red
colloid, hyalins—blue to purple to red
After HgCls:
elastic fibers and mucus—purple

Picro-Gomori Method (MENziEs, 1959)

FIXATION: mercuric chloride type best.
SOLUTIONS:
Hematoxylins, see page 124.
Orange G-picric acid:
orange G, C.I. 16230 1.0 emCc

picric acid, saturated aqueous 100.0 ml.

Light green-chromotrope:
light green SF, yellowish, C.I. 42095 0.3 em.
phosphotungstic acid ... 0.6 em.
chromotrope 2R, C.I. 16570 0.6 em.
glacial acetic acid .... 1.0 ml.
distilled water 95.0 ml.

Acidic water:
glacial acetic acid . 1.0 ml.
distilled water 100.0 ml.
PROCEDURE:

1. Deparaffinize and hydrate slides down to water. Remove HeCls.

9
2. Stain hematoxylin (Weigert’s, Mayer’s, etc.) for correct time de-
pending on hematoxylin used.
3. Stain in orange G-picric acid: | minute.
4. Wash in running water until erythrocytes are yellow = -5—10" min:
utes.

5. Stain in light green-chromotrope: 2 minutes.

6. Sharpen stain in acidic water, 3 changes: 1 minute each.
7. Rinse in 70% alcohol.
8. Hydrate, clear and mount.
RESULTS:
erythrocytes—brilliant orange yellow
fibrin—orange red or red
160 Connective Tissue (CHAP. 13)

smooth muscle—deep purplish red

epithelial cytoplasm—varies, red to green
collagen—clear green

COMMENTS:
This method gives excellent differentiation between smooth muscle,
collagen and fibrin.

Koneff’s Aniline Blue Stain (1936)

FIXATION: any general fixative, preferably containing HgCly.

SOLUTIONS:
Iron alum:
ferhicammeoniumsuliate. -aeenee =. foe 5.0 gm.
distilled :awaittet ii aes... Sea ee on 100.0 ml

Hematoxylins, see page 124.

Aniline blue solution:
anulhmenblue VY S; Cal 42780) ee oo an 0.1 gm
oxaligsacid) » ..s 5. OP e ess seee 2.0 gm.
phosphomolybdieacidh:::., Hee eos ote 15.0 gm.
CSIR Watery. 6.17...) solos a orem ees 300.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides down to water, remove HeCle.
2. Mordant in iron alum: 10 minutes.
3. Wash in distilled water: 10 minutes, several changes.
ip Stain in hematoxylin (Delafield’s or the like): 5-15 minutes. This
may be progressive or regressive.
Or Wash in running water: 5 minutes; rinse in distilled water.
SP Stain in aniline blue stain: 10-15 minutes. Koneff stains over-
night.
7. Rinse in distilled water; few dips.
8. Dehydrate quickly through 50 and 70% alcohol.
9 . Dehydrate in 95% alcohol, 2 changes: 3 minutes each.
0 . Finish dehydration in absolute alcohoi; clear and mount.

RESULTS:
collagen, reticulum, mucus, hyaline cartilage—blue
nuclei, red corpuscles—violet or purple blue
cytoplasm, myelin sheaths, muscle fibers—brown
elastic fibers—reddish brown or red
Trichrome Staining 161

Kornhauser’s Quad Stain (1943, 1945)

FIXATION: any general fixative, preferably containing HgClo.

SOLUTIONS:
Orcein:

OMCCN MS ep A ocak eihea soa 1d See ORT Roa ea! 0.4 em.
WtrICRICIG. CONC. ws? :.. iy. 64.0 D2e se eee Ose 0.4 ml.
OOP Aaethyl alcohol 4A... 0s. gkgegae
seh rae 100.0 ml.

Acid alizarine blue:

acia. alizarine blue BB, GC. 58610). 22. 2s ss cas - 030.9m,
aluminum ammonium sulfate ............... 5.0 gm.
acetic acid, N/10 (5.8 ml. glacial acetic acid
mage GO) UME ac. % «ls 1 eheeeere
ae See 82.0 ml.

Boil gently in Erlenmeyer flask covered with watch crystal: 10 min-

utes. Cool and filter.
Phosphotungstic-phosphomolybdic acid:
phosphominestie acd oo... sass ..2dene
eee eee 4.0 gm.
phosphomolybdiciacid 2.2: ..<5..0seaneeeeees 1.0 gm.
distilled WaLer 2.24. ga8o0 hs case Boke eee 100.0 ml.

Orange G-fast green:

oranpe GGA. 16230) oe iio o i ai est ta ge 2.0 gm.
fast Cree HCl Gea e0 08 ce sd at eee 0.2 gm.
glacial acetic acid ... aha - 2.0 ml.
Gistulled- Waler \ » v5.24 64-4 40 6acc aea epee ee 100.0 ml.

PROCEDURE:
1. Deparaffinize and run slides down to 85%/O alcohol. Remove HeCl.
fa) -
if present.
2. Orcein: 2-24 hours, until elastin fibers are red brown. May be
omitted if no elastin is present or is not to be stained.
3. Wash out excess orcein, two changes 85% alcohol.
4. Run down to water.
5. Acid alizarine blue: 5—10 minutes.
6. Decolorize and mordant in phosphotungstic-molybdic solution,
until collagen is destained: 10-30 minutes.
7. Rinse few seconds in distilled water.
8. Stain in orange G-fast green: 3-10 minutes.
9. Rinse in 50% alcohol: about 10 seconds.
10. Dehydrate, 95% and absolute alcohol, clear and mount.
162 Connective Tissue (CHAP. 13)

RESULTS:
elastin—red brown
nuclei, basophilic granules—blue
cytoplasm, muscle fibers—violet
collagen, reticulum, basement membrane—green
erythrocytes, myelin sheaths, acidophilic granules, plasmosomes—or-
ange

Movat’s Pentachrome Stain (1955)

FIXATION: formol-sublimate-acetic is best: 12—18 hours.

mercaric.chiloride: . os... 2 )..7 eee Se Ost 4.0 gm.

formalin: .: <> beige -sjigen thane ee. RY 20.0 ml.
Gistilled water: «ces. esc se See Cet de be 80.0 ml.
placial acetic acid) 3.4).5.5 1 ce aeeeeete
020s 5oe 5.0 ml.

Do not use a dichromate fixative; it diffuses the alcian blue staining.

If 10% formalin is used, after the sections are mounted on slides and
hydrated, post-fix them in formol-sublimate-acetic for 2-3 hours, or
overnight.

SOLUTIONS:
Alcian blue:

alcranabiue cGS,.Cile 7/474ON wees oso 1.0 gm.

distilled’ water’... 2... Seip 3 91d 4/2 a rrlrantntae 100.0 ml.
Filter, and add:
plaeimlFAGEITG*AClG. a> 0 tMeee we 1.0 ml.

Prepare fresh each time.

Weigert-Hart resorcin-fuchsin:
Weigert’s resorcin-fuchsin (stock solution):

basiciuchsin, (C.1.. 42500. -eeeaeee

eee pes er ae 2.0 gm.
FESOREINOL hs... noes Se Ee 2. ss ee 4.0 gm.
aistilled water. 24.5). 71. eee ce ant ee 200.0 ml.

Mix in a porcelain dish and after it comes to a boil cook for 1 minute.
A scum should form. Add 25.0 ml. of 29% aqueous ferric chloride.
Cool, filter, and leave precipitate on filter paper until dry. Cover and
leave overnight. Place precipitate in porcelain dish and add 200.0 ml.
of 95% ethyl alcohol. Bring to a boil on an electric hot plate. Add 4.0
mil. of concentrated hydrochloric acid (see Comment 1). Remove from
Trichrome Staining 163

hot plate and bring volume to 200.0 ml. by adding 95% alcohol. ‘The
solution keeps well.
Working solution:
JOO CUNY AICONO) o a5 oo BB ys Ghee BOO ies 94.0 ml.
Weigertsmesorcin-tuchsin ..,. san jy leas eis ees 5-0: mall.
hydrochloric acid (see Comment 1) ........... 1.0 ml.
Weigert’s hematoxylin, page 000.
Woodstain scarlet-acid fuchsin:
Stock solution A:
woodstainscavlet INS sic va.os ee SS 0.1 gm.
IStMCUOWALEH 64 2 nde ba ip rey gpa epee 22 99:5 mi.
placialsacee ACI 2.44 £6 s.i2i2 doama rear « 0.5 ml.
Stock solution B:
acid: fuchsin; Gil: 42685 ........de0<8eho.
ade 0.1 gm.
aistriled’ wate? ac. s Yas daa. an. doe ROSNSe eae 99.5 ml.
placialaCeMe AGIG! 424.2. hughes oad Renee Oe TE Os rmiks
Working solution:
Solution Aas oh ase ack oon O08 oes oe oe 40.0 ml.
DOMINO Roe ace one oo oe lnok ne oe oe 10.0 ml.

Phosphotungstic acid:
phosphotunestie acid. . .. .4.o1..s:.a.esanneee 5.0 gm.
custniied “Water .2.45..4...2..2:
04 sen eee 100.0 ml.

Alcoholic saffron:
SAUMON we 2 seg ee ay 2G a tk hw Does SEA 6.0 gm.
apsolube ethyl. alcohol 2h. 9 .4...s<)<s0s
eee eG 100.0 ml.

Place in tightly corked bottle to prevent hydration and place in incu-

bator, 58°C for 48 hours before use. Store in an airtight bottle.
PROCEDURE:
1. Deparafhinize and hydrate slides to water; remove HgCle.
2. Rinse thoroughly in distilled water.
3. Stain in alcian blue: 15 to 30 minutes.
4. Wash in running water: 3 minutes.
5. Place in alkaline alcohol: 2 hours: (Add a few drops of ammonia
to 95% alcohol until pH is over 8. This solution converts the
alcian blue into an insoluble pigment.)
‘Edward Gurr, London, England.
* Safran de Gatinais, Gritbler, may be obtained from Roboz Surgical Co., Washington,
Dic;
164 Connective Tissue (CHAP. 13)

6. Wash in running water: 10 minutes.

7. Rinse in 70% alcohol.
8. Stain in resorcin solution: 16 hours.
9. Wash in running water: 10 minutes; rinse in distilled water.
10. Stain in Weigert’s hematoxylin: 15 minutes.
11. Wash in running water: 10 minutes; rinse in distilled water.
12. Stain in woodstain-acid fuchsin: 5 minutes. (During its staining
action this solution differentiates the hematoxylin.)
13. Rinse in 0.59% aqueous acetic acid (0.5 ml./99.5 ml. water).
14. Differentiate in phosphotungstic acid: 10 to 20 minutes, or until
collagen is pale pink and ground substance bluish.
15. Rinse in 0.59% aqueous acetic acid.
16. Dehydrate thoroughly in 3 changes of absolute alcohol. Use fresh
and good-grade alcohol or the tissue will not take on the collagen
stain which follows.
17. Stain in saffron: 5 to 15 minutes. Use an airtight staining jar,
preferably screw-cap type of coplin jar, to prevent hydration of
saffron solution.
18. ‘Transfer through 3 changes of fresh, good-grade absolute alcohol.
19. Clear and mount.
RESULTS:
nuclei—blue to black
cytoplasm—red
elastic fibers dark purple to black
collagen and some reticular fibers—yellow to greenish yellow
ground substance (acid mucopolysaccharides) and some reticular
fibers—blue to bluish green
fibrinoid—intense red

COMMENTS:

1. Movat says: “It is important for these staining methods to prepare

solutions with HCl of specific gravity 1:124 (the offizielle Salzaure) and
not concentrated HCl as stated in some textbooks. It is prepared by
adding 50 cc. of distilled water to 100 cc. of concentrated HCl (specific
gravity 1.19).”
2. Movat includes other modifications of his method using orange G
and diphenyl fast red, but seems to favor the above procedure. He calls
his method a demonstration of all connective tissue elements in a single
section.
Picro-Ponceau Staining 165

Picro-Ponceau Staining
Picro-Ponceau with Hematoxylin (GuRR, 1956)
(Van Gieson Substitute, nonfading)
FIXATION: any general fixative.
SOLUTIONS:
9
Hematoxylins, see page 124.
Picro-ponceau:
ponceam,S AG. 1027195, 19%, aqueous 22 ket. .: 10.0 ml.
picric acid, saturated aqueous ............... 86.0 ml.
aeeme acid, 1, JAGMEOUS, «v0.2 2 eenema 4.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides down to water, remove HgCle.
2. Optional (see Comments): mordant with iron alum before Dela-
field’s (or similar) hematoxylin. Follow by washing in running
water.
3. Overstain in hematoxylin: 5-15 minutes.
4. Wash thoroughly in running water until slides are deep blue: 10
minutes or longer.
5. Stain in picro-ponceau: 3—5 minutes. (This may be too long for
some tissues; the stain acts, in addition to staining, as a destaining
agent on the hematoxylin.) Rinse for a few seconds in distilled
water and check under microscope. Continue to stain and destain
or differentiate in water until nuclei are sharp.
6. Dip several times in 70% alcohol.
7. Dehydrate in 95% alcohol, 2 changes, to insure complete removal
of excess picric acid. Only that which has acted as a dye must be
left in the tissue.
8. Dehydrate, absolute alcohol; clear and mount.
RESULTS:
nuclei—brown to brownish or bluish black
collagenous and reticular fibers—red
elastic fibers, muscle fibers, erythrocytes, epithelia—yellow
COMMENTS:
Weigert’s type of hematoxylin is excellent for this method, but it is
not necessary to precede it with iron mordanting. The iron mordant
is present in the staining solution.
166 Connective Tissue (CHAP. 13)

This method is so superior to the so-called Van Gieson stain which

uses acid fuchsin instead of ponceau S, that the Van Gieson method
has been omitted. Colors are identical but the Van Gieson proves un-
satisfactory because it fades rapidly.

Elastin Tissue Staining

Verhoeff’s Elastin Stain (MALLORY, 1944)
FIXATION: any general fixative.
SOLUTIONS:
Verhoeff’s stain:

on electric hot plate, dissolve 3 gm. hematoxylin in 66 ml. absolute

ethyl alcohol. Cool, filter and add 24 ml. of 10% aqueous ferric chlo-
ride (FeCl,) and 24 ml. Verhoeft’s iodine solution. Usefulness is lim-
ited to 1-2 weeks.
Verhoeff’s iodine solution:
potassium jedide (Ky). ). st eee eee 4.0 gm.
cistifled™water iS Atftiet, Fe eeemeeeeeensat?
0k Vv E 100.0 ml.
Dissolve, and add:
VOOM ee ree shit ee et ne 2.0 gm.

Ferric chloride solution, 10%:

PERTEGgCMLOTiGe ECC Ae ieee
Ns ice Foodie 10.0 gm.
GISHIMER Water, 250) sean jee Rae Ietorso ents ote 100.0 ml.

Ferric chloride solution, 2%:

NOE Acrertic ‘chloride: . “Saermeemerant
. ss nee 20.0 ml.
Gdismiled “water. oe eee eS 100.0 ml.

Picro-ponceau solution:
ponceauns, 'C.1. 27195,1 97 vammeous: . 4... 4.2% 10.0 ml.
Pleric.acid, ‘saturated aqmeausion 5...
1; scan. 86.0 ml.
acetic acid, 107, aGucOuUs me weieaa:
. + «ah. eelak 4.0 ml.

PROCEDURE:
1. Deparafhinize and run slides down to 70% alcohol. Removal of
HeCl, not necessary.
. Stain in Verhoeff’s stain: 15 minutes.
. Rinse in distilled water.
DO. Differentiate
©9
He in 2% ferric chloride: few minutes. Elastic fibers
Elastin Tissue Staining 167

should be sharp black, nuclei brown. If destained too far, return

slides to Verhoeff’s for another 5-10 minutes.
Transfer to sodium thiosulfate, 5% aqueous: 1 minute.
Wash in running water: 5—10 minutes.
“IDCounterstain
Oo in picro-ponceau: | minute.
ee
) Differentiate in 95% ethyl alcohol: few seconds in each of 2
changes.
9. Dehydrate in absolute alcohol; clear and mount.

RESULTS:
elastic fibers—brilliant blue-black
nuclei—blue to brownish black
collagen—red
other tissue elements—yellow
COMMENTS:
If elastic fibers stain unevenly, or not at all, Verhoeff’s solution is too
old.

Orcinol-New Fuchsin (FULLMER AND LILLIE, 1956)

FIXATION: any general fixative.
SOLUTION:

new, auchsia, Cl 42520": 23) 2 25 ane es 2.0 gm.

Ofranol 42.46 Ref Bau innec tis 356 to) bier eee 4.0 gm.
distilled Gwater ~ seis « see oe sso se eee 200.0 ml.
Boil for 5 minutes; add:
ferric.chloride;, FeCl; 29.197, aqueous ice <5 «-. 25.0 ml.
Boil for 5 minutes. Cool, collect precipitate on filter paper and dis-
solve in 100 ml. of 95% ethyl alcohol.
PROCEDURE:
1. Deparaffinize and transter slides into absolute alcohol.
2. Stain in orcinol-new fuchsin, 37°C, 15 minutes.
3. Differentiate in 70% alcohol, 3 changes, 5 minutes each.
4. Dehydrate, 95% and absolute alcohol, clear and mount.

RESULTS:
elastic fibers—deep violet
collagen—unstained by orcinol-new fuchsin
COMMENTS:
Slides can be counterstained with hematoxylin, safranin, or picro-
ponceau; of the three, safranin offers best color contrast.
168 Connective Tissue (CHAP. 13)

Aldehyde-Fuchsin (Gomori, 1950C) (MODIFIED By CAMERON AND STEELE,

1959)
FIXATION: any good fixative, preferably without dichromate. Bouin’s
and formalin produce a colorless background.

SOLUTIONS:
Potassium permanganate, 0.3%:
potassium permaneanate —.. (.. seats. . 0.3 gm.
distilled" water... 2... see 0-4 1S ee ee 100.0 ml.
sulfuric acrid) ‘concentrated . 284)
Soe eee 0.3 ml.

Sodium bisulfite, 2.5%:

Sodium ibisuliite 2% -. Fa. St. 2- 2 eectseee 2.5 gm.
distilledwaters. 4) arce : 5 Sie seen ee ee 100.0 ml.

Aldehyde-fuchsin:
Add 1 gm. basic fuchsin, C.I. 42500, to 200 ml. boiling water: boil 1
minute. Cool and filter. Add 2 ml. concentrated hydrochloric acid
and 2 ml. paraldehyde. Leave stoppered at room temperature. When
mixture has lost reddish fuchsin color and is deep purple (3—4 days),
filter it and discard filtrate. Dry precipitate on filter paper in an oven.
Remove and store in bottle. Makes about 1.9 gm. To make staining
solution, dissolve 0.25 gm. in 50 ml. of 70% alcohol. Keeps at least
six months.

PROCEDURE:
1. Deparafhnize and hydrate slides to water; remove HgCl,.
2. Oxidize in potassium permanganate: | minute.
3. Rinse in distilled water.
4. Bleach in sodium bisulfite until permanganate color is removed.
5. Wash in running water: 5 minutes.
6. Transfer to 70% alcohol: 2 minutes.
7. Stain in aldehyde fuchsin: 2—10 minutes.
8. Wipe off back of slide and rinse in 95% alcohol.
9. Differentiate in 95% alcohol until no more aldehyde-fuchsin
comes out of sections.
10. Dehydrate, absolute alcohol; clear and mount.

RESULTS:
elastin—deep purple
mast cells, chief cells of gastric mucosa, beta cells of pancreas, baso-
phils of pituitary and some kinds of mucin also stain purple.
Subcutaneous Tissue Staining 169

Orcein (ROMEIS, 1948)

FIXATION: any general fixative.

SOLUTION:
ORGCIOMpe eRe 40522 AGED 3 Paeieey, 4 2 Aleae tee 1.9 gm.
Oc sepiyi alcomol (Shih... 2k a) Was es tk Beales 100.0 ml.
hydrochloric acid (assay 37-38%) ........... 1.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
Stain in orcein: 30-60 minutes.
Wash briefly in distilled water.
Dehydrate in 95% alcohol: 2 minutes.
bo
oe
OLRDifferentiate in absolute alcohol until background is almost color-
less and elastin fibers are isolated.
6. Rinse in fresh absolute alcohol; clear and mount.
RESULTS:
elastin—red

COMMENTS:
Some disagreement appears in the literature concerning the solution
pH at which orcein is most effective. Wess (1954) writes that orcein
stains only from an acid alcoholic solution between pH 3 and 8, that
this puts orcein in a category between basic and acidic dyes, which
generally operate at the extremes of alkaline and basic pH. He there-
fore considers there is a formation of hydrogen bond between orcein
and elastin. Since this reaction takes place in acid alcohol, it 1s prob-
ably due to a uniquely low positive charge of elastin in such solutions
The use of alcoholic solutions is necessary to stabilize the positively
charged orcein fractions.
Darrow (1952), experimenting with orcein, found that pH 1-2.4
is best for elastin staining, that above pH 2.6 collagen stains as well.
A dye content of 0.4% is adequate for elastin staining; a higher con-
centration adds to collagen staining. For a specific elastin reaction
when collagen is present, therefore, it is advisable to check pH of acid
alcohol used for the solution and to reduce the dye content to 0.4 gm.
per 100 ml.

Subcutaneous Tissue Staining

Subcutaneous tissue, areolar tissue, and omentum (membranes) can
be easily fixed for staining. A simple way is to spread a piece with dis-
170 Connective Tissue (cHAP. 13)

secting needles on a slide, allow to slightly dry and immerse in fixative.

The tissue, in most cases, will remain adhering to the slide through
staining solutions.
If histologic rings are not available for spreading tissue, cut thick
filter paper into circles of a desired diameter and about 6-7 mm. in
width. Place the circle of filter paper under the omentum or other tis-
sue to be spread and press against the tissue. Cut out a ring of the tissue,
just beyond the outer edge of the paper circle and place in fixative.
Carry filter paper and tissue through staining procedures; connective
tissue stains can be applied as usual. Finally remove the paper by peel-
ing it off when the tissue is being placed on a slide preparatory to mount-
ing with cover glass.
Bits of loose connective tissue which are difficult to handle may be
dehydrated and infiltrated with nitrocellulose. Carefully spread the tis-
sue in a film of nitrocellulose on a slide, allow to slightly dry, and
harden in 70% alcohol. It can be stained in place on the slide as this
is carried through solutions like a nitrocellulose section. The latter
method usually gives more uniform staining.

Bone Staining
Decalcified (RoMEIs, 1948)
FIXATION: formalin, followed by decalcification (see pages 24—26).
SOLUTIONS:
Thionin:

thionin, C.I. 52000, saturated in 50% ethyl

UCGHOI 25.71 BARC eR bs os. napa 10.0 ml.
Gistilednwaterm ... 3. -.c0 eer ee 100.0 ml.

If the color does not set correctly, add 1—2 drops of ammonia to stain-
ing solution.
Carbol-xylol, see page 410.
PROCEDURE:
1. Deparaffinize and hydrate slides to water. If sections tend to loosen,
treat with 0.5-1% nitrocellulose (page 60).
Stain in thionin solution: 10 minutes.
Wash, distilled water: 20 minutes, change several times.
Treat with picric acid, saturated aqueous: 1 minute.
Se
ee Rinse in distilled water,
Bone Staining iB |

6. Differentiate in 70% alcohol: 5-10 minutes or more, until no

more color comes off.
7. Dehydrate in 95% alcohol, 2 changes: 3 minutes each.
8. Dehydrate and clear in carbol-xylol: 5 minutes.
9. Clear in xylene, and mount.

RESULTS:
lacunae and canaliculi—bordered with bluish black
background—yellow

COMMENTS:
Since occasionally paraffin sections loosen from the slides, some tech-
nicians prefer nitrocellulose embedding, but this takes time.

Hand Ground, Undecalcified Sections (ENLow, 1954)

PROCEDURE:
ie If necessary to remove organic materials and fat, treat in follow-
ing manner:
(a) Boil in soap solution: 3—4 hours; and wash in running water:
3—4 hours.
(b) Suspend over either or chloroform: 36—48 hours.
(c) Allow to thoroughly dry.
no. Cut slices as thin as possible without cracking them. A jeweler’s

saw is recommended.
. Grind on sharpening hones with finely powdered carborundum
or household cleansing powder. Keep surface wet with water.
Optical or metallurgical grinding and polishing equipment, if
available, can be used. The grinding can be done between two
stones, or the section can be held on a wet rubber or cork stopper
and ground against the hone. If this becomes difficult to manage,
glue the section on-a slide with Duco cement, and continue to
erind. When almost thin enough, loosen from slide with acetone,
turn section over, glue it down, and grind opposite side.
- Polish off both sides with fine leather strop.
Place in 95% alcohol: 5 minutes.
Air dry.
EDPlace in plastic solution, agitate to liberate air bubbles.

Ravloldin: 5 2 pee hes 2 ee Se. oye eee ee 28.0 gm.

butyl(or amyl) acetate « <c2.... nc ew chee eee 2DOLU mi.

Let stand until parloidin is completely dissolved. Stir thoroughly.

ly2 Connective Tissue (CHAP. 13)

8. Transfer to slide with a drop of solution.

9. Dry thoroughly; do not add more solution.
10. When completely dry, add mounting medium and cover glass.

COMMENTS:
To bring out density and distribution of the mineral in undecalcified
bone, see Frost (1959). He describes methods with basic fuchsin, silver
nitrate, alizarine red § and others.
Dowding (1959) uses methyl methacrylate as a plastic embedding
solution, and grinds down the bone as thin as 30 microns.
Hause (1959) describes a block for holding the bone while sawing
it and recommends a Razor Saw blade #35-ST (Lipshaw Manufac-
turing Company) as a good cutting instrument.
Kropp (1954) describes a plastic embedded method using heat and
pressure.
Yaeger (1958) uses freeze-drying and vacuum infiltration with butyl
methacrylate-ethyl methacrylate.
Norris and Jenkins (1960) describe a method using epoxy resin for
preparation of bone for radioautography. The resolution is good;
there are no chemicals in resin to produce artifacts in nuclear emul-
sions; the medium does not warp or chip when machined or abraded.
(Polyesters and methacrylate do distort when machined.) The design
of a metal and lucite mold for the embedding is included. Sections
can be made with a microtome (6-10 microns), with a circular saw
(50-100 microns) and ground to give sections as thin as 10-50 mi-
crons.

Alizarine Red S Method for Embryos (Bone Formation)

FIXATION: a hardening action.

Hollister (1934), 70% alcohol for fish, removal of scales desirable:
several days.

Hood and Neill (1948), 95% alcohol, organism preferably free of hair
or feathers: 3 days.

Richmond and Bennett (1938), 95% alcohol: 2 weeks.

Some specimens require decolorization. The best method is to lay

specimen in 95% alcohol in white tray. Place in direct sunlight for
24 hours each side. An Alpine sunlamp was used by Hollister on sun-
less days.
Bone Staining ] | co

SOLUTIONS:
Potassium hydroxide, 2%:
potassium hydroxide, white sticks ............ 20.0 gm.
GIS MMALENY oce.2 Meats 4a, 1 Seas Benn wee cee 1000.0 ml.
Alizarine stock solution: (Hollister, 1934)
alizarine red S, C.J. 58005, saturated solution in
DUP ACCHG ACG)? 2 2e ai vs os Bee
ee. add 5.0 ml.
CU CEN sire y ets ta. 3.4 DALE es os 7622 10.0 ml.
chlonal- hydrate; 197 aqueous “.¢ndorsaues ae 60.0 ml.
Alizarine working solution:
ALIZAPING SOCK SOLON... 4.1 « eda gener eee x5 Ome
1-2% potassium hydroxide in distilled water .. 1000.0 ml.

Make up at least 500 ml.; use at room temperature.

Clearing solution #1: (Hood and Neill, 1948)
2°, POtassiMM Wy OTORIE...< ..c5 ps Yaa eee a os 150.0 ml.
O20 POUMRANN 5 reing.Atcha bs ole os «8 eee eres hoes 150.0 ml.
PIVECIOUGS § 56 Mia aecha yes 4 i «od dee See oo 150.0 ml.
Clearing solution #2: (Hood and Neill, 1948)
OG Potassiuin My GnOKIGe. . 2.4... seeaaee es 100.0 ml.
CIV CELOW” . cA rey edit VS = 4 eee ee 400.0 ml.
PROCEDURE:
1. From hardening solution, rinse few minutes in distilled water.
2. Leave in 2% potassium hydroxide until skeleton shows through
musculature: 2—4 hours for small embryos, 48 hours or longer for
larger forms. (St. Amand and St. Amand, 1951, warmed solution to
38°C for quicker action.)
3. When clear, transfer to alizarine working solution: 6-12 hours or
longer depending on specimen. Skeleton should be deep red.
Large specimens may require fresh changes of dye solution.
4. Transfer directly to 2% potassium hydroxide: | day or longer un-
til soft tissues are destained. The KOH may be 1.0-0.5°% for small
specimens. Sunlight or lamp speeds up process.
5. Clear in solution #1: 2 days, room temperature. Then in solution
ape. bday:
6. ‘Transfer to pure glycerol with thymol added as preservative.
7. Store in sealed tubes or bottles. Mount on glass rods and seal in
museum jars, or embed in plastic.
174 Connective Tisswe (CHAP. 13)

COMMENTS:
Cumley et al. (1939) gradually replaced the glycerol with 95% alco-
hol, absolute alcohol, and finally toluene. Then the specimens were
transferred to toluene saturated with naphthalene, and stored in
anise oil saturated with naphthalene. This method is supposed to
produce greater clarity than the glycerol storage.
 Chapter 14

Silver
Impregnation |
RETICULUM TECHNICS

Silver Impregnation
According to Baker (1958) methods under this heading depend upon
the local formation within tissues of a colored substance which is not a
dye. Impregnation applies to a condition developing when an un-
reduced metal (silver, etc.) is taken up from a solution of salt or other
compound and deposited.in a colloidal state on a tissue element. Follow-
ing impregnation, the tissue is removed to a reducing solution of a
photographic type and the metal is reduced to the elementary state,
probably in the form of a black deposit. ‘Thus the tissue itself does not
reduce the metal, but some extraneous reducer is required to perform
the reaction.
Silver staining goes back as far as 1843 when Krause tried small pieces
of fresh tissue in silver nitrate. The Golgi method appeared in 1873.
Ramon y Cajal (1903, 1910) first experimented with silver nitrate in
1881 and tried reducing it. Also in the early 1900s protargol (Bodian
method) and other organic silver compounds were introduced as sub-
stitutes for silver nitrate. The albumen fraction in the organic com-
pounds is considered to act as a protective colloid which prevents too

L79
176 Silver _Impregnation I (cHAP. 14)

rapid reduction by formaldehyde, thereby inducing the formation of

finer-grained deposits of silver.) The next step was experimentation
with silver on tissue sections, leading to a literature full of modifications
of Ramon y Cajal, Bielschowsky, and others.
Ammoniacal silver is the familiar complex, and when reduced by
formaldehyde to metallic silver forms a colloidal solution containing
negatively charged particles. These may be precipitated out by oppo-
sitely charged surfaces which can be changed either to repel or to absorb
the silver. Thus, the negatively charged silver (formed by reduction) is
deposited on positively charged surfaces and allows selective impregna-
tion of neurofibrils, reticulum, Golgi, etc. The charges of tissue ele-
ments may be effected by the fixation and dehydration coagulating the
proteins and leaving them positively or negatively charged. ‘The pH of
the silver solution is a strong factor in determining charges and depends
on whether an ammonium or sodium hydroxide or sodium or lithium
carbonate is used (Ramon y Cajal and del Rio-Hortega methods).
As already mentioned, protective colloids, such as gum arabic and
mastic, and the use of protargol slow down the reduction of the silver
to produce a finer grain (Liesegang method). Dilute reducing reagents,
combined with the above, can have the same effect (Ramon y Cajal and
Bielschowsky methods). Temperature is a factor in this respect, because
it increases the kinetic energy of the particles and permits a greater
number of collisions of the particles against the tissue surfaces. In some
methods, copper is added to the silver solution supposedly to speed up
impregnation by initiating the reduction to metallic silver. At the same
time too heavy a deposit is prevented by removal of some of the silver
from the solution. ‘Thus various applications of the principles can be
used to control the impregnation of different kinds of tissue elements
(Silver, 1942). Chemical properties of tissues and their responses to these
conditions all help to determine the place and amount of deposition.
The types of silver impregnation (all ammoniacal) can be classified in
the following manner: (1) ammoniacal silver nitrate, (2) ammoniacal
silver hydroxide, and (3) ammoniacal silver carbonate. In all of these
solutions the silver is present largely in the form of a complex silver
ammonia cation [Ag(NH3)2]*. In (1) ammonia alone is used to form the
precipitate, the chief product in solution being silver diammino nitrate.
In (2), the Bzelschowsky method (also modified by Ramon y Cajal),
sodium hydroxide is used to form the precipitate and ammonia to re-
dissolve it, the chief product being silver diammino hydroxide. The
difference between the Bielschowsky and the Ramon y Cajal methods
lies in the way the silver is applied and the reduction is performed. The
Silver _Impregnation ly? |oe

Ramon y Cajal type uses a single ammoniated silver impregnation fol-

lowed by reduction in pyrogallol, hydroquinone, or one of the amino
phenols. The Bielschowsky type uses double impregnation (silver nitrate
followed by ammoniated silver) and reduction in formalin. In (3), the
del Rio-Hortega method, sodium carbonate (sometimes lithium car-
bonate) is used to form the precipitate and is followed by ammonia, the
chief product being sélver diammino carbonate. (Kubie and Davidson,
1928)
The reactions of these solutions to various conditions should be
understood:
1. The ammoniacal silver nitrate is most stable, least sensitive to
light, least readily reduced, and combines least easily with tissues.
During formalin reduction, a cloud of finely divided gray dust slowly
develops and the staining is slow. This method is rarely used in prefer-
ence to other solutions.
2. Ammontiacal silver hydroxide lies to the other extremes; it is least
stable, most sensitive to light, most readily reduced, and combines most
easily with tissues. Almost instantly a heavy black cloud appears. It com-
bines almost at once with the tissue, the solution darkens quickly, and
a precipitate begins to form. Since silver nitrate is not reduced in an
acid solution, but reduces readily in an alkaline solution, it stands to
reason that the ammoniacal silver hydroxide solution is the most
sensitive of the three.
3. The ammoniacal silver carbonate solution has properties lying be-
tween the above two. Its precipitate forms more promptly than that of
(1) ammoniacal silver nitrate, but not as fast as that of (2) ammoniacal
silver hydroxide. It is darker than that of (1) but not to the degree of
(2). Its reaction begins within 5-10 minutes and reaches optimum
color before the solution begins to darken. Although solutions number
2 and 3 are used interchangeably, the carbonate solution has the ad-
vantage that its hydroxide (OH)~ ion concentration is not high enough
to render it as unstable and oversensitive as the hydroxide solution, and
the presence of buffer salts makes the reduction proceed steadily and
evenly. As the acid, HNOs, is formed during the reduction the buffer
absorbs it and blocks its effect; thus the reduction is not lessened. In
addition, the presence of CO; ion buffers the formalin and prevents for-
mation of formic acid, also an effective stop to further reduction. Foot
(1929) buffered his formalin and prolonged the reducing action, making
his results darker and more intense. He warned, however, to keep the
buffer to a minimum so the reaction will not become too intense.
Equimolar silver solutions produce the most uniform results. In most
178 Silver Impregnation I (cHaAP. 14)

cases, it is wise not to dissolve completely the precipitate which is first

formed; otherwise results can be inferior.
The use of pyridine, a fat solvent, precedes some methods, removes
lecithin,
myelin, mitochondria, galactolipids, etc., and makes subse-
quent penetration of the silver easier. (This is particularly true for
connective tissue.) ‘Toning with gold chloride is optional in many
methods, it may yield a more desirable color and may improve contrasts.
The timing apparently is variable, actually only long enough to make
the desired change in color, usually a few seconds. If the reaction is slow,
then the solution has weakened. ‘The final fixing in sodium thiosulfate
(hypo) is necessary to remove all unreduced silver.
It is readily seen by virtue of the many factors involved to make it
specific for certain tissue elements, that silver impregnation can be
complex.
Corked bottles should be avoided, since cork extractives may be a
disruptive factor resulting in little selective impregnation of tissue
elements (Deck and DeSouza, 1959). In all silver methods, take care that
metal instruments do not come in contact with the silver solution; a
black precipitate may dribble down the surface of slides handled in such
a manner. Coat forceps with paraffin, or use horn or wooden instru-
ments. All glassware for metallic stains (silver, gold, etc.) must be acid-
clean. Soak in cleaning solution (page 412), wash thoroughly in running
water to remove cleaning solution, and rinse 4 or 5 times with distilled
water.
Sections loosening from slides is a common problem during silver
impregnations. Davis and Harmon (1949) use a rinse (0.5 ml. of 2%
acetic acid, aqueous, in 50.0 ml. of water) before reduction in sulfite and
hydroquinone in the Bodian method (page 203). Many technicians find
that the Masson gelatine fixative is superior to Mayer’s albumen for
afhxing sections for silver processes. Transferring unmounted paraffin
sections through all solutions and mounting and deparaffinizing at the
conclusion of the method is feasible.
Smith (1943) warns that ammoniacal silver hydroxide solutions can
become explosive on standing. Several laboratories have experienced
explosions when such solutions have been stored for some time; vio-
lently explosive silver amide has formed. Prepare ammoniacal silver
solutions just before use.
References: Baker (1958); Beech and Davenport (1933); Bensley (1959);
Foot (1929); Kubie and Davidson (1928); Long (1948); and Silver
GIES)
Silver Impregnation for Reticulum 179

Silver Impregnation for Reticulum

Bielschowsky-Foot Method (MALLory, 1938)
FIXATION: 10% formalin or Zenker-formol.

SOLUTIONS:
Ammoniacal silver solution:
In acid-clean glassware place 20 ml. of 10% silver nitrate (10 gm./100
ml. water); add 20 drops of 40% sodium hydroxide (40 gm./100 ml.
water). Dissolve precipitate which forms with ammonia, drop by drop,
with shaking until the precipitate is almost dissolved. Filter out re-
maining granules and make up to 80 ml. with distilled water. Make
up fresh each time.
Gold chloride:
gold chloride, 1% stock solution (1 gm./100 ml.
WANGCI)) oe er ei de. oo eae ess 1.0 ml.
distilled’ Water 2.00.4 2.0 dex «++ ac gettin Oi) 09:00 mal:

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HeCly.
2. Rinse in distilled water.
3. Oxidize in potassium permanganate, 0.25% (0.25 gm./100 ml.
water): 5 minutes.
4. Wash in running water: 3 minutes.
5. Bleach in oxalic acid, 5% (5 gm./100 ml. water) till clear.
6. Wash in running water: 5 minutes. Rinse in distilled water.
7. Treat in silver nitrate, 2% (2 gm./100 ml. water): 48 hours, in
subdued light, not dark.
8. Rinse in distilled water.
9. Impregnate with ammoniacal silver: 30 minutes.
10. Rinse very quickly in distilled water.
11. Reduce in formalin 5% (5 ml./95 ml. water): 30 minutes.
12. Rinse in tap water.
13. ‘Tone in gold chloride until fibers take on blackish grey color.
14. Rinse in distilled water.
15. Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3 minutes.
16. Wash in running water: 5—10 minutes.
17. Counterstain if desired.
18. Dehydrate, clear, and mount.
180 Silver Impregnation I (cHAP. 14)

RESULTS:

Reticulum—dark violet to black

del Rio-Hortega Method (MALLORY, 1938)

FIXATION: any good general fixative.
SOLUTIONS:
Ammoniacal silver carbonate:

To 10 ml. of 10% silver nitrate add 10 ml. of saturated lithium

carbonate, aqueous. Shake, allow precipitate to settle. Decant and
wash precipitate with distilled water 5 times. Add 25 ml. of distilled
water. Almost dissolve precipitate with ammonia (28% reagent),
drop by drop, with shaking. Allow a few grains of precipitate to
remain. Add 95% alcohol up to 100 ml. Filter. Warm uncovered at
50°C for 20 minutes. Ready for use. Can be reused, but heat to 50°C
and filter before doing so. Keep in brown bottle.
Formalin solution:
fonmialisiy, }.. 4 sees eeciae See ee 2s oe 10.0 ml.
distilled water 4..° i243. 2p eee dart el 50.0 ml.
Gold chloride:
gold chloride 1% stock solution (1 gm./100 ml.
WUGI) ft. Peis 2 ee Oe ee ec aa [2:0 ele
distilled" water’. oo. 2.2. ee a. ee 50.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Treat with potassium permanganate, 0.2% (0.2 gm./100 ml.
water): 3 minutes.
3. Wash in distilled water: 2 minutes.
4. Bleach in oxalic acid, 5% (5 gm./100 ml. water): 3 minutes.
5. Wash thoroughly in distilled water, several changes: 10 minutes.
6. Impregnate with silver solution in 37°C oven: 15-30 minutes.
Do not expose to bright light. Keep metal instruments out of
solution.
Rinse quickly in distilled water.
Reduce in formalin solution: 3 minutes.
ecincWash in distilled water: 3 minutes.
oe
Gold tone until yellow color turns to purplish grey.
Rinse briefly in distilled water.
Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3 minutes.
|
ae
ee — . Wash
coo
©
Nn in running water: 5 minutes.
Silver Impregnation for Reticulum 181]

14. Counterstain (if desired) in hematoxylin and picro-ponceau (van

Gieson), page 165.
15. Dehydrate, clear, and mount.

RESULTS:
reticulum—black
collagen—red to rose
nuclei—black, blue or brownish
cytoplasm—greyish yellow
muscle fibers, elastin—light yellow
COMMENTS:
To prevent loosening of sections, Mallory (1938) substituted an
alcoholic for the aqueous silver solution of the original method. See
also German (1939).

Gridley’s Method (1951)

FIXATION: any good general fixative.
SOLUTION:
Ammoniacal silver hydroxide:
To 20.0 ml. of 5% silver nitrate add 20 drops of 10% sodium hy-
droxide. Add fresh 28% (reagent) ammonia drop by drop until pre-
cipitate which forms is almost redissolved. Add distilled water up to
60.0 ml.
PROCEDURE:
1. Deparafhinize and hydrate slides to water. Remove HegCl», if
present.
2. Treat with 0.5% periodic acid (0.5 gm./100 ml. water): 15
minutes.
3. Rinse in distilled water.
4. ‘Treat with 2% silver nitrate (2 gm./100 ml. water): 30 minutes,
room temperature.
5. Rinse in 2 changes of distilled water.
6. Impregnate in ammoniacal silver solution: 15 minutes, room
temperature.
~I Rinse rapidly in distilled water.

8. Reduce in 30% formalin (30 ml./70 ml. water): 3 minutes. Agi-

tate gently. .
9. Rinse in 3 or 4 changes of distilled water.
10. ‘Tone in gold chloride (10 ml. 1°% stock solution /40 ml. water)
until yellow brown color has changed to lavender-grey.
182 Silver Impregnation I (cHap. 14)

11. Rinse in distilled water.

12. Fix in 5% sodium thiosulfate (5 gm./100 ml. water): 3 minutes.
13. Wash in running water: 5 minutes.
14. Counterstain if desired.
15. Dehydrate, clear, and mount.

RESULTS:
reticulum fibers—black
other tissue elements—depends on counterstain
COMMENTS:
Gridley uses periodic acid for oxidation in preference to potassium
permanganate and oxalic acid because the latter combination fre-
quently causes the sections to detach from the slides.

Laidlow’s Method (1929)

FIXATION: Bouin’s recommended. Formalin-fixed can be Bouinized 3
days to produce almost Bouin’s results.

SOLUTIONS:
Ammoniacal silver carbonate solution:

In a clean 125 or 250 ml. glass-toppered graduate, dissolve 6 gm. of

silver nitrate in 10 ml. distilled water. Add 115 ml. saturated solution
of lithium carbonate in distilled water. Shake well. Settle precipitate
and pour off solution to leave about 35 ml. of precipitate. Wash well
with distilled water 4 or 5 times. Allow to settle, pour off wash water
and gradually add ammonia, shaking occasionally until fluid is almost
clear. A few grains of precipitate should remain. Add distilled water
to a total of 60 ml. Shake and filter into brown stock bottle. Keeps for
several months.

Gold chloride:
gold chloride stock solution, 1% (1 gm./100 ml.
Walle) Age tt: 7 e Oe 2 en 10.0 ml.
distilled: water’ 7) s ne wareeertoe= 8. PNR eae 40.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCl, if pres-
ent.
2) Oxidize in potassium permanganate, 0.259% (0.25 gm./100 ml.
water): 3 minutes.
3. Wash in running water: 3 minutes.
Silver Impregnation for Reticulum 183

Bleach in oxalic acid, 5% (5 gm./100 ml. water); 3 minutes or

until clear.
Wash in running water: 10 minutes.
Wash in distilled water, 3 changes: 5-10 minutes total.
Impregnate in ammoniacal silver carbonate, 50°C: 5 minutes.
Rinse in distilled water, several dips.
Reduce in formalin, 1% (1 ml./99 ml. water), 2 changes: 3
minutes total.
. Rinse in distilled water, few dips.
. Tone in gold chloride until sections turn purplish erey.
Rinse in distilled water, few dips.
Fix in sodium thiosulfate, 5°% (5 gm./100 ml. water); 3 minutes.
. Wash in running water: 5-10 minutes.
Dehydrate, clear, and mount.
RESULTS:
reticulum—black
collagen—reddish purple
formalin-fixed nuclei—black; cytoplasm—colorless
Bouin-fixed nuclei—colorless; cytoplasm—blackish

Lillie’s Method (1946)

FIXATION: any good general fixative.
SOLUTIONS:
Ferric chloride:

ferric chloride, official solution (29.1%) ....... 1.0 ml.

distilled Water 4:2 %25:5: ee es 49.0 ml.
Ammoniacal silver hydroxide:
Use acid-clean glassware. Place 1 ml. ammonia (28% reagent) in small
flask; add 7—8 ml. silver nitrate 10% (10 gm./100 ml. water) rapidly.
Continue to add silver nitrate drop by drop, shaking between each
addition until a faint turbidity remains (approximately 9-10 ml.
silver nitrate). Dilute resultant solution with equal amount of dis-
tilled water.
PROCEDURE:
L; Deparaffinize and transfer slides to absolute alcohol.
Coat with celloidin; drain; harden in 80% alcohol.
Hydrate slides to water; remove HgCly.
ta) Oxidize
seein in potassium permanganate, 0.5% (0.5 gm./100 ml.
water): 2 minutes,
184 Silver Impregnation I (cuap. 14)

5. Wash in running water: 5 minutes.

6. Bleach in oxalic acid 5% (5 gm./100 ml. water): 2 minutes or
until sections are clear.
7. Wash in running water: 5-10 minutes.
8. Treat with ferric chloride solution: 2 minutes.
9. Wash in running water: 3 minutes.
10. Wash in distilled water, 2 changes.
11. Impregnate in ammoniacal silver hydroxide solution: 3 minutes.
12. Rinse briefly in distilled water.
13. Reduce in formalin, 10%: 2 minutes.
14. Wash in running water: 3 minutes.
15. Tone in gold chloride: few seconds or until color has turned to
purplish grey.
16. Rinse briefly in distilled water.
17. Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3 minutes.
18. Wash in running water: 5 minutes.
19. Counterstain if desired.
20. Dehydrate, clear, and mount.
RESULTS:
reticulum—black
other tissue elements—depends on counterstain
COMMENTS:
Lille warns that hematoxylin counterstaining may not offer enough
contrast with reticulum, because presence of ferric chloride makes the
hematoxylin take black.

Wilder’s Method (1935)

FIXATION: any good general fixative.
SOLUTIONS:
Phosphomolybdic acid, 10%:
phosphomolydicj acid” aera.cn: sels 10.0 gm
distilled: water... fee eee.= <2 ce 100.0 ml.
Uranium nitrate:
WANIUIN: Neate. . 6a eeon Re 1.0 gm.
distilled water) = awa.) Weems
5 |. ia 100.0 ml.
Ammoniacal silver nitrate:
Add ammonia (28% reagent), drop by drop to 5 ml. of 10% silver
nitrate (10 gm./100 ml. water) until precipitate which forms is almost
Silver Impregnation for Reticulum 185

dissolved. Add 5 ml. of 3.19% sodium hydroxide (3.1 ¢m./100 ml.

water). Barely dissolve the resulting precipitate with a few drops of
ammonia. Make the solution up to 500 ml. with distilled water. Use
immediately. Glassware must be acid-clean.
Reducing solution:
GistiMlediwaten’ 22% 1.4 «<0 nate yee eine ee ee 50.0 ml.
HeTenTM AANA eee ee as 2% 4. bots eel. etd ce c 05m:
uranium nitrate, 1 (above), 2.7 seee heeds. },5emal:
Make up fresh each time.
Gold chloride:
gold chloride stock solution (1 gm./100 ml.
PVAUCT ID ashy deh Oy <2 reso 2-4 yt As Me = OLD) sale
Is D WAtET, yo oct u4 3 okey oo bee om 40.0-80.0 ml.

PROCEDURE:
1. Deparafhnize and hydrate slides to water; remove HeCle.
Wash thoroughly in distilled water.
Treat with phosphomolybdic acid: 1 minute.
Wash in running water: 5 minutes.
Treat with uranium nitrate: 5 seconds or less.
Rinse in distilled water.
Impregnate with ammoniacal silver nitrate solution: 1 minute.
MID
Go
GR
Ge1 Dip quickly in 95% alcohol and immediately into reducing solu-
tion: | minute.
9. Wash in distilled water: 2-3 minutes.
10. ‘Tone in gold chloride until yellow colors turn purplish grey.
11. Brief rinse in distilled water.
12. Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3-5
minutes.
13. Wash in running water: 5 minutes.
14. Counterstain if desired.
15. Dehydrate, clear, and mount.

RESULTS:
reticulum fibers—black
other tissue elements—depends on counterstain

COMMENTS:
Phosphomolybdic acid replaces potassium permanganate as an Oxi-
dizer; the former shows less tendency to loosen sections. Sensitization
with uranium nitrate reduces the time and eliminates the heat re-
186 Silver Impregnation I (cHap. 14)

quired by some reticulum methods. Lillie (1946) disagrees that

uranium nitrate is a sensitizer; claims it is an oxidizer.

Reticulum, Collagen, and Elastin. (HUMASON AND LUSHBAUGH 1960)

FIXATION: any good general fixative.
SOLUTIONS:
Ammoniacal silver carbonate:

To 10 ml. of 10.2% silver nitrate (1.2 gm./100 ml. water) add am-
monia (28% reagent) drop by drop until the precipitate, which forms,
is almost dissolved. Add 10 ml. of 3.1% sodium carbonate (3.1
gm./100 ml. water) and distilled water to make 100 ml. Filter.
Buffered formalin:
RO GAOT
HNOCRD beAe oe © ee eR oh 50.0 ml.
sodium carbonate, 1% (1 gm./100 ml. water) .. 1.0 ml.
Orcein solution:
once io eat Siar eee estet. via 0.5 gm.
702%, ethyl alcoholbers. 1-4: aes reid 100.0 ml.
hydrochloric acid, concentrated). < 4.43048 23% 3. 0.6-1.0 ml.
Aniline blue solution:
Aniline blue IVS; Gl 27 30 eeetas. sh. ae 0.1 gm.
OncalRER ACN ger ep atyin hart a am Reo occ, a a 2.0 gm
phesphomolvibdiclacid seems os 5'sLe ee 15.0 gm.
Gastilled. wWatele ot25 << se aE Sos5.2 Seep See 300.0 ml.

PROCEDURE:
1. Deparaffinize and rinse off xylene in absolute alcohol.
2. Pyridine-glycerol (2:1), room temperature: overnight, or 15%
aqueous pyridine: 15 minutes.
3. ‘Treat with 95% alcohol: 3 minutes.
4. Wash in running water: 5 minutes. Remove HgCl, if present.
5. Treat with periodic acid, 0.5% (0.5 gm./100 ml. water): 15
minutes.
op) Wash in running water: 5 minutes; rinse in distilled water.
~J. Impregnate with ammoniacal silver carbonate solution, 37°C:
].5—2.0 hours.
8. Rinse in ammoniated distilled water (1 drop ammonia/100 ml.
water) a few seconds to remove excess silver precipitates. Rinse
in distilled water.
9. Reduce in buffered formalin: 5 minutes. Use fresh solution.
Silver Impregnation for Reticulum 187

10. Wash in distilled water: 2-3 minutes.

Qo

11. ‘Tone in gold chloride (10 ml. 1% stock/80 ml. water) until yel-
low color turns purplish grey.
12. Rinse briefly in distilled water.
13. Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3 minutes.
14. Wash in running water: 5 minutes; rinse in 70% alcohol.
15. Stain in orcein solution, 37°C: 15 minutes, or at room temper-
ature: 1 hour.
16. Rinse in 70% alcohol, followed by distilled water.
17. Treat with phosphomolybdic acid, 5% (5 gm./100 ml. water):
10-15 minutes.
18. Briefly rinse in distilled water.
19. Stain in aniline blue solution: 2—3 minutes.
20. Rinse off some of blue in distilled water, and transfer to acidu-
lated water (1 ml. glacial acetic acid/100 ml. water): 5 minutes or
longer.
21. Dehydrate, clear, and mount.

To modify with the addition of orange G, proceed after step 19 with

a rinse in distilled water and continue as follows:
20. Stain in orange G (G.I. 16230) 2% (2 gm./100 ml. water): 1-2
minutes. Extended time dulls aniline blue.
21. Rinse in distilled water and treat with acidulated water.
22. Dehydrate, clear, and mount.

RESULTS:
reticulum—black
elastin—red
collagen—blue
colloid—brown or grey
nuclei—brown-black
erythrocytes—orange
smooth muscle—beige
striated muscle—grey-blue or mauve
epithelium—light grey-brown
COMMENTS:
The orange G can be added to the aniline blue solution but separate
solutions offer better individual control of each color intensity. If
sections tend to loosen, follow step 14 by dehydrating slides to abso-
lute alcohol, apply a coat of celloidin, harden it in 70% alcohol and
proceed to step 15.
188 Silver Impregnation I (cHAP. 14)

The method was developed for use in pathological problems con-

cerning vascular invasion and capsular infiltration by carcinoma and
degenerative diseases of the connective tissue and blood vessels.
Any of the other silver impregnation solutions and methods for
reticulum may be substituted for the above step 7.
 Chapter 15

Silver
Impregnation II
NEUROLOGICAL TECHNICS

Briefly, the neurological elements may be grouped as follows:

]. SUPPORTING ELEMENTS; the neuroglial fibers and cells, also some con-
nective tissue
a. Astrocytes—support the neurons
b. Oligodendroglia—help to form and maintain the myelin
c. Microglia—part of the defense system of the nervous system
+. NERVE CELL BODIES (neurons), including various cytoplasmic struc-
tures
a. Nissl’s granules
b. Neurofibrils, fine cytoplasmic fibers
c. Mitochondria and Golgi (sometimes present; see p. 259)
d. Melanin and lipofuscins (see p. 241)
3. NERVE FIBERS (axons and dendrites)
a. Myelin—a glistening fatty material that coats the axons in the
outer layer of the developing spinal cord
4. NERVE ENDINGS
a. Pericellular end bulbs, sensory
b. Motor end plates
When impregnating tissues from the central nervous system certain
facts should be kept in mind. If the hydroxide method is used, astro-
cytes and microglia are not specially impregnated. If the carbonate
method is used, the opposite effect takes place. Use body temperature
for impregnation, not hot solutions. For nervous tissue, frozen sections
iat a
190 Silver Impregnation II (cHav. 15)

actually are best; the alcohol and xylene embedding processes may
remove lipids, which can be an essential part of the tissue. Lipid extrac-
tion may result in no impregnation of oligodendroglia and weakened
microglia, since their impregnation depends on lipid complexes. Also,
periodic and chromic acid oxidation weaken the reaction of oligoden-
droglia and microglia. (The reverse is true for connective tissue, when
pyridine, periodic, or chromic acid treatment is desirable to suppress
nervous tissue elements from confusing the picture.)
Neurological technics, as perhaps has become evident, so often neces-
sitate highly specialized methods that many technicians prefer to avoid
them. Precise attention to all details, however, can produce beautiful
and exciting slides. Carefully follow directions for fixation—the solu-
tion composition and the duration of fixation, and whether fixation is
or is not followed by washing. Always wash in distilled water unless tap
water is specified. For making silver and other special solutions use
double (glass) distilled water if possible and clean glassware—cleaned
in cleaning solution (page 412), washed well in running water, and
rinsed 4 or 5 times in distilled water. All chemicals should be at least
reagent grade. Use no corks in containers and no metal instruments in
silver solutions. If in doubt about the age of solutions, make fresh ones.
If, during toning with gold chloride, the tissue retains a yellow or
brownish hue, the gold chloride is weakened. Prepare a new solution.
Sometimes artifact precipitates are difficult to avoid, but strict adher-
ence to procedure details will reduce them to a minimum.
Embedding and sectioning will depend on the impregnating or
staining technic to follow. Paraffin and nitrocellulose methods are used
at times, but as mentioned above frozen sections usually are more satis-
factory. Section thickness frequently is thicker than for other tissues —
7 to 20 microns, or even more.
When fixing an entire brain, do not allow it to rest on the bottom
of the container. Carefully insert a cord under the circle of Willis on
the underside of the brain and support the two ends of the cord on the
sides of the container. The brain, hanging upside down, should be free
of the bottom of the vessel but remain completely submerged in fixative.
The spinal cord can be supported in a graduated cylinder filled with
fixative. Run a thread through one end of the spinal cord and tie the
thread around an applicator stick supported on the edges of the cylin-
der. For a perfusion method for the brain, see pages 22.
Neurological Staining io

Note: In neurological technics the methods sometimes employ section-

staining and sometimes employ block-staining. The letters $ and B will
be used in the headings of this chapter to indicate which type of staining
is being described.

Neurological Staining

Heidelberger’s Victoria Blue (MODIFIED BY PROESCHER, 1934) S

FIXATION: 10% formalin.

PROCEDURE:
. Cut frozen sections, 10—15 microns.
Place sections in distilled water. If not stained at once they may
be stored in 10% formalin for 24 hours, but no longer.
Stain in Victoria blue B, C.1. 44045, saturated aqueous solution:
12-24 hours.
Wash rapidly in distilled water and mount on albumen-coated
slides. Blot with filter paper and dry in air.
Expose to ultraviolet light: 30 minutes.
Immerse in N/20 iodine solution (aqueous): few seconds.
Remove from iodine, blot, air dry for 10 minutes.
Differentiate in xylene-aniline (1:1), clear in 2 or 3 changes of
xylene, and mount.

RESULTS:
glia cell bodies and fibrils—light to deep blue
nerve cell bodies, dendrites, and axis cylinders—faintly stained

COMMENTS:
1. Staining time can be shortened to 30-35 minutes at 56°C.
2. Proescher experimented with oxidizing agents—potassium chro-
mate and dichromate and hydrogen peroxide—and found they
could be substituted for ultraviolet. Potassium dichromate was
most successful and can be used as a 0.59% aqueous solution for 30
minutes.
3. Victoria blue may be replaced by methyl violet 2B, ethyl violet, or
crystal violet.
192 Silver Impregnation II (cuap. 15)

Nassar and Shanklin’s Silver Impregnation (1951) S

FIXATION: 4 days in:
FORMA echt date. 7 3 2 ae. 70.0 ml.
aniMOnior bromides: -) . 22)AZo em eo . - 14.0 gm.
austilled* water 28..208.
20". eee 680.0 ml.

WASHING, EMBEDDING, and SECTIONING:

Il. After fixation, wash tissue blocks in distilled water
plus strong
ammonia (50 drops ammonia to 100 ml. water): 10 hours. Keep
container covered to prevent escape of ammonia.
Wash in 2 changes of distilled water: 1—2 hours each.
. Dehydrate, clear, and embed in paraffin. Cut sections at 10-15
microns and prepare slides.

SOLUTION:
Ammoniated Silver (Lillie, 1946):
To 1 ml. of concentrated ammonia rapidly add 7-8 ml. of 10%
silver nitrate, aqueous. Continue to add 10% silver nitrate drop by
drop, shaking each time, until only a faint turbidity remains (9-10
ml .). Dilute with an equal amount of distilled water.

PROCEDURE:

. Deparaffinize and hydrate to 3 changes of distilled water: 1-2

minutes each.
Sensitize in 5% sodium sulfite (5 gm./100 ml. water): 2 hours.
Transfer quickly through 3 changes of distilled water.
Impregnate in ammoniated silver solution: 2—5 minutes, room
temperature.
Dip in distilled water: 1—2 seconds.
. Reduce in 2% formalin (2 ml./98 ml. water): 1 minute. Agitate
gently, and change formalin solution 3—4 times.
~I. Wash in distilled water: 1 minute.
. Tone in gold chloride (1 gm./500 ml. water): few seconds to |
minute, or until section turns grey.
Ke) Fix in 5% sodium thiosulfate (5 gm./100 ml. water): 1-2 min-
utes.
10. Wash in running water: 5 minutes.
le Counterstain if desired. Dehydrate, clear, and mount.

RESULTS:
neuroglia dark grey to black
Astrocytes 193

COMMENT:
del Rio-Hortega’s silver carbonate solution also can be used in this
method.

Astrocytes
Ramon y Cajal’s Gold Chloride Sublimate Method (MALLory, 1938) S
FIXATION: Fix thin slices of tissue in:
POVIM AMMAN Cee ee ce von tn oan gt aad AG GR 15.0 ml.
ammonimny bromide \ >... ..4.mee
Seria es ae 2.0 gm.
Gastilled: water oe 6 feed. . ood 6p oes otiel e 85.0 ml.

27°C? L day
SECTIONING: frozen sections, 15 microns thick.

SOLUTIONS:
Gold chloride sublimate solution:

mercuric chloride crystals (not powder) ....... 0.5 gm.

poldichloride, 19% aqueous ......5...2 45244 ie 6.0 ml.
distilled water 2 ois ocho oe pow aca, Ree ee ee 35.0 ml.

Prepare fresh, using acid-clean glassware. Pulverize mercuric chlo-

ride; add to distilled water. Heat gently. When dissolved, add gold
chloride, and filter.
del Rio-Hortega’s carbol-xylol-creosote mixture:
CYEOSOLC 28 rere tee ede, os omg 1th Se 10.0 ml.
phenol (canbolic acd), melted ...... 4:425a5ece 10.0 ml.
RPMI Sree macs ama Was Gt in <b >be SE 80.0 ml.

PROCEDURE:
1. Place sections in 1% formalin.
2. Wash quickly in distilled water, 2 changes.
©9 Place in freshly prepared gold chloride (in acid-clean glassware).
Flatten sections, keep in dark at room temperature: 4—6 hours.
When sections appear purplish, examine for astrocytes.
4. Wash in distilled water: 5-10 minutes.
5. Fix in 5% sodium thiosulfate (5 gm./100 ml. water): 5-10 min-
utes.
6. Wash in distilled water, several changes.
Float on slide, dehydrate with 95% alcohol.
Loe Silver Impregnation II (cuar. 15)

8. Clear with del Rio-Hortega’s solution.

9. Blot and mount.
RESULTS:
astrocytes and processes—black
background—unstained or light brownish purple
nerve cells—pale red
nerve fibers—unstained
COMMENTS:
Extra sections after being fixed in hypo can be preserved in 1%
formalin for long periods.

Oligodendroglia and Microglia

Penfield’s Modification of del Rio-Hortega’s Silver Carbonate Method
(MCCLUNG, 1950) S
FIXATION: 10% formalin at least one week; longer fixation also will
give excellent results, or fix in:
101g
07 1) 2 i ener ay Oi ip a x 14.0 ml.
aMMORIM PLOMIde. 22. Ree.
. ce te 2.0 gm.
distilled water (4) 4... SFUUae.
S|) a ite 86.0 ml.

SECTIONING: frozen sections, 20 microns.

SOLUTIONS:
Globus’ hydrobromic acid:
4077, NyGQropromic, aAciG. 2 <omngeee sf: ee 5.0 ml.
GistilledsWatel fo. vos. pee tos ls leew 95.0 ml.

Silver carbonate solution:

Combine 5.0 ml. of 10% silver nitrate and 20.0 ml. of sodium car-
bonate. Add ammonium hydroxide, drop by drop, until precipitate is
just dissolved. Add distilled water up to 75 ml. Filter. The solution
keeps for long periods if stored in a dark bottle.
PROCEDURE:
1. Place sections in 1% formalin or distilled water.
2. Transfer to distilled water plus 1% of ammonia. Cover to prevent
escape of ammonia, and leave overnight.
3. Transfer to hydrobromic acid, 38°C: 1 hour.
4. Wash in 3 changes of distilled water.
Nissl Substance 195

5. Mordant in 5% sodium carbonate (5 gm./100 ml. water): 1 hour.

‘Tissue may remain in this solution 5—6 hours with no ill effect.
6. Impregnate in silver solution: 3—5 minutes or until sections turn
a smooth grey when transferred to reducer. Try single sections
at 3 minutes, 5 minutes, or longer.
7. Reduce in 1% formalin. Agitate during reduction.
8. Wash in distilled water.
9. ‘Tone in gold chloride (1 gm./500 ml. water) until bluish grey.
10. Fix in 5% sodium thiosulfate, 3 minutes.
11. Wash in running water: 5 minutes. Dehydrate, clear, and mount.
RESULTS:
oligodendroglia and microglia dark grey to black
COMMENTS:
If only the oligodendroglia are to be shown, cut fixation time down
to 2 days. Long fixation tends to increase the staining of the microglia
and astrocytes.
Procedure for oligodendroglia:
1. Follow fixation by treatment with 95% alcohol: 36 & 48 hours.
2. Wash, freeze, and cut sections.
©9 Stain in a stronger silver solution by diluting the above solution to
only 45 ml.:
15 minutes or more, until the sections begin to turn
brown.
4. Wash for a few seconds in distilled water.
5. Reduce with agitation, wash, and tone as above.
6. Dehydrate, clear, and mount.

RESULTS:
oligodendroglia—black

Nissl Substance
Cresyl Violet (KELLER, 1945) S
Nissl substance is characteristic of fixed nerve cells. It is found in a
granular form distributed through the cytoplasm and stains_bril-
liantly with basic aniline dyes.
FIXATION: Bouin’s recommended; others are satisfactory; Zenker’s not
recommended.
SECTIONING: Paraffin method, 10 microns; thinner sections of no ad-
vantage.
196 Silver Impregnation II (cHap. 15)

SOLUTIONS:
Cresyl violet:
CLESYI VIOLEL-ACCLALE B82) a oy 0.5 gm.
eIstitled: Water-coe See ete)... See.«athe « 100.0 ml.
Resin-xylene:
MOUNTING, FESiMes Aa Goss See See 50.0 ml.
xylene! 4 Ase pene: tae ft) eee deel |. 50.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate to water; remove HgClo.
Stain in cresyl violet: 3-5 minutes.
Rinse in distilled water, 2 changes: few seconds each.
Dehydrate in 95% alcohol: 30 seconds.
Dehydrate in absolute alcohol: 30 seconds.
Transfer to xylene: 1 minute.
Treat with resin-xylene: 2 minutes.
GoID Differentiate in absolute alcohol: 10-30 seconds. Check
10
go
OCR under
microscope.
9. Clear and mount.
RESULTS:
Nissl substance—purple

COMMENTS:
Steps 7, 8, and 9 may be repeated several times until desired differen-
tiation is reached.
Banny and Clark (1950) recommend Coleman and Bell for the best
cresyl violet for Nissl staining.

Thionin (CLARK AND SPERRY, 1945) S

FIXATION: Bouin’s recommended; other’s are satisfactory; Zenker’s not
recommended.
SECTIONING: 10 microns; thinner sections of no advantage.
SOLUTIONS:
Lithium carbonate, 0.55%:
inchium).car bonaté- gs -2..s.ctas seers ot ee 5. Ti.
distilled: water, acs. wis. ae ee eb Oe 1000.0 ml.
Thionin:
thiomim Ais 52000. y290 se AN ad ee 0.25 gm.
0:069ss thiuny carbonate ft. Me oer ses tile oe: 100.00 ml.
Neurofibrils 197

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Treat with lithium carbonate solution: 5 minutes.
3. Overstain in thionin: 5—10 minutes.
4. Rinse in distilled water.
5. Dip in 70% alcohol: few seconds.
6. Dehydrate in butyl alcohol, 2 changes: 2—3 minutes in each.
7. Clear and mount.

RESULTS:
Nissl substance—bright blue
COMMENTS:
If differentiation is necessary, briefly rinse slides in 95% ethyl alcohol
(following step 5) and place in aniline, then in lithium carbonate
saturated in 95% alcohol. Proceed to step 6.

Gallocyanin (EINARSON, 1951) §

FIXATION: any general fixative.
SECTIONING: 10 microns.

SOLUTION:
Pallocyanin S24, « odd casts oan ee 2 2 0.15 gm.
chrome alum (chromium potassium sulfate) |. . 5.0 gm.
distilled! water 25642 2s. aks Fie ee Ae 100.0 ml.

Dissolve chrome alum in warm water, add gallocyanin and _ boil

gently for 10 minutes. Keeps well for about | week.
PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCly.
2. Stain in gallocyanin: overnight.
3. Wash in running water: 5 minutes.
4. Dehydrate, clear, and mount.
RESULTS:
Niss] substance—blue

Neurofibrils
Ramon y Cajal’s Method (MopIFIED FROM FAVoRSKY, 1930) B
FIXATION: cut slices perpendicular to organ surface, about 5 cm. thick.
Place in 70% alcohol, plus .5% glacial acetic acid: 6 hours.
198 Silver Impregnation II (cHAP. 15)

PROCEDURE:
l. Transfer to 80% alcohol: 6 hours.
2 Treat with ammoniacal alcohol: 24—36 hours.
(a) For cerebrum, cerebellum, spinal cord, or ganglia add 4 drops
of ammonia to 50 ml. of 95% alcohol.
(b) For medulla, add 9 drops of ammonia to 50 ml. of 95%
alcohol.
. Wash in distilled water, several changes, until pieces sink.
. Treat with pyridine: 1—2 days.
~~

. Wash in running water: overnight, followed by distilled water,

several changes.
. Blot on filter paper and place in relatively large volume of 1.5%
silver nitrate, aqueous (1.5 gm./100 ml. water): 5 days in dark,
go-G:
. Rinse in distilled water and place in following fluid: 24 hours.
Ramon y Cajal’s reducing fluid:
pyrogallic acid or hydroquinone ............. 1.0 gm.
clus tulle Gewallet 05. 2b asc oe ae ies os 100.0 ml.
neutnal formal 20422 +66 eee ols 15.0 ml.

. Rinse in distilled water, several changes over a period of at least

1 hour.
. Dehydrate and embed in paraffin, celloidin, or double embed.
. Make sections perpendicular to surface of organ and about 15 or
more microns thick.
ll. Afhx to slides, dry, remove paraffin with xylene and mount. Lay
celloidin sections on slides and mount in resin.

RESULTS:
neurofibrils—black
background—brownish yellow

Nerve Cells and Fibers

Golgi’s Rapid Method (mMALLory, 1938) B
FIXATION: fix small pieces in:
potassium dichromate, 2.5% aqueous ......... 40.0 ml.
osmile acid, LY, saqueons :4 traits).
Sede te 10.0 ml.

Place cotton in bottom of container so fixing fluid has access to all

sides; 1-7 days. Renew fixative if it becomes turbid.
Nerve Cells and Fibers 199

STAINING:
1. Dry pieces of tissue with filter paper, place in 0.75% aqueous silver
nitrate (0.75 gm./100 ml. water): 24-48 hours. If solution turns
yellow, renew it.
2. Transfer to 40% ethyl alcohol, several changes: 1—2 hours.
3. Transfer to 80% and 90% alcohol: 1 hour each.
EMBEDDING and SECTIONING:
1. Dehydrate in absolute ethyl alcohol: 12 hours.
2. Transfer to absolute alcohol-ether (1:1): 2—4 hours.
3. Infiltrate with 4% celloidin or nitrocellulose: 1—2 days.
4. Embed and cut thick sections, 20-25 microns, or more if desirable.

MOUNTING:
1. Wash sections carefully in 80% alcohol to remove excess silver.
2. Dehydrate in absolute alcohol: few minutes. (Add few ml. chloro-
form to absolute alcohol to prevent loss of nitrocellulose.)
oo. Clear in clove oil, terpinol, or the like.

4. Mount on slides, press with filter paper. Add a drop of thick

mounting medium. Warm to drive off solvent and just before resin
cools, add cover glass.

RESULTS:
nerve cells and processes—deep black
background—light yellow
COMMENTS:
If tissue has been fixed in 10% formalin, blot off excess formalin and
chromate for 1—2 days, room temperature, in one of the following
fluids (Davenport and Combs, 1954).
]. potassium dichromate, 5% aqueuos ....... 100.0 ml
glacial acetic acid ...... memereeres2)! 0.15 7Pak 6.9m.
2. ine or cadmium chromate... .. 4epzuahay: 1.5 gm.
distilled Water cos). giee a « ao Os sy fylmee mene eos 25.0 ml.
placial acetic ACIG \)..6 5,026 6s «12> Dea eee © 6.0 ml.
when chromate has dissolved, add:
potassium dichromate, 59% aqueous ........ 65.0 ml.
5»; poOtassim, dichromate, :.. 0. «+ «3 ate Been 3.0 gm.
potassium acetate, anhydrous ............. 1.8 gm.
Gistilled Swateiy ses oii. eons {54s cnt geen es case 100.0 ml.
4, A. cadmium nitrate (4H,0) or zinc nitrate
(OTLOt ox hen... S > 98 yt a eRe, - 3.0 gm.
a@istulled-water dg cs, oe ee eee 25.0 ml.
200 Silver Impregnation II (cHAP. 15)

B. potassium dichromate, 5% aqueous ..... 65.0 ml.

glacialgacetre*acicdl ! 1/4) Weeds?)
SELPRe. 6.0 ml.
Mix A and B.

Remove tissues from any of above fluids, blot and place immediately
in silver solution: 18-24 hours. Proceed into step number 2 above
(under Staining).
Moliner’s Modification (1957)
FIXATION: fix 3-5 mm. pieces, 3 days, change daily.
potassium dichromate, 6% aqueous .......... 40.0 ml.
potassiumchlorate, 5%, aqueous) Were.)
: «2. =A 20.0 ml.
chilorall hydrate, 209, "aqueotisitiawaee
ot: . ake 30.0 ml.
fOFMaliM: SVORTo. OF teselieal ey | RPE ss fees 10.0 ml.

STAINING:
1. Transfer tissue blocks to 3% aqueous potassium dichromate: 3
days, change twice a day.
ho. Transfer to 1% aqueous silver nitrate (lgm./100 ml. water): 3
days, room temperature.

SECTIONING AND MOUNTING:

1. Cut frozen sections, 50-100 microns. Sections can be kept in abso-
lute alcohol until mounted.
2. Mount on slides and clear. Synthetic mountants can be used with
a cover glass. (Before synthetic mountants were developed, Golgz
preparations had to be mounted in balsam without a cover glass.)

Ramon y Cajal’s Pyridine-Silver Method (MODIFIED BY DAVENPORT ef

al., 1934) B
FIXATION: as soon as possible in:
apsoluexethiyl caleohol @ ic sceeesees
2. 5. ces 98.0 ml.
AMiinOlia:, CONCeMtrated i. peeeet
so .. eee ees oe 2.0 mal:

Fix for 1-6 days, preferably no longer.

PROCEDURE:
1. Treat tissue blocks in 5% aqueous pyridine: 24 hours. This is
recommended; but Davenport e¢ al. say it can be optional.
2. Wash in distilled water: 2—6-hours; change every half hour.
3. Impregnate in 1.5—2.0% aqueous silver nitrate (1.5-2.0 gm./100
ml. water), 37°C: 2 to 3 days, or longer depending on size of tissue
blocks. A minimum time yields the best differentiated tissue. The
Nerve Cells and Fibers 20]

longer the tissue is left in silver nitrate, the greater the tendency
for everything to stain. This time will have to be determined by
trial and error.
4. Wash in distilled water: 20 minutes to 1 hour. This 1s a critical
step. The amount of silver washed from the tissue depends on
whether the water is changed often or whether the tissue is shaken
in the water. Change every 10 minutes or use a large volume of
water and shake every 10 minutes. Equal staining of the periphery
as well_as central parts of the tissue determines correct washing.
A light periphery indicates too much washing.
5. Reduce in 4% aqueous pyrogallol solution: 4 hours.
6. Dehydrate, clear, embed, and section.
7. Mount on slides, deparaflinize, and clear. Add mountant and cover
elass.
RESULTS:
nerve cells—yellow to brown
neurofibrils—brown to black
axis cylinders of myelineated fibers—yellow to brown
axis cylinders of nonmyelinated fibers—black
COMMENTS:
If the preparation tends to be too light, reduce the time in pyridine
and washing.
If the preparation is too dark, omit the pyridine and wash 48 hours
after fixation, or reduce the concentration of the silver nitrate solu-
tion and wash longer between impregnation and reduction.

Bielschowsky Method (MopIFIED By DAVENPORT ef al., 1934) B

FIXATION: 10°% formalin: 2 days. For embryos add 0.5% of trichlor-
acetic acid to 10% formalin.

SOLUTION:
Ammoniated silver:
Add 5.0 ml. of concentrated ammonia to 40.0 ml. of 2% sodium hy-
droxide (aqueous). Mix well. Add slowly from a burette or pipette
with shaking 8.5% aqueous silver nitrate until an opalescence re-
mains in the solution (about 40.0 ml.). Add 0.5-1.0 ml. of ammonia.
Dilute with about 5 parts of distilled water. (Dilution is not critical.)
PROCEDURE:
1. After fixation, wash for | hour.
2. Transfer to 50% aqueous pyridine: 1-2 days.
202 Silver _Impregnation IT (cuar. 15)

3. Wash in distilled water: 2-6 hours depending on size of block.

Change every half hour.
4. Impregnate with I—-1.5% aqueous silver nitrate (1.0-1.5 gm./100
ml. water): 3 days, 37°C.
5. Wash in distilled water: 20 minutes to 1 hour, depending on size.
Change every 10 minutes or use a large volume of water and
shake every 10 minutes. Periphery and central portions must be
equally stained.
6. Impregnate in ammoniated silver solution: 6—24 hours.
7. Wash in distilled water: 15 minutes to 1 or 2 hours, depending
on size.
8. Reduce in 1% formalin: 6—12 hours.
9. Wash in running water: 10-15 minutes.
10. Dehydrate, clear and embed. Section and mount.
11. Deparaffinize, clear, and cover.

Alternate method, for gold toning:

(a) Hydrate sections to water.
(b) Gold tone and fix.
(c) Dehydrate, clear, and mount.
RESULTS:
nerve fibers, neurofibrils—brown to black (no gold toning)
—erey to black (with gold toning)

Fluorescent Method (zEIcER et al., 1951) S

FIXATION: 95% ethyl alcohol.
SECTIONING: paraffin method.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2.
Se) Stain in 0.1% acridine orange (C.1. 46005): 6 minutes.
3. Differentiate in 95% ethyl alcohol: 2 seconds.
Ds . Blot with filter paper and mount in fluorescent mountant.

RESULTS:
unmyelinated fibers—bluish gray
myelinated fibers—brownish orange
COMMENTS:
Fresh tissue can be cut frozen and stained in acridine orange made
up in physiological saline (page 410) or in Ringer’s solution (page
411).
Nerve Fibers and Endings 203

Nerve Fibers and Endings

Bodian Method (1936, 1937) S
FIXATION: Bodian recommends:

HOTA AME Meta Wap, os oa.od ame She Phe He 5.0 ml.
PlaGialkaCehe ACIGi 5). 5.4. sna adie ate kao 5.0 ml.
COrZ sethyl alcohol. i ws. 4 us neque pas He a 90.0 ml.

Not suitable are chromates, chromic and osmic acid, or mercuric

chloride. 10% formalin causes excessive staining of connective tissue.
SECTIONING: paraffin method.

SOLUTIONS:
Protargol solution:
|
U0 6) A a cree
ot 0 a Rm 1.0 gm.
distilled! water «ccandd
«> >os yea wan Bho cwen 100.0 ml.

Sprinkle Protargol on surface of water in a wide dish or beaker. Do

not stir, this is critical. Keep on hot plate at 37°C until Protargol is
dissolved.

Reducing solution:
NY GNOCMINONE 222.0 ob ta.c . sm aocne « call ne
OVCHUUDCOUR. FUL
0.Ct i. a nner
aii <- CeCe 5.0 gm.
dustilled fwater 2s. 2.65. ¢4 hc one on nee be bee

Gold chloride:
POLCCMOTI 5.gts kw Sapa. 6.8 a soa, 1.0 gm.
MistilGd Water ...v Aacucacse
< ae. a ac a0 Pe 100.0 ml.

Acidified water:
Placa aCe ACG jae: ¥i.o. Ge. oo... SR ae 0.5 gm.
Gistillediwater, v< - c ccoxsccteial o 4 wis.sv. ds oo eee 100.0 ml.

PROCEDURE:
1. Deparafhnize and hydrate slides to water.
2. Impregnate in Protargol solution plus 5 gm. clean copper (wire,
shot or sheet, cleaned with | part HNO3/3 parts HCl) to 100 ml.
of solution, 37°C: 12—24 hours.
1S) Wash in distilled water, several changes.
4. Reduce: 5—10 minutes.

* Roboz Surgical Instrument Co., Washington, D.C,

204 Silver Impregnation II (cHAP. 15)

5. Rinse in distilled water, 3 changes: 1 minute total.

6. ‘Tone in gold chloride: 4 minutes.
7. Rinse in distilled water, 3 changes: | minute total.
8. Develop in 2% oxalic acid (2 gm./100 ml. water). Check under
microscope until background is grey and fibers are sharply de-
fined: approximately 3 minutes.
9. Wash in distilled water, 3 changes: 1 minute each.
10. Fix in 5% sodium thiosulfate (5 gm./100 ml. water): 5 minutes.
11. Wash in running water: 5-10 minutes.
12. Rinse in distilled water.
13. Counterstain if desired.
14. Treat with acidified water: 5 minutes.
15. Dehydrate, clear, and mount.

RESULTS:
nerve fibers—black
background colors—depending on counterstain
COMMENTS:
Foley (1943) considers counterstaining essential, claiming that it
serves as contrast for nervous and non-nervous tissue and adds to
transparency of the sections. Follow step 11 with:
2 Stain in gallocyanin (page 138): overnight.
or Wash thoroughly running water: 5-10 minutes.
14. Mordant in 5% phosphotungstic acid (5 gm./100 ml. water): 30
minutes.
13x Transfer directly to dilution (20 ml./30 ml. water) of following
stock solution: | hour.

amtline blue WS. CAL 42780reee ro... cose 0.1 gm.

taster cee, Cols 42g. sarees ein). ee 0.5 gm.
onange JG, ION VO2ZG0) sic! pees Geos + ss, 1g eee 2.0 gm.
GU STU DCO 21 Ce eget RENE oy, of, Nena 92:0rmil:
eilacial acetic AGid! x. ieee css 5 eae: 8.0 ml.

16. Differentiate through 70% and 95% alcohols.

Wie Dehydrate, clear, and mount.

Ungewitter’s Modification (1943)

PROCEDURE:

le Deparaffhinize and hydrate sections to water.

92 Treat with Protargol solution (plus copper), 37°C: 12-24 hours.
Nerve Fibers and Ending 205

3 . Rinse in distilled water, 2 changes.

4 . Reduce: 5-10 minutes, agitating for first minute:
AREY 2 oe aaene ee ae 0.2 gm.
sodium sulfate, anhydrous. 42 seh.
ses aoa dee = 10.0 gm.
Nya nGqUuInONe. 4 oh jz.5 5 .<oeteherneies eee 0.5 gm.
SOGMIMBDOT ALE. 2. s3onii3 -<2:59 Ab SES eRe SE 3:tO) 0.1 gm.
Gisciled “Water 2 cts s vac ceeda ees bend oes wee 100.0 ml.

Wash in distilled water, 4—5 changes.

Treat with silver nitrate, 1% aqueous (1 gm./100 ml. water):
10-20 minutes, room temperature or 37°C.
Wash in distilled water, 2—3 changes.
CO . Reduce (step 4 above).
Wash in distilled water, 4 changes. Examine. If nerve fibers show
distinct differentiation, dehydrate, clear, and mount. If not well
differentiated, repeat steps 6, 7, 8, and 9 until desired results are
reached.
10. Toning may be done in 0.2% gold chloride (0.2 gm./100 ml.
water) until greyish in color. Fix, dehydrate, clear, and mount.

Chloral Hydrate-Silver Method (NoNwwEz, 1939) B

FIXATION: 24 hours or longer (up to 3 days):
chloral hydrate) Ac, co. ba05< aes eee eee 25.0 gm.
BOY, -ciliyl MGOROl 14 a eet. te bn ees 100.0 ml.
PROCEDURE:
1. Wipe off excess fixative and put blocks in:
9peZ ethylalcohWols, 82 ¢4..4%- .. sama eiss 60.0 ml.
ammonia, concentrated .......<0:.+.ss4es44
ea: 4 drops

Leave for 24 hours, but change at least once, particularly if blocks

are large or contain a large amount of fat.
2. Rinse in distilled water: 5 minutes.
3. Place in 2% aqueous silver nitrate (2 gm./100 ml. water) in dark,
37—40°C: 5-6 days. Replace silver solution after 2 days, or as soon
as it becomes brownish in color.
4. Rinse in distilled water: 2—3 minutes.
5. Reduce for 24 hours in:
PNROSANOL sc cnWe tee fea Y ict decedent 2.5-3.0 gm.
formalin Deter da, BMA leBro ae a Ave’a 2 ce Ss 8.0 ml.
distilled water grma> & & this «0 ioctl eee 100.0 ml.
206 Silver _Impregnation II (cHar. 15)

. Wash in several changes of distilled water: 2—3 hours.

. Transfer to 50% alcohol, change twice over a period of 24 hours.
. Dehydrate, clear, and embed.
. Section, mount on slides, deparaffinize, clear, and cover.

RESULTS:
nerve fibers and endings—brown to black
COMMENT:
Gold toning does not improve impregnation.

Pericellular End-Bulbs (or Boutons)

Chloral Hydrate-Silver Method (DAVENPORT, 1933) B
FIXATION: Perfusion (page 22) with 10% chloral hydrate is a desirable
procedure, followed by fixation in 10% chloral hydrate: 1—2 hours.
(After perfusion cut whole organ into tissue blocks, 5-8 mm. thick.)
PROCEDURE:
In Treat tissue blocks with 20-40% pyridine (aqueous): 2 days.
2. Wash in distilled water: 1-2 hours, no more. Change water every
half hour.
3. Impregnate with 1.5% aqueous silver nitrate (1.5 gm./100 ml.
water): 3—7 days. (Time is not critical within these limits.)
4. Wash in distilled water: 20-30 minutes, change water once.
5. Reduce in 2% aqueous pyrogallol (2 gm./100 ml. water): 5 hours.
6. Dehydrate, clear, and embed.
7. Section (15—20 microns) and mount on slides.
8. Deparaffinize and gold tone if desired, or clear and cover.

RESULTS:
nerve fibers and endings—black
cells pale yellowish brown
COMMENTS:
Davenport suggests that the chloral hydrate is perfused into the aorta
and can be pumped through the system for about 1-3 minutes before
the heart stops.
The washing after the silver impregnation is critical. If not suffi-
ciently washed the periphery will be overstained. One-half hour
leaves the superficial as well as the central zones well stained. Five
hours of washing produces pale staining and leaves the end bulbs
unstained. Cat tissue gave excellent results.
Pericellular End-Bulbs (or Boutons) 207

Nauta and Gygax Method (1951) B

FIXATION: 10% formalin: 2 weeks to 6 months.

SECTIONING: frozen sections, 15-20 microns.

SOLUTIONS:
Silver solution A:

UVTI AMAL CoDySoothes, £ 2.c.0g clea eee OF 2 ie 1.5 gm.

Gistuiledtw acer (2.5 h Pile. 2c. Sat en Ree a Sa 100.0 ml.
Poy CU Greg 2k thn cht yon ee ee Ree a eee 5.0 ml.

Silver solution B (ammoniated silver):

Dissolve 0.45 em. silver nitrate in 20.0 ml. distilled water. Add 10.0
ml. of 95% ethyl alcohol. From calibrated pipettes add 2.0 ml. am-
monia (concentrated) and 2.2 ml. of 2.5% sodium hydroxide. Mix
thoroughly and keep the container covered to prevent escape of am-
monia.
Reducing solution:
10g, ethyl alconol osc 2. : 2.4 ieeeee 45.0. ml.
NOS, aormmalamt 4.2 oaad = sd au PS on FA 201ml.
LO, SOC ACG Sa yes 2 2c s es aR oe 1.5 ami;

PROCEDURE:
1. Demyelinate sections in 50% ethyl alcohol plus 1.0 ml. ammonia
per 100 ml.: 6-12 hours. A longer time has no ill effect.
2. Wash in distilled water, 3 changes: few seconds each.
3. Impregnate in silver solution 4: 12—24 hours.
4. With no washing, transfer into silver solution B: 2—5 minutes.
5. Transfer directly into reducing solution until the sections turn
gold in color.
6. Transfer to 2.5% sodium thiosulfate (2.5 gm./100 ml. water): 1-2
minutes.
7. Wash in distilled water, at least 3 changes.
8. Dehydrate rapidly, clear, and mount.

RESULTS:
nerve fibers and endings—black
cells pale yellowish brown

COMMENTS:
This method is nonselective and stains normal as well as degenerat-
ing axons. For degenerating axons see following methods.
208 Silver _Impregnation II (cHAP. 15)

Degenerating Axons
Nauta and Gygax (1954) S
FIXATION: perfusion method seems to be preferred.
Anderson (1959):
1 Perfuse with 0.9% sodium chloride (intracardiac and intra-aortic
canulation) until fluid escaping is clear.
Xe) Perfuse with 500 ml. of 5% potassium dichromate and 2.5% po-
tassium chlorate. This is peculiar to Anderson; others perfuse
with the fixing solution.
. Remove tissue and fix in 10% formalin neutralized with carbon-
ate: 1 week or longer. Nauta and Gygax recommend 1-3 months
as best.
EMBEDDING AND SECTIONING:

L. If embedding is necessary use gelatine. Wash slices (5-10 mm.

thick) in running water: 24 hours. Incubate in 25% gelatine, 37°C
(cover tightly): 12-18 hours. Wipe off excess gelatine and immerse
in cool formalin: 6 hours or longer.
2 . Frozen sections. Cut sections 15-25 microns and place in 10%
formalin, room temperature or cooler. Can be stored in formalin,
preferably in refrigerator. Process only a few sections (6-10) at
one time.
Adey et al. (1958) prefer a dual-freezing block method, using dry
ice evaporated in 70% alcohol, over CO, freezing. This produces
more even freezing. Rapid freezing and thawing with CO, may rup-
ture some of the fibers (Freon equipment eliminates some of this
problem).
SOLUTIONS:
Ammoniacal silver nitrate:
Silver MUGnate) 2 2.3.05 oe2 os uo 0.9 gm.
distilleduwater. 4.5070 sae ee eee 20.0 ml.
When dissolved add:
absolute ethyl alcohol i. eer
= 2. tae 10.0 ml.
aimimonia. concentrated. neers.
es eee I-Siamnll:
sodium hydroxide, 2.59% “aqueous”... 157152
a0" 1.5 ml.
Reducing fluid:
distilledswater’ <x: 2Sie A us Bet ee SE 400.0 ml.
absolute: ethyl alcoholy sca2 sera: ces tase 45.0 ml.
Degenerating Axons 209

formalin fee a ee fi hte a mae 13.5 ml.

Cline acids ale raqueous .2 05.282 PYM 13:5 mal,

PROCEDURE:
1. Soak sections in 15% alcohol: 30 minutes.
2. Wash briefly in distilled water and soak in 0.5% phosphomolyb-
dic acid (0.5 gm./100 ml. water): 15-20 minutes.
3. No wash; transfer to 0.59% potassium permanganate (0.5 gm./100
ml. water): 4-10 minutes. Can be critical; make trial runs at 5, 7,
and 9 minutes. Turn sections over occasionally.
4. Rinse in distilled water.
5. Decolorize in equal parts of 1% hydroquinone (1 gm./100 ml.
water) and 1% oxalic acid (1 gm./100 ml. water): 1-2 minutes.
6. Wash thoroughly in distilled water, 3 changes.
7. Treat with 1% silver nitrate (1 gm./100 ml. water): 20-30 min-
utes.
8. Transfer sections individually; a brief wash in distilled water
and then into freshly prepared ammoniacal silver: 1 minute.
9. ‘Transfer to reducing solution, allowing sections to float on sur-
face of solution. Brown color within | minute. If too light, add
a little sodium hydroxide to ammoniacal silver solution; if too
dark add some ammonia.
10. Wash briefly, fix in 1% sodium thiosulfate (1 gm./100 ml. water;
2 minutes.
11. Wash in distilled water, 3 changes.
12. Mount, Albrecht’s (1954) method:
(a) ‘Transfer into equal parts of 1.5% gelatine (1.5 gm./100 ml.
water) and 80% alcohol: 5 minutes or longer.
(b) ‘Tease section onto slide, withdraw from fluid with slide tilted
allowing rapid drainage of excess fluid. Wipe around section,
but do not allow it to completely dry.
(c) Blot gently with smooth filter paper. If section sticks to paper,
first moisten paper with 95% alcohol.
(d) Immerse rapidly in 95% alcohol to congeal gelatine.
13. Dehydrate, clear, and mount.

RESULTS:
disintegrating axons—black
normal axons various shades of brown

COMMENTS:
See also Nauta and Gygax (1951); Nauta and Ryan (1952); Wall
(1950); White (1960); and additional references included therein.
210 Silver _Impregnation II (cHap. 15)

Hamlyn (1957) and Guillery et al. (1961) describe methods for paraf-
fin-embedded sections that are stained after mounting.

Marchi Method B
FIXATION: 10% formalin plus 1% of potassium chlorate: 24—48 hours,
no longer. (Swank and Davenport, 1934)
SOLUTION:
Marchi fluid (Poirier et al., 1954):

osmire acid, 0.597, aqueous... 00: os. eee 11.0 mi:

potassium chlorate 1°, aqueous... ..... 7 ames 16.0 ml.
BOPIVALTINY 09% Beal Pace Ns Cong) A ae Ae 3.0 ml.
aceticvacia, LOS, aqueous...) ie. .ere. Ae 3.0 ml.
esstilled «water 10 IMake, sof... ee ae Go eee 100.0 ml.

PROCEDURE:
1. Cut tissue into thin slices—about 3 mm. for easier impregnation.
2. Chromate, 2.5% potassium dichromate, aqueous, in dark: 7-14
days, change twice.
3. Transfer directly to Marchi fluid, a volume 15-20 times that of
tissue: 1-2 weeks depending on size of tissue. ‘Turn tissue over
every day to improve penetration.
4. Wash in running water: 24 hours.
5. Dehydrate and embed. If using celloidin, keep embedding steps
to a minimum. If using paraffin, avoid xylene and its solvent ac-
tion on osmic acid. Chloroform is safer; also keeps steps to mini-
mum.
6. Deparaffinize slides with chloroform and mount in chloroform-
resin.
RESULTS:
degenerating myelin—black
background—brownish yellow
neutral fats—black

COMMENTS:
The principle behind the Marchi method is based on the fact that
myelin of medullated nerves oxidizes more easily than degenerated
myelin. Normal myelin is oxidized by chromating and will not react
with osmic acid. Degenerate myelin contains oleic acid which does
not oxidize during chromating and therefore reduces the osmic acid
and stains black.
Motor End Plates 211

References: Culling (1957); Lillie (1954); Mettler (1932); Mettler and

Hanada (1942); Poirier et al. (1954); and Swank and Davenport (1934
A,B and 1935 A,B).

Motor End Plates

Cole Method (1946) (Whole Mounts)
FIXATION: none, carry fresh tissue directly into step 1.
PROCEDURE:
i Remove striated costal muscle and place in 10% citric acid in
physiological saline (page 410): minimum time 10 minutes, maxi-
mum 30 minutes.
Transfer directly to 1% aqueous gold chloride (1 gm./100 ml.
water). Make up day before used. Keep in dark: 60 minutes or
until tissue turns dark yellow. (If pieces are larger than 4 mm. in
width a longer time is required.)
. Transfer directly to 20% aqueous formic acid (20 ml./80 ml. wa-
ter): 10-20 hours in dark. Do not use metal forceps, coat them
with paraffin or use glass rods.
. Rinse in tap water.
Or Transfer to 95% methyl alcohol-glycerol (1:1): several hours.
Then remove top of container and allow alcohol to evaporate.
Transfer to pure glycerol.
~I To mount: with a sharp razor cut muscle strips into small pieces.
Place a piece in a very small drop of glycerol (usually, amount
carried over by the piece is sufficient) on a round cover glass. Lay
a smaller size cover glass over it. Spread the muscle fiber to single
fiber thickness by using gentle pressure and strokes at right angles
to the fibers. Turn cover glasses over and mount in Permount (or
the like) on a glass slide. (Double cover glass mounting, page 123.)
RESULTS:
muscle fibers—red-blue
motor end plates and medullated axons—black
muscle fiber nuclei—unstained

Carey’s (1941) modification:

Carey used undiluted fresh filtered lemon juice in place of the
citric acid. He claimed that if longer than 12 hours was required in
the formic acid (step 3), the gold chloride technic was faulty. The
color of the tissue should be gold, not brown,
212 Silver Impregnation IT (cHAp. 15)

Warren's (1944) modification:

Warren writes that large pieces of muscle give better results than
small ones because in the latter the muscle fibers may become too
dark. He removed 5 or 6 ribs from a rabbit with the muscle and ten-
dons intact. In this case the timing in solutions will have to be ex-
tended.
PROCEDURE:
1. Treat with citric acid: 2 hours.
ho Wash in 2 changes of distilled water: 5 minutes each.
3. ‘Transfer to gold chloride: 2 hours.
— . Transfer to formic acid: 24 hours; change solution at end of first
12 hours.
5. Wash in 3 changes of distilled water: 5 minutes each.
6. ‘Treat with pure glycerine: 24 hours, then change to a fresh solu-
tion.
7. To mount: cut the muscle from between the ribs, cutting close to
the bone. Cut the muscle in small strips 5 mm. long. Keep strips
stored in the glycerine while preparing them for mounting. Pre-
pare thin mounts as in technic above.
COMMENTS:
The author has not used this method, but makes the following sug-
gestion: to prevent swelling of the tissue, do not go directly from
water (step 5) into glycerine (step 6). First treat with 50% alcohol-
glycerine (1:1) several hours or overnight, then proceed with step 6.

Myelin
Luxol Fast Blue (MARGOLIS AND PICKETT, 1956) S
FIXATION: any good general fixative.
EMBEDDING: paraffin method.
SOLUTIONS:
Luxol fast blue:

Ieuexol fast blue JIBS?) <cme aee. . 6 sce 0.1 gm.

So mealcOhol: | Us heya eee Ms) ROUMaar
HGcHe wea, 10% AGueous. «eee... cae 0.5 ml.

Keeps indefinitely.

*E. 1, DuPont de Nemours and Company.

Myelin 213

Periodic acid, 0.5%:

PEMlOGIG ACNE 6 ns. so ascsaeeee
Ot ane ee ee 0.5 gm.
aisnilled= watery... 225...
ee ee 100.0 ml.

Lithium carbonate, 0.05%:

Leeann camDOMAte 4.65 ua. oda aw ye ns east ace 0.5 gm.
distifledMwaten fee 25 ky esaa He Ser exe one he 1000.0 ml.

Schiff’s reagent, see page 294.

Sulfurous acid:
sodium metabisulfite, 109% AGUCOUS) £5 36004 es 6.0 ml.
distilled water ...... eyre eC eee 100.0 ml.
INGE (See pape: 408): ..... .¥du sew emared
oat 5.0: mi:

Mayer's hematoxylin, see page 125.

PROCEDURE:
ly Deparaffinize and run slides down to 95% alcohol, but remove
HegCl, if present.
Stain in Luxol fast blue, 60°C: overnight.
Rinse off excess stain in 95% alcohol.
oo
Hm
bo Rinse in distilled water.
Dip in lithium carbonate: 15 seconds.
Differentiate in 70% alcohol: 20-30 seconds.
Rinse in distilled water.
Place in lithium carbonate, second solution: 20—30 seconds.
Differentiate in 70% alcohol: 20-30 seconds.
re
Rinse in distilled water. If differentiation is not complete, repeat
steps 8 and 9.
. Oxidize with periodic acid: 5 minutes.
. Wash in distilled water, 2 changes: 5 minutes.
. Treat with Schiff’s reagent: 15-30 minutes.
. Treat with sulfurous acid, 3 changes: 2 minutes each.
. Running water: 5 minutes.
. Stain in Mayer’s hematoxylin (or the like): 3 minutes.
. Wash in running water: 5 minutes.
. Dehydrate, clear, and mount.

RESULTS:
myelin—blue-green
PAS positive elements—rose to red
nuclei—blue
214 Silver Impregnation II (cHAr. 15)

COMMENTS:
Margolis and Pickett include methods for following luxol fast blue
with phosphotungstic acid-hematoxylin to differentiate neuroglia
from myelin, and oil red O to distinguish degenerating myelin from
normal myelin.
Dziabis (1958) describes a method for gross brain sections.

Luxol Fast Blue—Holmes Silver Nitrate (MARGOLIS AND PICKETT, 1956) S

FIXATION: 10% formalin preferred.
EMBEDDING: paraffin method.
SOLUTIONS:
Silver nitrate, 20%:

SHIVER “ANUrate! Phat ee | oe tis Foe eee ae ee 20.0 gm.

Gustilled ‘waters. - sii col oa Seeeee 100.0 ml.

Boric acid:
IDOTTC. aC Ul ee oO ane 2 ee eel ot ee aie SR Le 12.4 gm.
Gistilled water g'85c. Le. see
ee eee 1000.0 ml.

Borax:
DOTA yee Soko cha sneer oe ts, 5 teem eee Oe ee 19.0 gm.
distilled: water) «03. Oe J) AO eee 1000.0 ml.

Impregnating fluid:
In 500 ml. cylinder mix 55 ml. boric acid solution and 45 ml. borax
solution. Dilute to 494 ml. with distilled water. With pipette, add 1
ml. of 1% aqueous silver nitrate (1 gm./100 ml. water). With another
pipette add 5 ml. of 10% aqueous pyridine (10 ml./100 ml. water).
Mix thoroughly.
Reducer:
hydroquinone 2 20) MIME VG). ie eeee ee 1.0 gm.
sodiuni sultites (crystals): 2th 4. Sat een 10.0 gm.
distilled water’ =... a2... 52254. oo Oe Oe 100.0 ml.

Can be used repeatedly, but only for a few days.

Luxol fast blue—see page 212.
PROCEDURE:
1. Deparaffinize and hydrate sections to water.
2. ‘Treat with 20% silver nitrate in dark, room temperature: | hour.
Prepare impregnating fluid.
Myelin 215

3. Wash in distilled water, 3 changes: 10 minutes.

Impregnate, 37°C: overnight.
5. Shake off superfluous fluid and place in reducer: 2 minutes.
6. Wash in running water: 3 minutes; rinse in distilled water.
7. Tone in gold chloride, 0.2% (0.2 gm./100 ml. water): 3 minutes.
8. Rinse in distilled water.
9. Treat with oxalic acid, 2% (2 gm./100 ml.): 3-10 minutes; when
axons are thoroughly black, remove.
10. Rinse in distilled water.
11. Fix in sodium thiosulfate, 5% (5 gm./100 ml.): 3 minutes.
12. Wash in running water: 10 minutes.
13. Rinse briefly in 95% alcohol.
14. Stain in Luxol fast blue: overnight.
15. Rinse in 95% alcohol; rinse in distilled water.
16. Treat with lithium carbonate, 0.05% (.5 gm./1000 ml. water):
15 seconds.
17. Differentiate in 70% alcohol: 20—30 seconds.
18. Rinse in distilled water.
19. Repeat steps 16 and 17 if necessary. (The author finds that usu-
ally 3 repeats is necessary for sharp differentiation.)
20. Dehydrate, clear, and mount.

RESULTS:
axis cylinders—black
myelin sheaths—green blue

COMMENTS:
Margolis and Pickett write that slides may be left in step 6 until time
to prepare them for Luxol fast blue staining. This tends to encourage
loose sections. The author has lost no sections by carrying the slides
through to step 13. Transfer them into absolute alcohol and coat
them with nitrocellulose. Harden the nitrocellulose and store the
slides in 70-80% alcohol until ready to stain in luxol fast blue. Slides
have been left in this condition for over the weekend with completely
satisfactory results. The nitrocellulose protection also permits freer
agitation of slides in the differentiating fluids than without this coat-
ing.

Ora’s Method (1958) S

FIXATION: 10% formalin.

SECTION: at 17 microns, paraffin method.

216 Silver Impregnation II (cuap. 15)

SOLUTIONS:
Mordant, modified Marchi fluid:

potassium clichinOMabe 2k eeeao 25.0 gm.

SOT UMS PARC ee ee ins: < ¥ ys CR hae 1.0 gm.
distillecdiawatteiee 2... .). 2: tae eee 100.0 ml.
Add 1 ml. of above to:
ferric alum, 5% aqueous (3 gm./60 ml. water)... 60.0 ml.

Hematoxylin:
GMA ONVANINA oe,555k cog.) ns eG aig he ee yeeS CIEE 1.0 gm.
Apsolmbexethyl alcghol) .. 4ae cool. eee eee 10.0 ml.
Dissolve hematoxylin in alcohol and add:
Gesciled walter. (5. .g Gove tans) ones ney Seer 90.0 ml.
lithium carbonate, saturated aqueous ......... 4.0 ml

Mix thoroughly. Prepare fresh each time.

Differentiating solution:
potassium) permanganate... ocu9.4e
eb ta ee 0.5 gm.
distilledaweaten ~ oa Pru ye ee tae ee 100.0 ml.

Thin celloidin:
gum mastic, saturated in absolute alcohol ..... 2:0 mit
celloidim or mitrocellulose: 109% 22...eee 7.0) ail.
Cther,- amliydrOusy. tio. 2 es enh ed does 45.0 ml.
absolute ethyl alcohol 3.2 oy. ceo: 2 ote 45.0 ml.
ACE EOIN ete ate Phe eeTe S eset en I Re st ee 10.0 ml.

PROCEDURE:
1. Deparaffinize and run slides into absolute alcohol: 2 minutes.
2. Dip twice in thin celloidin, blot lightly with filter paper and
harden in 95% and 80% alcohol: 2 minutes each.
3. Hydrate down to water.
4. Mordant, 60°C: 30 minutes.
5. Preheat hematoxylin, 60°C, add slides, stain: 30 minutes.
6. Wash in running water until sections blue: 2 minutes.
7. Differentiate with potassium permanganate until myelin fibers
are jet black and background is light yellow: maximum 30 sec-
onds.
8. Wash in running water: 2 minutes.
9. Dehydrate, clear, and mount.
Myelin 247,

RESULTS:
myelin sheaths—black
background—light yellow

Mahon Method (1937) S

FIXATION: 10% formalin. Cut paraffin sections 15—20 microns. Not good
for frozen sections.
EMBEDDING: parafhin method.

SOLUTIONS:
Mordant:
ferric ammonium sulfate (ferric alum) ........ 4.0 gm.
US CUTE CSWater 1s tien hos sd coe teat abana Gas 100.0 ml.
Hematoxylin:
lithium carbonate solution (to 93 ml. distilled
water add 7 ml. of saturated aqueous lithium
carbonate... 22 Ae: 132 304 ee 604, o00Lanl:

COMON c,h Pret rece BE bac on MRE ate 10.0 ml.

Mix immediately before use.

PROCEDURE:
1. Deparafhinize and hydrate slides to water.
2. Mordant: 15-30 minutes.
3. Rinse in 2 changes distilled water: | minute.
4. Stain in hematoxylin: 30-60 minutes.
5. Wash in running water: 2 minutes.
6. Treat with lithium carbonate, 7% (7 gm./100 ml. water) until
myelin is dark blue and background is colorless: 15-30 minutes.
Watch carefully that too much dye is not extracted; also, pro-
longed exposure to lithium carbonate may loosen sections.
7. Wash thoroughly in running water: at least 5 minutes.
8. Dehydrate, clear, and mount.
RESULTS:
myelin—dark blue
background—colorless
 Chapter 16

Hematologic Elements
and Related Tissues

Blood is a fluid (plasma) that contains suspended in it cells and frag-

ments of cytoplasm. ‘The fragments are platelets and the cells are either
red cells (erythrocytes) or white cells (leukocytes). The erythrocytes are
nonnucleated, but 90% of their solid content is made up of hemoglobin,
a protein. Leukocytes are all nucleated and are classified as five different
kinds. ‘The granular leukocytes have a granular cytoplasm and include
the neutrophils, eosinophils and basophils. The nongranular leukocytes
have nongranular cytoplasm and are the lymphocytes and monocytes.
The granular leukocytes are named according to the affinity of their
granules for certain types of stains—neutral, acid (eosin), and basic. ‘The
term polymorphs is often applied to neutrophils because of their many-
formed or many-lobed nuclei.
Bone marrow is a hemopoietic (blood forming) tissue and is described
as either red or yellow. The red marrow is red due to great numbers of
red blood cells in various stages of formation; it is actively producing
blood cells. Yellow marrow contains large quantities of fat; it is not
actively manufacturing blood cells but is storing fat. Yellow marrow
nonetheless retains the potential to resume production of blood cells.
(References Elam, 1957)
Blood cells and fluid may become parasitized in various ways, as with
malaria, trypanosomes, inclusion bodies of various diseases, such as rick-

218
Blood Smears 919

ettsia and psittacosis. These foreign elements, in most cases, are best
demonstrated with a Romanowsky type of stain.

Blood Smears

Preparation for Thin Smears

Slides must be clean for a uniform smear. Handle slides at the edges,
keeping fingers off the clean surface. Prick the finger and when a small
drop of blood appears, wipe it away. ‘Touch the next drop of blood to
the clean surface of the right end of the slide. Place the narrow edge of
another slide at about a 20° angle on the first slide and to the left of the
drop blood. Pull to the right until the slide touches the blood. As soon
as the blood has spread along the line of contact, push the right hand
toward the left. Push steadily until all the blood disappears or the other
end of the slide is reached. This method drags the blood but does not
run over it and crush some of the cells. The hand can be kept from
shaking by resting it on the table. Also do not use a slide with a rough
edge to leave streaks in the smear. If the blood seems thick, reduce the
20° angle to feed it out at a slower rate. With thin blood increase the
angle. (See Pequeno, 1960, for a clever method using a pen to make
thin smears.)
Dry the slides rapidly in the air; waving them facilitates drying, and
prevents crenation (notching or scalloping of edges) of the red cells.
Preferably blood smears are stained immediately or within 24 hours.
If they must be stored, place them in a tight box away from dust and
flies.
Blood smears are commonly stained with a Romanowsky type of
stain, or neutral stain (page 110), Neutrality is essential, and therefore
dilution usually is made with a buffer solution of a known pH. Dis-
tilled water often is too acid, and tap water too alkaline. The smears
require no fixation in the usual sense, since Wright’s stain (1902) in-
cludes both fixing agent (methyl alcohol) and stain.

Thin Smear Method

SOLUTIONS:
Wright’s stain:
The stain may be purchased in three forms: (1) by the bottle, 10, 25
or 100 gms.; (2) in capsule form, 0.1 gm. per capsule; or (3) in solu-
tion, 4, 8 or 16 ounces.
 20
ho Hematologic Elements and Related Isswes (CHAP. 16)

The powder (bulk or capsule) is ground up with methyl alcohol (0.1

em. to 60.0 ml.). The alcohol must be labeled “‘neutral” and “acetone
free.’’ Grind thoroughly in a glass mortar, and pour off supernatant
(surface-floating) liquid. If undissolved powder remains, pour back
the liquid and grind again.
Buffer solution, pH 6.4:

monobasic potassium phosphate ............. 6.63 gm.

anhydrous dibasic sodium phosphate ......... 2.56 gm.
SGU CRM ALCE: 3 eos) 57.08 2 hap eRe eee ee 1000.0 ml.

PROCEDURE:
1. With wax pencil draw 2 marks across slide delineating region to
be stained (length of 40 mm. cover glass).
2. Cover with 10-12 drops of Wright’s stain: 1-2 minutes.
©9. Add an equal amount of buffer: 2-4 minutes.
4. Rinse in distilled water, 1 or 2 dips. Precipitate deposit on the
slide can be avoided by flushing the slide with a pipette, or carry
the stain with the slide into the wash water; do not drain it off
first.
5. Blot with 2 sheets of filter paper; press but do not rub.
6. Allow slide to thoroughly dry before applying cover glass.
RESULTS:
erythrocytes—pink
nuclei—deep blue or purple
basophilic granules—deep purple
eosinophilic granules—red to red-orange, bluish cytoplasm
neutrophilic granules—reddish brown to lilac, pale pink cytoplasm
eranules of monocytes—azure
lymphocyte granules—larger and more reddish than monocyte gran-
ules, sky blue cytoplasm
platelets—violet to purple
COMMENTS:
Precipitate formation can be troublesome; the dark granules obscure
the blood cells and are confusing in malarial smears. In addition to
poor washing in step 4, another case of precipitate may be evaporation
during exposure to undiluted stain. Methyl alcohol is highly volatile
and readily lost by evaporation. In dry warm weather use more
Wright’s, or shorten the time a bit, or cover the slides with a petri
dish. Rapid evaporation is easily detected, and more stain can always
be added.
Blood Smears 22k

The longer the washing with water, the more stain is removed from
the white cells. Usually only a dip or two is sufficient, but if the white
cells are overstained, differentiate them by longer washing.
If the slides are overstained, the erythrocytes are too red, the white
cells too pale, or the stain has precipitated, the slides can be recov-
ered: (1) Cover the entire slide almost to excess with additional
Wright's stain: 15-30 minutes; (2) rinse with distilled water; (3) dry.
(Morrison and Samwick, 1940)

Preparation of Thick Blood Films

This type of film concentrates a relatively large quantity of blood in a
small area, thereby increasing the possibilities of finding parasites. ‘The
concentration and timing of staining are adjusted so the action is
stopped at the point when the leukocytes have stained, some hemo-
globin has been dissolved and the red cell membranes have not yet
begun to stain. At this point the leukocytes, platelets and protozoa only
are stained and lie on an unstained or very lightly stained background,
yellowish from remaining hemoglobin. Freshly prepared films stain
better than films one or more days old.
Puncture the skin deep enough to form a large drop of blood. On a
slide cover a space the size of a dime with enough blood (about 3-4
average drops) to spread easily. Too much blood will crack and peel
off when dry. Smear it by circling the slide under the finger without
making actual contact. Some find it easier to swirl the blood with a dis-
secting needle or the corner of a slide. Practice will determine the best
method and how much blood to use. ‘Too thin a film has no advantage
over a thin smear. An ideal film is several cell layers thick in the center,
tapering off to one cell thickness at the periphery.
Allow the slide to dry in a horizontal position; if tilted the blood will
ooze to one edge of the film. Protect from dust and flies, and do not use
excessive heat for drying.

Thick Film Method

SOLUTIONS: Field (1941)
Solution A:
methylene DinerE. 1652015) 24. . 1 5 on PGE ee, 0.8 gm.
azure As Co DZ00D 2ato i ets as =o : a Rees 0.5 gm.
dibasic sodium phosphate, anhydrous ........ 5.0 gm.
monobasic potassium phosphate ............. 6.25:-em.
distilled water. 254.0 4 2 obo os eee ee: 500.0 ml.
Pa af Hematologic Elements and Related Issues (CHAP. 16)

Solution B:
Cost Wi Gaieet oes as 4: i. | Xe AER Bee gs eet 1.0 gm.
dibasic sodium phosphate, anhydrous ........ 5.0 gm.
monobasic potassium phosphate ............. 6.25 gm.
cistilled# water anes... 20.11 22.20). Ae, PET 500.0 ml.

Dissolve phosphate salts first, then add stains. (The azure will go into
solution by grinding in glass mortar with a small amount of phos-
phate solvent.) Set aside for 24 hours. Filter.

PROCEDURE:
1. Dip slides in solution A: | second.
2. Rinse gently in clean distilled water: few seconds, or until stain
ceases to flow from film.
3. Dip in solution B: | second. Use this solution with care; it tends
to decolorize the leukocytes stained with methylene blue-azure,
and accelerates the dissolving of hemaglobin.
4. Rinse gently in distilled water: 2—3 dips.
Or. Dry, do not blot. Stand slides on end and allow to air dry.
6. When completely dry, add mounting medium and cover glass.

RESULTS:
There will be a thicker central area of partially laked blood. This
may not be well-suited for examination. But the surrounding area
and especially at the edge toward which the hemaglobin drained will
be creamy, sometimes mottled with pale blue. This is the best area
for study.

leukocytes:
cytoplasm—pale blue, poorly defined
nuclei—dark blue, well defined
eosinophilic granules—bright red, large, well defined
neutrophilic granules—pale purple pink, small indistinct
basophilic granules—deep blue with reddish cast
platelets—pale purple or lavender

parasites:
cytoplasm—blue
chromatin—purplish red or deep ruby red
pigment—unstained yellow granules of varying intensity
Blood Smears 223

SOLUTIONS: Wilcox (1943)

Wright-Giemsa:
GICMsa POWER 65.48 oS saven foes Ce Ree 2.0 gm.
Slycenolymamaern ceil. 2'2 0. BEAM ae teak 100.0 fale

Heat in water bath, 55-60°C: 2 hours, stirring at intervals. Avoid ab-

sorption of moisture by covering mouth of flask with double thickness
of paper secured with rubber band.
Add: aged Wright’s staining solution (2.0 gm.
per 1000:0imil, methyl alcohol)...7)24 «2.2000: 100.0 ml.
Let stand overnight: add additional Wright's
Staming solution \. 2)... ...-.5« sae a segeees eos oe 800.0 ml.

Filter; ready for use.

Working solution:
Ny ientHGacmsa Stock SOLUTION 4.5052 on 1 part
Newtral’ distilled water (buffer) 2)... sss0% 253 9 parts

Butter, PH 7.0:
Solution A:
dibasic sodium phosphate, anhydrous ......... 9.5 2m,
dastilledy Water: i 2% e246 okie 4 a oe ee 1000.0 ml.

Solution B:
monobasic potassium phosphate ............. 9.7 gm.
Gastilicd Water mds oy. eds ov oe 4s eee ees 1000.0 ml.

Working Solution:
SOME OMA oo icnce doe a oe ene no ee eens 61.1 “mil.
DOMMIOM He = 2 eee bee weeae es 6. Be 38.9 ml.
distilled water's 44.24% «sunt ¢snndeemee 900.0 ml.

PROCEDURE:
Short Method:
1. Stain film: 10 minutes.
2. Flush scum from top of solution with natural water to avoid
picking up precipitate on slides.
3. Remove slides to neutral distilled water: 1 minute.
4. Dry slides standing on end. Do not blot. Mount.
Long Method (a more brilliant stain):
1. Stain: 45 minutes in dilution of 1 to 50 of neutral water.
224 Hematologic Elements and Related Isswes (CHAP. 16)

2. Flush off scum and transfer slides to neutral distilled water: 3—5
minutes.
3. Dry as above.
Note: Thin smear and thick film can be stained on same slide. Fix thin
portion in methyl alcohol for 1-2 minutes, taking care that thick film
does not come in contact with alcohol. Stain as above (short method),
but shorten washing time to 2 or 3 dips so thin smear is not too light;
this shorter washing time will leave a deeper background in the thick
film.
MODIFICATIONS:
Fenton and Innes (1945).
Manwell (1945).
Modified Wright’s stain, Stel (1936).
STAINING OLD BLOOD SMEARS:
Old smears will stain more brilliantly if treated 5-10 minutes in al-
cohol-acetic solution—10 drops glacial acetic acid to 60 ml. absolute
alcohol.

Blood Tissue Elements and Inclusion Bodies*

Giemsa Stain, Wolbach’s Modification (MALLORY, 1944)
FIXATION: any good general fixative.
SOLUTIONS:
Acid alcohol:

OSL: pee aC OM ON. erie Mie erventas 21S eee 100.0 ml.
gl MCECTC ACI” oi Ashe Reale
doth oc Runt 0:5=1-0 mi:

Giems ck solution:
G SAN POWGEIW e's. PROSPER
ot acs 2 yaks Bec e 1.0 gm.
me yl alcohol, neutral, acetone-free ......... 66.0 ml.
elgpeerol 2345) \0ly oe Pe ee chee 2 2 66.0 ml.
Work Giemsa powder into glycerol and place in 60°C oven for 2
hours. Add methyl alcohol and stopper tightly.
Working solution:
StocteGiemsa. solution <->. 246s: se 2.5 ml.
methylealcohol >..cad 5 -cten-s2 Rae Pees 7s ee 100.0 ml.
disuilledawater..; {0d ce deja ke Pt 3.0 ml.

*See p. 306.
Blood Tissue Elements and Inclusion Bodies 225

Rosin stock solution: (Colophonium)

white wood rosin ........ Dei J ee a 10.0 gm.
aDSolmtemalcoMmOlirss, c65 2-024 st eee 2 ee 100.0 ml.
Working solution:
LOSI StOCKeSONIEION. 2; 24s oo ssa dakt ce eal 5.0 ml.
DOVARC OW PALCONO! 25... io: stiketsALS 40.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water. Remove HeCls.
2. Treat in acid alcohol: 5 minutes (not necessary if tissue has been
subjected to acid decalcification).
3. Wash, running water: 5 minutes.
4. Rinse in distilled water.
5. Place in freshly mixed Giemsa working solution: | hour.
6. Transfer to second jar of Giemsa working solution: overnight.
7. Rinse in distilled water.
8. Differentiate in rosin alcohol.
9. Rinse in 95% alcohol.
10. Dehydrate in absolute alcohol, 2 changes; clear and mount.
RESULTS:
nuclei—reddish purple
other tissue elements—similar to Wright’s stain
inclusion bodies—blue to purplish blue
COMMENTS:
For other stains for inclusion bodies, see pages 326-328.

Staining for Malaria

Ri

Jenner-Giemsa (MCCLUNG, 1939, MODIFIED MAY-GRUNWAI

FIXATION: Smears may be fixed during staining process. ‘T * sections

in any good general fixative, preferably containing merci.:si/chloride
and alcohol. iis
SOLUTIONS: :
Jenner’s solution:
Jenners: stain. BUiih Cita Uy p22 2 te eee 0.2 gm.
methyl alcohol, neutral, acetone free ........ 100.0 ml.
Giemsa stock solution:
AGiemsa POWGER |! 95.05.26 oc. ene ooo Dee 3.8 gm.
methyl alcohol, neutral, acetone free ......... 75.0 ml.
PIV CCI ar eran et end « 44% sk che 304 eee 25.0 ml.
226 Hematologic Elements and Related Isswes (cHAP. 16)

Work stain into glycerol, warm for 2 hours, 60°C oven. Add methy]
alcohol.
Working solution:
Giemsatstackesoiition! : 2... Sas 10.0 ml.
Gistilleewaer ye a:\k. a... sft eee LO 100.0 ml.
or 1.0 ml. to 10.0 ml. water for flooding a slide.

PROCEDURE:
1. Deparaffinize sections and run down to 50% alcohol. (Remove
HegCl, if present.)
2. (a) Sections—flood with ample amount of Jenner’s solution and
add an equal amount of distilled water, or mix equal amounts of
Jenner’s solution and water and flood slide or stain slide in coplin
jar: 4 minutes.
(b) Smears—flood with Jenner’s solution: 3 minutes. Add equal
amount of distilled water: 1 minute.
3. Pour off Jenner’s; no rinse.
4. Flood with diluted Giemsa solution or place in coplin jar of
solution: 15-20 minutes.
5. Rinse off stain with distilled water and continue to differentiate
with distilled water. If too blue, eosin color can be brought out by
rinsing in 1% acetic acid in water.
6. Dehydrate in following sequence of acetone and xylene. Do not
use alcohol; it extracts the stain.
acetone 95 parts: xylene 5 parts
acetone 70 parts: xylene 30 parts
acetone 50 parts: xylene 50 parts
acetone 30 parts: xylene 70 parts
acetone 5 parts: xylene 95 parts
7. Clear in xylene and mount.
RESULTS:
malaria:
chromatin—red or purplish red
cytoplasm—blue (other elements’ color similar to Wright’s stain)
pigment—yellow brown to black
Schiiffner’s stippling—red
COMMENTS:
A beautiful clear stain for any blood picture, particularly blood
parasites.
Bone Marrow Staining 22)

Brooke and Donaldson (1950) recommend the use of Triton X-30,

a nonionic liquid detergent to prevent the transfer of malarial para-
sites between films when mass staining.

Bone Marrow Staining

Maximow’s Eosin-Azure Stain (BLOCK e¢ al., 1953)
FIXATION: Zenker-neutral formalin: 2 hours. Neutralize formalin by
adding 2 em. lithium or magnesium carbonate to 500 ml. of formalin.
Excess of carbonate should be present. Wash in running water 1-24
hours before preparation for embedding. (See Comment 4)

SOLUTIONS:
Solution A:

Cosine. Gel, ADSBO sekecic is ws ng oceans

Or ne BS x 0.1 gm.
@ishilled Water aces odd+a- «cs auev ee ooo den 100.0 ml.

Solution B:
MUTE p20 cece vac a oes ai 4 5 Gogh VRE Oe NOs 0.1 gm.
austiiled?watern:.. 22 ee . 4... Ace eee — 100.0 ml.

Working solution:
distilled: water \ 5 7G ales «hc wees ee 85.0 ml.
Solution A 15.0 ml.

Stirring vigorously, add gradually

Solution B 10.0 ml.

Fresh solutions are best; their action deteriorates after 3 or 4 weeks.

Working solution should appear deep violet in color, and a precipi-
tate should not form in it for an hour or more. If a precipitate forms
on the slides, the stain mixture was improperly made; solution B was
added to solution A too rapidly or without stirring. If the eosin loses
its brilliance, solutions are old.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCl». If tissue
was fixed in a fixative without potassium dichromate, chromate
slides overnight in 2.5-3% aqueous potassium dichromate. Wash
thoroughly in running water: 15 minutes. Proceed to step 2.
ho. Stain in Mayer’s hematoxylin: 30-45 seconds, no more. (See Com-
Mene “3:)
 Hematologic Elements and Related Issues (CHAP. 16)

Wash in running water: 5—10 minutes.

Wash in distilled water: 5 or more minutes.
Stain in eosin-azure overnight.
Differentiate in 95% alcohol: may require 2—3 hours. In stubborn
cases of differentiation (old solution) a brief immersion in colo-
phonium alcohol (page 225) may help to sharpen the colors, but
not as well as a new solution. The over-all appearance of the slide
should be blue with a slightly greenish tinge, but differentiation
must be done under the microscope.
vi Dehydrate, absolute alcohol; clear and mount.
RESULTS:
nuclei—dark purple blue
erythrocytes—light pink
eosinophilic granules—brilliant red
cytoplasm—pale blue

COMMENTS:
L After 2 hours fixation, cells in center are not as well fixed as
those at periphery, but fixation longer than 2 hours produces
granular cytoplasm in eosinophilic erythrocytes and erythroblasts.
ho Block embeds in celloidin and mounts with clove oil, page 77
and stains with a weak solution of Delaefield’s hematoxylin, 2
drops/100 ml. distilled water, overnight, or in a mixture of 10-15
drops/100 ml. for approximately 5 minutes, or till nuclei are faint
blue; check under microscope. ‘The author has had good results
with the above-scheduled method (step 2), but take care not to
stain too long, never over 60 seconds.
Bone marrow when collected should have an anticoagulant added
to it. Gardner (1958) and many other workers mix the bone mar-
row in a tube containing 0.5 mg. heparin powder. Kniseley? wets
his syringe with d-potassium EDTA as an anticoagulant. Smears
can be made with some of the aspirated (drawn out by suction)
marrow. The remaining material is poured into a small funnel
lined with “Filtrator Coffee Paper” (Zbar and Winter, 1959).
Rinse marrow, while in funnel, several times with saline, or until
the marrow has lost most of its color. ‘This also helps to wash the
material into one mass at the apex of the funnel. Partially clotted
marrow will be broken up and washed free of blood, leaving excel-
lent clear particles of marrow. Fold in the filter paper, place in
? Ralph M. Kniseley, Chief of Clinical Research and Training, Oak Ridge Institute of
Nuclear Studies. Personal communication.
Bone Marrow Staining 229

tissue receptacle and carry through fixation, washing, dehydration

and infiltration without removing. No marrow will be lost.
Minute amounts of marrow may prove difficult to recover from
the filter paper without loss of material. ‘he author has avoided
loss by allowing the paraffin around the marrow (on the filter pa-
per) to congeal slightly. Then carefully scrape paraffin and marrow
together and remove to melted parafhn in mold. The congealed
paraffin containing the marrow will sink to the bottom of the mold
or can be pressed down into desired position. This method keeps
the marrow concentrated reasonably well.
Gude and Odell (1955) recommend for dilution vinisi_ (Abbott
Laboratories), a 3.5% solution of polyvinylpyrrolidone (PVP) in
normal saline.

Endicott (1945) used plasma serum as a diluting fluid to thin

smears. The serum can be kept on hand for several months if
stored in refrigerator.
5. For a fluorescent method, see Werth (1953).

Phloxine-Methylene Blue (THoMAs, 1953)

FIXATION: any general fixative.
SOLUTIONS:
Phloxine solution:

phioxine BeGd.ApalO). ou... /) is em goers 0.5 gm.

GIStMMea Waters we vers css ences ao 2 eee 100.0 ml.
placiall ACEC ACIG'y ycday ss 2b. SS. > peer ein 0.2 ml.

A slight precipitate will form. Filter before use. (The author has
found this of no importance; the precipitate settles at bottom of
bottle and does not collect on the tissue.)
Methylene blue-azure solution:
methylene blue ryGil, 52015) 2. 2s ics 5 sees 0.25 gm.
azune 1. Cr or ONO! oo oss cee oe os Lee eo 0.25 gm.
DOLANG Bh oh hae he pee h Mes 6) ee ws oh ee 0.25 gm.
Mustilled: WATE 45% Peed oh an i wee 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCly.
2. Stain in phloxine: 1—2 minutes.
3. Rinse well in distilled water.
230 Hematologic Elements and Related Issues (CHAP. 16)

4. Stain methylene blue-azure: 0.5—-1 minute.

5. Partially destain in 0.2% aqueous acetic acid.
6. Complete differentiation in 95% alcohol, 3 changes.
7. Dehydrate, absolute alcohol; clear and mount.

RESULTS:
nuclei—blue
plasma cell cytoplasm—blue
other tissue elements—shades of rose and red

COMMENTS:
Thomas substitutes azure B for azure II of other methods because of
the uncertain composition of the latter. It is unnecessary to use
colophonium alcohol as in other methods. The author recommends
Thomas’ method over all others she has tried. It is practically fool-
proof.
Delez and Davis (1950) make up their phloxine with oxalic acid;
1% phloxine in 0.05% aqueous oxalic acid.
This staining method frequently is used for general staining in
place of a hematoxylin-eosin method.

Staining for Fibrin

Ledrum’s Acid Picro-Mallory Method (CULLING, 1957)
FIXATION: any good general fixative.

SOLUTIONS:
Celestin blue:

celestin bless Ca aha so ee 0.25 gm.

ferric alum, 5%, (5 gm. (100 milwater) 0... 50.0 ml.
Boil 3 minutes. Cool and filter. Add:
BIN GEOR: 3. eye aed yar ca ee cea a ke 7.0 ml.

Keeps for several months.

Mayer’s hematoxylin, see page 125.

Picro-orange:
picric acid saturated in 80% alcohol ......... 100.0 ml.
orange G, C.J." 16230) 2.5. 7 ea eek, ee 0.2 gm.
Staining for Fibrin 2 1Soe)—

Acid fuchsin:
acig wuchsime Gaeta cOB: 2.6). Sed bbeag cs oak se tat 1.0 gm.
dusted tet ats oo ic s.2 Gn as Soles awe am Be 100.0 ml.
frichiloracemGsaciGs ois icine Ova emeae Se ar ak 3.0 gm.
Phosphotungstic acid:
PMOsPMOMINeSiC AIC asi .0ruan eae ene eee 1.0 gm.
Gas tC aater 8 2 be Sod Seis are Bene a cgay aes 100.0 ml.
Aniline blue:
aniline blue, WS, GC.1. 42780 ; 2.2.8. 026..26.25 2.0 gm.
distilled waters, «code Ss fo hd es Meee ae Shee 100.0 ml.
Glacial ACEC ACI 27.3 hss ac oc ee a te Beet 2.0 ml.

PROCEDURE:

Deparaffinize and hydrate slides to water; remove HgCle.

Stain in celestin blue: 3—5 minutes.
Rinse in tap water.
Stain in Mayer’s hematoxylin: 5 minutes.
Wash in running water: 5 minutes.
Rinse in 95% alcohol.
Stain in picro-orange: 2 minutes.
Stain in acid fuchsin: 5 minutes.
Rinse in distilled water.
—OF
Soo
mm
oP
N
ont
— Dip in equal parts of picro-orange and 80% alcohol: few seconds.
11. Differentiate in phosphotungstic acid: 5-10 minutes, until colors
are Clear.
12. Rinse in distilled water.
13. Stain in aniline blue: 2—10 minutes.
14. Rinse in distilled water: 2-3 minutes.
15. Dehydrate; clear and mount.
RESULTS:
fibrin—clear red
erythrocytes—orange
collagen—blue
nuclei—blue-black

COMMENTS:
This method is specific for fibrin, setting it off sharply from other
tissue elements. See also Phosphotungstic acid-Hematoxylin (page
136), and Gram-Weigert (page 307).
 Chapter |'{

Pigments and
Minerals

Pigments are a heterogeneous group of substances containing enough

natural color to be visible without staining. Sometimes, however, color is
added to give them more intense differential staining. Some pigments
are artifacts, such as formalin pigment (page 7). Others, exogenous
pigments, are foreign pigments that have been taken into the tissue in
some manner. Carbon is a common pigment found in the lungs of city
dwellers, particularly of people from coal-burning cities. Endogenous
pigments are found within the organism and arise from nonpigmented
materials. Iron-containing hemoglobin can become broken down into
iron-containing pigment, hemosiderin, and a noniron-containing pig-
ment, hematoidin, or bilirubin (brown) which can be oxidized to
biliverdin (green). Normal hemoglobin, when not broken down into
hemosiderin, will not show a positive Prussian blue reaction (page 233).
The iron is masked or occult, and the organic part of the hemoglobin
molecule must be destroyed, the iron must be unmasked. Hydrogen
peroxide has become the reliable reagent for this purpose, producing
ferric oxide, and finally a fairly good Prussian blue reaction.
Melanin (brown or black pigment) is found normally in the skin,
hair, and eye, but may occur pathologically anywhere in the body.
Lipofuscin, sometimes known as the “wear and tear’ pigment, can be
found in the heart muscle, adrenals, ganglion cells, and liver. Melanin
and lipofuscin are brownish pigments stainable by fat dyes and some
g) 3 9
am
Testing for Iron 239

basic aniline dyes such as fuchsin. They are metachromatic (page 110)
with methyl green and give a positive Schiff reaction after periodic
acid treatment (page 298). Reference: Ham (1957).

‘Testing for Iron

According to Baker (1958) the iron reaction is an example of local

formation of a colored substance which is not a dye. It is a type of histo-
chemical test wherein a tissue is soaked in a colorless substance. Certain
tissue elements react with the substance and become colored. The well-
known test is the Berlin blue, Prussian blue or Perl’s reaction, in which
the iron is dissolved from hemosiderin by hydrochloric acid, and then
reacts with potassium ferrocyanide to form the Berlin blue precipitate,
ferric ferrocyanide.
Sometimes fading occurs in slides due to the reduction of the Berlin
blue to colorless ferro ferrocyanide. The mounting resin probably takes
up oxygen while drying and deprives the sections of oxygen, thereby
reducing them to the colorless condition. If this takes place and it is
essential to recover the slides, treat them with hydrogen peroxide. The
newer synthetics are not as prone to reduce Berlin blue as former resins;
also Lustron 2020 is recommended (page 118).

Tron Reaction (Gomori, 1936)

FIXATION: 10% buffered formalin; other general fixatives are satis-
factory but acid content may dissolve some of the hemosiderin (iron
bearing pigment, Lillie, 1954B).
SOLUTIONS:
Hydrochloric acid, 20%:
hydrochloric acid (sp.gr. 1.188-1.192, 37-38%
2 (6) en re 4 20.0 ml.
castiled iwater sc ie% sole etn sks ee 80.0 ml.
Potassium ferrocyanide, 10%:
potassium ferrocyanide, K,Fe(CN),-3H,O .... 10.0 gm.
Mistilleg@ water ba. e o.oo bees
bn oo ees 100.0 ml.

Safranin:
SairaninvOnG:1, SOTO! 4 6s 5-24 casa Pine Os 0.2 gm.
distilled water .__.. said a Ate tc,& cst dayteuat 2 _ 100.0 ml.
glacial acetic acid ..... Rn 20 8.2 70-44. eee 1.0 ml.
234 Pigments and Minerals (cHaP. 17)

PROCEDURE:
l. Deparaffinize and hydrate slides to water; remove HeCly.
2 Wash in distilled water: 1—2 minutes.
3. Place slides in equal parts of hydrochloric acid and potassium
ferrocyanide mixed immediately before use: 20 minutes. Do not
handle slides with metal forceps.
. Wash thoroughly in distilled water (not tap water).
. Counterstain with safranin (or other red nuclear stain): 2-3
minutes.
. Rinse in 70% alcohol.
. Dehydrate, clear, and mount.

RESULTS:
iron pigment—brilliant greenish blue
nuclei and other tissue elements—shades of rose and red

COMMENTS:
For masked or occult iron (Glick, 1949), pre-treat slides with 30%
hydrogen peroxide alkalized with dilute ammonia (1 drop/100 ml.
peroxide): 10-15 minutes. Wash well and proceed to step 3. If the
hydrogen peroxide tends to form bubbles under the sections and
loosen them, keep the solution and slides cool in the refrigerator dur-
ing the treatment.

Tron Reaction (HUTCHISON, 1953)

FIXATION: Hutchison recommends:
SOGMTEI SME ATCs Behe he ee eine cd 12.0 gm.
placialgacetic acta (04) as. ne ie oe A A ee 33.0 ml.
fisting sed, - eae tug gases ee SS oc, ec 40.0 ml.
distilledinvatersto make me.)
age ee 200.0 ml

Go directly into 70% alcohol from fixative.

SOLUTIONS:
Ferrocyanide-hydrochloric acid solution:
(@)y potassium: ferrocyanide Wks re hs osha: 2.0 gm.
distilled WatCiys-gpakio came ok. Stl 50.0 ml.
(a) Chydrachlomejgacid: 5,jee serm ete tse ice 2.0 ml.
distilled .waketa tigre eee es, A ci 50.0 ml.
Freshly mix above solutions, warm slightly and filter. Place in
paraffin oven, 56°C a short time before using.
Safranin, see page 233.
Testing for Iron 230

PROCEDURE:
1. Deparafhnize and hydrate slides to water.
2. Wash in distilled water: 3 minutes.
3. Treat with ferrocyanide-hydrochloric acid, 56°C: 10 minutes.
4. Rinse, several changes distilled water: 5 minutes.
5. Counterstain with safranin (or other red nuclear stain): 2--3
minutes.
6. Rinse in 70% alcohol.
7. Dehydrate, clear, and mount.

RESULTS:
iron pigment—brilliant greenish-blue
nuclei—red
other tissue elements—shades of red and rose

COMMENTS:
Hutchison claims that the warm solution is the most important step
in this method. Do not leave the slides in the solution longer than 10
minutes, and if they have been well washed in distilled water, pre-
cipitate seldom forms on the sections. In the author's experience, this
method produces deeper, more brilliant colors than Gomori’s
method.

Turnbull Blue Method for Ferrous Iron (PEARSE, 1953)

FIXATION: 10% buffered formalin; other general fixatives satisfactory if

acid is absent.
SOLUTIONS:
Ammonium sulfide: a saturated solution (NHy4).S, 20-30% content,
analytical reagent.
Potassium ferricyanide:
potassium ferricyanide, K3Fe(CN), ........... 20.0 gm.
distilled water .. totes A000 ani?
Hydrochloric acid:
hydrochloric acid (sp.gr. 1.188-1.192, 37-38%
jel C)| oe poe ae en Oe oe 1.0 ml.
distilled water ...... Se re ee 100.0 ml.

Safranin, see page 233.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCly.
2. Wash in distilled water.
236 Pigments and Minerals (cHar. 17)

3. Treat with yellow ammonium sulfide: 1-3 hours.

sae Rinse in distilled water.
5. ‘Treat with equal parts of potassium ferricyanide and hydrochloric
acid, freshly mixed: 10—20 minutes.
6. Rinse in distilled water.
7. Counterstain with safranin (or other red nuclear stain): 2-3
minutes.
8. Rinse in 70% alcohol.
9. Dehydrate, clear, and mount.

RESULTS:
ferrous iron and ferric iron converted to ferrous iron—deep blue
other tissue elements—shades of rose and red

Iron Reaction: Di-nitrosoresorcinol (HUMPHREY, 1935)

FIXATION: 10°% buffered formalin, general fixative without acid.
SOLUTIONS:
Ammonium sulfide, 30% analytical reagent.
Di-nitrosoresorcinol (resorcin green), saturated aqueous solution, or
a 3% solution in 50% alcohol.
PROCEDURE:
1. Deparaffinize and hydrate slides down to water.
2. Treat with ammonium sulfide: 1 minute.
3. Rinse in water.
4. Transfer to di-nitrosoresorcinol: 6—24 hours.
5. Wash in water (or 50% alcohol).
6. Dehydrate, clear, and mount.

RESULTS:
iron—dark green
background—brown
COMMENTS:
Reagent is more effective if solution has aged for a few days. Undis-
solved chemical should be present in the saturated aqueous solution.
Author’s reaction—a disappointingly dull slide.

Testing for Calcium

Von Kossa, modified from Mallory (1944)
FIXATION: 10% formalin or alcohol, although other fixatives give reason-
ably good results.
Testing for Calcium 237

SOLUTIONS:
Silver nitrate:
STAVE I Mal HAC INO tage (esa cx. d es Re ed 5.0 gm.
GistilledGwaltetaese 2 Bon 2-8... 55 Gi cecliaind Asela eee Me 100.0 ml.

Fresh solution always best; never use one more than | week old.
Safranin, see page 233.

PROCEDURE:
1. Deparaffinize and hydrate slides to distilled water; remove HgClo.
Treat with silver nitrate in dark: 30 minutes.
NO Rinse
Oo thoroughly in distilled water.
4. Expose slides (in distilled water) to bright light (75-100W_ bulb
satisfactory): 1 hour. Lay them over a white background to expe-
dite reaction.
5. Wash thoroughly in distilled water.
Counterstain in safranin (or other red nuclear stain): 2—3 minutes.
Rinse in 70% alcohol.
omaDehydrate, clear and mount.

RESULTS:
calcium deposits—dark brown to black
nuclei and other tissue elements—shades of red and rose

COMMENTS:
If iron blocks out the calcium, treat 10-15 minutes with .005M_ so-
dium EDTA (ethylenediaminetetraacetic acid) in normal saline.
Wash in distilled water and proceed to step 2. (McGee-Russell, 1958)
This reaction actually demonstrates phosphates and carbonates
rather than calcium itself, but since soluble phosphates and carbon-
ates are washed out, the calctum phosphates and calcium carbonates
remain to react with the silver (Lillie, 1954B) and the test can be
regarded as sufficiently specific (Pearse, 1953).
Renaud (1959) demonstrates that a high percentage of alcohol (at
least 80% content) in the fixing fluid is necessary in order to detect
calcium in some tissues (i.e., heart and coronary vessels). Water and
even buffered formalin can dissolve out some small deposits of cal-
cium salts.

Alizarine Red S, PEARSE (1953)

FIXATION: preferably one containing 80% alcohol, for maximum preser-
vation of calcium.
238 Pigments and Minerals (cHap. 17)

SOLUTIONS:
Alizarine red S:
alizanine reas) Gal: 580051. 6 Ae 1.0 gm.
Clistalliecna ater cc aks. it eR ea es 8 100.0 ml.

Toluidine blue:
toluidmetbiue,O, C.1. 52040 2 see.
-see: 0.1 gm.
GIStIMICU WATER: © 2.5.2... 2 Saas PS ee ce 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
. Stain in alizarine red: 3—5 minutes.
. Rinse in distilled water.
. Stain in toluidine blue: | minute.
bO.
OO
#®
Or Rinse in distilled water.
6. Dehydrate, clear and mount.
RESULTS:
calcium—orange-red
nuclei—blue
inorganic iron (not hemosiderin)—purple
COMMENTS:
Any counterstain should be kept light to provide contrast for the
alizarine red color.

Hemoglobin Staining
Benzidine Method (rRALPH’s, 1941; GLick, 1949)
FIXATION: alcohol or formalin.

SOLUTIONS:
Benzidine reagent:
DENZIdING*, 55.0) Ss Noe y. 2 ace eee ae Ps 1.0 gm.
absolute amethyl alcohol: Bettie.
ce tee on: 100.0 ml.
Peroxide reagent:
peroxide (reagent 3°97), 2 ie eee aoe tee 25.0 ml.
WOCZ ethyl alcoholism es ice toe 75.0 mil.
PROCEDURE:
1. Deparaffinize sections and hydrate to water. Not necessary for
smears, which will be fixed by benzidine reagent.
2. Flood with benzidine reagent: 1-2 minutes.
Hemoglobin Staining 239

Drain, and flood with peroxide reagent: 1.5 minutes.

ierWash in distilled water: 15 seconds.
Counterstain if desired with a red nuclear stain.
oe.Dehydrate, clear, and mount.

RESULTS:
hemoglobin—dark brown
For hemosiderin, see iron reactions, page 233.

Cyanol Reaction (DUNN, 1946)

FIXATION: formalin, preferably buffered to pH 7; fix no longer than 48
hours. Dried smears may be methyl alcohol fixed and then proceed to
step 2 below.
SOLUTIONS:

Cyanol reagent:
Stock solution:
CV AION sc. afha tere sce = 5G sad es HES 6 3 1.0 gm.
Gastillied water :.<\.42 03. ss2<.~ <n e ee ieee es es 100.0 ml.
TMC POWOED, OP: f aen oo0h. Sass ioe es eee aa 10.0 gm.
Placa ACCC AGM: 2 6 2 ess oes 2 eee na 2.01mi.

Bring to boiling point. In short time the solution is decolorized.

Stable for several weeks.
Working reagent:
Filter 10 ml. of stock reagent, and add:
Placiall ACEC ACA. i 4d 2s) bee) ae ee 20 1a,
hydrogen peroxide, epracreusall: oe A ore a 1.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides down to water.
2. Treat with working reagent: 3—5 minutes.
3. Rinse in distilled water.
4. Counterstain in red nuclear stain.
5. Dehydrate, clear, and mount.

RESULTS:
hemoglobin—dark blue to bluish grey
COMMENTS:
Gomori (1952) uses 1 gram acid fuchsin instead of cyanol and pro-
duces a red hemoglobin. Counterstain with hematoxylin or other
blue nuclear stain.
240 Pigments and Minerals (cHAp. 17)

Bile Pigment Staining

Glick (1949)
FIXATION: formalin or alcohol: approximately 6 hours.
SOLUTIONS:
Tincture of iodine:
OGUIITC MR OI A Toter. a!eo a che,Se, ee Ee eee 10.0 gm.
POLAssmMMTHOCICE >,» 2.8 es tae een ; 6.0 gm.
clistilec watery.” soc. Wo Asai. Aerie a eee 5.0) mle
Soe aCeuyl AlCOMOM ogee. achat Sats es 95.0 ml

Lugol’s solution:
HOROMENS os 0. A ae eae ae ee 1.0 gm.
potassium, LOGE ar. ..5-ak AIM ee eee 2.0 gm.
elistilled watering 2aLee. che eae ee 100.0 ml.

Dissolve potassium iodide in water first, then add iodine.

Stein’s working solution:
timcture of aodinel peak cue ieee oy eee 10.0 ml.
Lugol's; solution, uittuseepte:
Bopeon 3 ee 30.0 ml.

Sodium thiosulfate, 5%:

sodium tiiosuliates .4 i524: eee teen ee 5.0 gm.
distilled ..water (s--.0>. 4 OS eee 2 100.0 ml.

Safranin, see page 233.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. ‘Treat with Stein’s iodine solution: 6—12 hours.
3. Wash in distilled water: 5—10 minutes.
4. Bleach in sodium thiosulfate: 5—10 minutes.
5. Wash in distilled water: 2—3 minutes.
6. Counterstain in safranin (or other red nuclear stain): 2—3 minutes.
7. Rinse in 70% alcohol.
8. Dehydrate, clear, and mount.

RESULTS:
bile pigment—emerald green
nuclei—red
Melanin and Lipofuscin Staining 24]

COMMENTS:
This reaction can not be considered reliable at all times because the
reactants are diffusible; also the final color can spread from the
original site. The test is based on oxidation of the bile pigment
(bilirubin) to green biliverdin by the iodine solution.

Melanin and Lipofuscin Staining

Lillie (1956 A, B), Nile Blue Method
FIXATION: any good general fixative.
SOLUTION:
Nile blue A:
Nile bine AG SIS) .< sacar ax 0.05 gm.
sulfuric acid, 1% (1 ml. conc. H,SO,4/99 ml. dis-
tilled: water) .....:%.. : 3 aa, aR,ie ook. 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HeCl..
2. Stain in Nile blue 4 solution: 20 minutes.
3. Wash in running water: 10-20 minutes.
4. Mount in glycerol jelly.
RESULTS:
lipofuscins dark blue or blue green
melanin—pale green
erythrocytes—greenish yellow to greenish blue
myelin—green to deep blue
nuclei—poorly stained
Alternate Method:
PROCEDURE:
Steps 1 and 2 as above.
3. Do not wash in water. Rinse quickly in 1% sulfuric acid to remove
excess dye.
4. Dehydrate at once, acetone, 4 changes: 15 seconds each.
5. Clear in xylene and mount.
~

RESULTS:

cutaneous, ocular, meningeal melanins dark green

mast cells—purple red
lipofuscins—unstained but appear yellow to brownish
 242 Pigments and Minerals (cHap. 17)

muscle, myelin, erythrocytes—unstained

nuclei—greenish to unstained
COMMENTS:
Lipofuscins stain with Nile blue by two mechanisms: a fat solubility
method operating at PH below 1.0; and an acid-base mechanism oper-
ating at levels above pH 3.0. When stained by the second method,
they retain a green stain after acetone or brief alcoholic extraction,
but when the first mechanism is used, they are promptly decolorized
by acetone or alcohol. (Lillie, 1956 B)
Melanins stain with basic dyes at pH levels below 1.0 and retain
the stain when dehydrated and mounted. (Lillie, 1955)

Ferric-Ferricyanide Method (LILLIE, 1957)

FIXATION: avoid chromate fixatives, others are satisfactory.

SOLUTIONS:
Ferrous sulfate:
ferrous) sulfaten(FeSO.- 7H5O) ie lie. hac ae 2.5 gm.
distilled: water "a? ..f206 00 50) CONSE Rey 15 aT 100.0 ml.
Potassium ferricyanide:
potassium ferricyanide (K3Fe(CN),) .......... 1.0 gm.
Gistilledipweatcrurcct seen aus tke penal. 2a ieee 99.0 ml.
slaciailaacetic acide <0 5554. enemies. eee 1.0 ml

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. ‘Treat with ferrous sulfate: 1 hour.
3. Wash in distilled water, 4 changes: total 20 minutes.
4. ‘Treat with potassium ferricyanide: 30 minutes.
5. Wash in 1% acetic acid (1 ml./99 ml. water): 1-2 minutes.
6. Counterstain if desired, picro-ponceau satisfactory, do not use
hematoxylin.
7. Dehydrate, clear, and mount.

RESULTS:
melanin—dark green
background—faint greenish or unstained; with picro-ponceau, col-
lagen stains red and muscle and cytoplasm will be yellow and brown
COMMENTS:
Lillie says this method is highly selective. No other pigments react
in this procedure, except occasionally hemosiderin.
 Removal of Pigments 243

Morton ef al. (1960)

FIXATION: any general fixative, or fresh frozen sections.
SOLUTION:
sodium-2,6-dichlorobenzenone-indophenol .... 0.1 gm.
DOS etlivl AICONOL 24 face. 3 4p aeeyy ena sce 100.0 ml.
Prepare just before use. Filter and add:
1% hydrochloric acid (1 ml./99 ml. water) until color of solution
is red (approximately 1:5) or until pH is 2.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. ‘Treat with HCl-indophenol: 5 minutes.
3. Dip in tap water several times until background loses color.
4. Mount in glycerol jelly or Apathy.
RESULTS:
lipofuscins—red
erythrocytes—blue
other tissue elements—colorless to blue or slight pink
COMMENTS:
Avoid alcohol; it removes color immediately. Stain fades in about 1
day.

Removal of Pigments
Melanin pigment (PEARSE, 1953)
This will appear as brown, greyish, or almost black granules.
Permanganate Method of Removal
1. Hydrate slides to water.
0.1% potassium permanganate, aqueous: 12-24 hours.
Wash in running water: 5 minutes.
bo 1% oxalic acid, aqueous:
CO
Hm | minute.
5. Wash in running water: 10 minutes, and proceed to stain.
Performic or Paracetic Acid Methods of Removal
Lillie (1954 B) claims that the above method can be unpredictable
and that the best bleaching is done with performic and paracetic acid:
1-2 hours:
PERFORMIC ACID: to 8 ml. of 90% formic acid, aqueous, add 31 ml.
of 30% H2O. (undiluted reagent) and 0.22 ml. of concentrated
244 Pigments and Minerals (CHAP. 17)

H.SO,. Keep at or below 25°C. About 4.7% performic acid is formed

within 2 hours, but it deteriorates after a few more hours.
PERACETIC ACID: to 95.6 ml. glacial acetic acid add 259 ml. of 30%
H,O, (undiluted reagent) and 2.2 ml. concentrated H,SO,. Let stand
1-3 days. Add 40 mg. disodium phosphate as stabilizer. Store at
0.5°C. Keeps for months in refrigerator.
Chlorate Method of Removal
Treat sections 24 hours in 50% alcohol plus small mount of potas-
sium chlorate and a few drops of HCl (concentrated). Wash 10
minutes before staining.
Bromine Method of Removal
1% bromine in water: 12-24 hours. Wash well before staining.
Chromic Acid Method of Removal
Mixture of equal parts of 1% chromic acid and 5% calcium chlo-
ride, aqueous: 8—12 hours. Wash well.

Peroxide Method of Removal

10% H2Oz: 24-48 hours. Wash well before staining.
The peroxide method is considered by some to be the best; it is
specific for melanin; other pigments resist longer than 48 hours. ‘The
permanganate and chromic acid methods bleach all pigments, which
allows for no differentiation among them.

Formalin pigment
Brown and black crystalline granules and artifacts, produced by
formalin, are found in and around blood.
Baker (1958) Method of Removal
1% potassium hydroxide in 80% alcohol, or picric acid dissolved
in alcohol, until precipitate is removed.
Murdock (1945) Method of Removal
3% HeOvc, 25 ml.; acetone, 25 ml.; and 1 drop ammonia, until
precipitate is gone: 1—2 hours.
Barrett (1944) Method of Removal
Saturated solution of picric acid in alcohol: 10 minutes to 2 hours.

Malarial pigment
This appears as amorphous brown-black granules.
oS
Removal of Pigments 245

Gridley (1957) Method of Removal

Method 1:
1. Slides down to water.
2. Bleach for 5 minutes in:
AECCUO MG MeReeth OR Kas, 2.5 sa anne Gaeta 50.0 ml.
BU/g ails
Obert a a.2 Aen g 2m oo puke eke on., —00h anil:
28-29%, ammonia (conc.) ........ 7 1.0 ml.
or overnight in 5% aqueous ammonium sulfide (diluting 20%
analytical reagent 4:1).
3. Wash thoroughly, running water: 15 minutes or longer.
Method 2: 39, Yi2O,> 2 hours.

Carbon

Carbon (opaque black) usually appears in the lungs and adjacent

lymph nodes, sometimes in the spleen and liver. If it is necessary to
distinguish it from malarial pigment, iron, or some other pigment or
precipitate, carbon is black and is insoluble in concentrated sulfuric
acid, in which other pigments will dissolve.

Hemosiderin

This yellowish, brownish, or greenish brown pigment resists

bleaching and does not dissolve in alkalis or acids. It can be identified
by Perl’s, test, pagee925
233.

Bile pigments
These are yellowish green and resist bleaching, but can be con-
verted by H2Oz, Lugol’s solution, nitrous acid, or dichromates into
greenish biliverdin-emerald green. Use Lugol’s solution (page 410):
6-12 hours. Decolorize with sodium thiosulfate.
Glick (1949) does not consider this reliable, because of diffusibility
of the reactants.
For extensive consideration of pigments and other organic sub-
stances, see: Glick (1979); Gomori (1952); Gurr (1998).
 Chapter 1

Cytoplasmic
Elements

Protein Staining
Protein demonstration is of minor importance, but sometimes it
becomes necessary to determine the protein or nonprotein nature of
eranulation in cells. The Millon reaction is one of the oldest and most
reliable methods but actually is specific for phenols and for the one
amino acid, tyrosine.

Millon Reaction (Gomori, 1952)

FIXATION: any good general fixative.
SOLUTIONS:
AL.: MET CUE GACELALE pNee Pe cin. ets een 5.0 gm.
distilledicowater. 02 see ete... ci. ee et a oe 100.0 ml.
trichloracetic-;acith mrmimetis
ss7Seat Me.) 15.0 gm.
B. sodiumypminite, 197 aAqgMeeuse. oS. yaa aN 10.0 ml.

PROCEDURE:
1. Run sections down to water. (Cut sections at least 10 microns
thick.)
2. Incubate sections in solution A, 30—37°C; 5-10 minutes.
3. Add solution B: incubate another 25 minutes.
4. Rinse in 70% alcohol.
5. Dehydrate, clear and mount.
 The Argentaffin Reaction ho—— ~I

RESULTS:
tyrosine proteins—pink to brick red

Ninhydrin Reaction (SERRA, 1946)

FIXATION: 10% formalin.
SOLUTION:
Ninhydrin:
phosphate buffer, pH 6.98, page 418.
ninhydrin, 0.4% aqueous (triketo-hydrindene-hydrate)
Mix equal amounts for staining.

PROCEDURE:
1. Deparaffinize and hydrate slides to water, or use frozen sections.
2. Place slides on a rack over boiling water, cover with stain, and
steam for 1—2 minutes.
3. Drain stain off the slide and mount in glycerol jelly.
RESULTS and COMMENT:
Blue or violet color indicates the presence of amino acids, peptides,
and proteins. The slide should be examined at once because the color
diffuses readily and begins to fade within a day or two.

The Argentafhn Reaction

The argentaffin reaction should not be confused with silver impreg-

nation (page 175). In argentaffin reactions some substances (ascorbic
acid, aldehydes, uric acid, polyphenols, and others) reduce silver solu-
tions under specific conditions. This reaction with the tissue itself,
therefore, can be used histochemically to identify these substances.
The only source of error in the method can be found in calcification
areas, but only if these are in large masses. Most silver phosphates and
carbonates will be dissolved out during the the process. If not, the
slides can be treated with 0.2 to 0.59% nitric acid or hydrochloric acid
in absolute alcohol, 2-3 minutes before step 3 below. Wash off the acid
in absolute and 95% alcohol and then proceed with step 3.

Uric Acid Staining

Argentaffin Method (Gomori, 1952)

FIXATION: 95% or absolute alcohol.
248 Cytoplasmic Elements (CHAP. 18)

SOLUTIONS:
Methenamine silver solution:
silver nitrate, 5°% aqueous (5 gm./100 ml. wa-
tr) cee ee es. Ss os eee es 5.0 ml.
methenamine, 3°% aqueous (3 gm./100 ml. wa-
CE) rene re coos od 2 gee se 100.0 ml.
Mix and shake. Precipitate which forms redissolves. Keep in cool
dark place. Good for several months.
Buffer solution, pH +9.0:
M/5 boric acid (12.368 gm./1000 ml. water) ... 20.0 ml.
M/20 borax (19.071 gm./1000 ml. water) ..... 80.0 ml.

Working solution:
methenamine silver Solution 2.127% 0275.24 30.0 ml.
bniter solutions 4% .h 5... se face eee 8.0 ml.

PROCEDURE:
1. When mounting sections on slides, float on water only a few sec-
onds or try 95% alcohol.
Deparafhnize and run slides down to 95% alcohol.
ND Treat
oS in methenamine silver working solution, prewarmed to
37°C: 30 minutes.
Rinse in distilled water.
Fix in sodium thiosulfate, 5% aqueous: 3 minutes.
Wash in running water: 5 minutes. (Gold toning optional)
ee
PE Counterstain if desired.
8. Dehydrate, clear, and mount.
RESULTS:
uric acid crystals—black crystalline fine granules

Enterochromaffin (EC) Cell Stain

Fontana Method (cuLLING, 1958), and Methenamine Silver
(Gomor!I, 1952; 1954B)
FIXATION: Zenker formol preferred; do not use alcohol—it dissolves
argentaffin granules.
During embedding, do not expose to paraffin longer than 30 minutes.
SOLUTIONS:
Silver solution A: (Fontana Method)
To 25 ml. of 10% silver nitrate (10 gm./100 ml. water) add ammonia
The Argentaffin Reaction 249

(28%), drop by drop until the precipitate which forms is almost dis-
solved. Add 25 ml. distilled water. Store for 24 hours in brown bottle.
Filter before use. Good for 2 weeks.

Methenamine silver solution B: (Gomori’s Method)

Solution TI:
methenamine, 3% (3 gm./100 ml. water) ...... 100.0 ml.
silver mitrate, 59, (6 gm. /100 mili -water)i..- 2.26. 5.0) mil.
Shake until white precipitate disappears. Keeps for several months
if stored in a cool dark place.
Solution IT:
M/5 boric acid (12.368 gm./1000 ml. water) ... 80.0 ml.
M/5 borax. (19,071/1000 ml. water) 4... &.< 20.0 ml.

pH should be 7.8 to 8.0

Working solution:
SHOCKS OMITIOID Das 8rd ies: 5, LA ee eee ats 30.0 ml.
Stock soliton 432) Se 2 acc 2s a oO Sie wee 24 8.0 ml.

Gold chloride:
gold chlonde stock solution, 1% «4.¢.«5:86++- 10.0 ml.
@ISGTEM GWaAtee op 52he dc erscess « ok hee Lae: 100.0 ml.

Safranin:
safranin O, C.T. 50240 ..........0..c000ec0.- 1.0 gm.
Gismled water: 20.3 ses oguls loc cts be keen 100.0 ml.

Add a few drops of glacial acetic acid.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. ‘Treat with Lugol’s solution (page 410): 30 minutes to 1 hour.
3. Wash in running water: 3 minutes.
4. Bleach in sodium thiosulfate, 5°% (5 gm./100 ml. water): 3 min-
utes.
Or Wash in running water: 5 minutes. The above treatment sup-
presses background staining.
6. ‘Treat with silver solution 4 in dark, room temperature: 18—48
hours.

Alternate Method. Use Methenamine silver solution B, 60°C: 3.5

hours; or at 37°C: 12-24 hours—until EC cells stand out black.
Then continue:
250 Cytoplasmic Elements (cHAP. 18)

7. Rinse in several changes of distilled water.

8. ‘Tone in gold chloride: 10 minutes.
9. Rinse in distilled water.
10. Fix in sodium thiosulfate, 5% (5 gm./100 ml. water): 3 minutes.
11. Wash in running water: 5 minutes.
12. Counterstain in safranin (or other red nuclear stain).
13. Rinse in 70% alcohol: few seconds.
14. Dehydrate, clear, and mount.

RESULTS:
argentafin granules—black
melanin—black
other tissue elements—reds and pinks
grayish to blackish background—slide is overstained

COMMENTS:
Chromaffin material is found only in the adrenal medulla and the
gastrointestinal tract, and received its name because of its reaction
with chromium salts and certain other metals to produce a yellowish
to brown color, a brownish chromium oxide. A dichromate fixative
should be used to preserve this material, or post-chromating with po-
tassium dichromate, page 23. Chromaffin is also argentaffin and is
blackened in the above method. Melanin, also brownish, is not a
chromaffin substance.

Diazo-safranin Method (LILLIE ef al., 1953)

FIXATION: 10% formalin buffered with 2% of calcium acetate: 1-3 days.
SOLUTION:
Diazotized safranin:
Stock solutions:
Acid safranin:
Safranin Oza: (50240) cae erat)
racers takite 3.6 gm.
q@ustilled swatety. s.,23 4 Sees
ose as ee 60.0 ml.
INGHIGI?. wre on |. RRR ER re ie eh ps 30.0 ml.

Stable for several weeks.

N sodium nitrite:
Socmonn mitrite, NaIN@s ne
tes ore ee 6.9 gm.
GISEMMNEG: Water ... 3. ace Cake ieee taco). ae 100.0 ml.

Keep in refrigerator, stable for 3 months.

The Argentaffin Reaction 2 wt —

Disodium phosphate, M/10:

disodium phosphate, Na,HPO, anhydrous 14.2 gm.
distilled water ...... eee eR ss Ue core 1000.0 ml.

Working solution: (do not prepare until immediately before use,

see step 2 below)
To 4.5 ml. of ice-cold safranin solution add 0.5 ml. of sodium nitrite
solution. The resulting mixture turns deep blue and foams. Keep at
0 to 5°C for 15 minutes for diazotization. Dilute | ml. of the solution
with 40 ml. of ice-cold disodium phosphate solution and use imme-
diately. (pH should be about 7.7)
PROCEDURE:
1. Deparaffinize and hydrate slides to distilled water.
2. Place slides in a previously chilled coplin jar and pour over them
the freshly prepared diazo-safranin solution: 5 minutes.
3. Decant stain and wash slides in 3 changes of N/10 aqueous hydro-
chloric acid or acid-alcohol (1 ml. conc. HC1/99 ml. 70% alcohol):
10-30 seconds total, to remove most of adherent stain. Longer ex-
traction lightens the colors but does not improve contrast.
4. Wash off acid with water (if N/10 HCl is used) or 95% alcohol
(if acid-alcohol is used).
5. Dehydrate, clear, and mount.
RESULTS:
enterochromaffin granules—black
eastric gland, chief cell granules—dark red
Paneth cell granules—red
mucin—unstained
nuclei and cytoplasm—pink to red

Ferric-Ferricyanide Method (Modified Schmorl Technic)

(LASKY AND GRECO, 1948; LILLIE AND BURTNER, 1953)

FIXATION: 10% formalin buffered with 2% of calcium acetate: 1—3 days.

SOLUTION:
Ferric-ferricyanide:
potassium ferricyanide, 1% aqueous ......... 10.0 ml.
termic calonde, 17, 2Queous <5: .22 .. +98.
oaee 75.0 ml.
Gistled WateIiG eidee seal es biol oven ieee 15.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Stain in freshly prepared ferric-ferricyanide: 5 minutes.
 hdOoXe) Cytoplasmic Elements (CHAP. 18)

3. Rinse in 3 changes of distilled water.

4. Counterstain in safranin, or like.
5. Dehydrate, clear, and mount.

RESULTS:
enterochromaffin granules dark blue
nuclei—red

COMMENTS:
Lillie and his co-workers have been devoting several years to research
on the enterochromaflin granules along with melanins, etc. A few of
these references are: Glenner and Lillie (1957); Lillie (1955, 1956A,
B, ands@, 1957A,. B, and-C, 1960; 1961); Lallie eh ala(1953)"balieves
als(San); Lillie’ et.al. (1961).

Azo-Coupling Method (curr, 1958)

FIXATION: formalin or Bouin’s.

SOLUTION:

Garnet reagent:
Garnet GB Grater ee eee”
eee 0.5 gm.
distilledwater® w+. eer eee 100.0 ml.
borax, saturated aqueous! (2288
sees. < ee 2.5 mil:

If it is necessary to purify the garnet GBC, see page 346.

PROCEDURE:
1. Deparafhinize and hydrate to water.
Transfer to garnet solution: 30-60 seconds.
Wash in running water: 30 seconds.
Stain in Mayer’s (or other) hematoxylin: 3 minutes.
Wash in running water: 5 minutes.
1S
oo
Doe Dehydrate, clear and mount.

‘ RESULTS:
argentaffin granules—red
nuclei—blue
background—light yellow

* Imperial Chemical Industries, Manchester, England, is best source, Other sources: Dajac
Laboratories or American Cyanamid Company.
Fat Staining 253

Fat Staining
Lipids (synonyms: lipoids, fatty substances) include a large number
of substances grouped together because of their solubility properties.
They are insoluble in water and soluble in fat solvents: alcohol, ether,
chloroform pyridine, benzene, and acetone, to name a few. Classifying
the lipids is not to be undertaken here, but a few familiar groups can
be mentioned: carotenoids (vitamin A), fatty acids, triglycerids (neutral
fats), phosphatids, and lipid pigments (lipofuscins—page 232).
For the fixation of fats, formalin is best, particularly if 1% of calcium
chloride is added to make the phospholipids insoluble (Gomori, 1952).
Because of the use of fat solvents, the tissue cannot be embedded in
paraffin or nitrocellulose, but it can be embedded in carbowax. Frozen
sections are simpler and most frequently used. During any processing
alcohol higher than 70% must be avoided. (Concerning the fixation of
lipoids, see Elftman, 1958.)
The dying of fats is one of the simplest forms of staining; the color-
ing agent merely dissolves in a fluid contained within the tissues. In
addition, it should be emphasized that a dye solvent should be used
which does not dissolve the lipid itself. The dye, therefore, must meet
certain requirements:
1. The dye must be strongly colored.
2. It must be soluble in the substance which it is intended to show, but
must not be soluble in water, the major constituent of cells.
3. It must not attach itself to any tissue constituents except by solution.
4. It must be applied to tissues in a solvent which will not dissolve the sub-
stance to be dyed, and must be less soluble in the solvent than in the
substance.
Baker (1958) suggests the name “‘Lysochromes” for these dyes that
dissolve in the tissue elements to be colored. The name is derived from
the Greek lusis meaning solution. The Sudans were the first synthetic
dyes of this sort, followed by the Nile blues and reds.
These dyes are used in saturated solutions and often introduce a
problem of dye precipitate on the tissue. Vlachos (1959) makes a sensi-
ble proposal that the precipitate probably is formed by the solution be-
coming oversaturated and that perhaps a saturated solution is unneces-
sary. He suggests two alternatives:
1. Make up the concentration below the saturation point; for example, 0.25
gm. Sudan IV per 100 ml. 609% alcohol.
 54
ihe) Cytoplasmic Elements (CHAP. 18)

i) Desaturate the solution by: (a) dilution of a saturated solution with equal
volumes of 60% alcohol, or (b) by refrigeration. When the solution has
been refrigerated long enough to have acquired the refrigerator tempera-
ture, filter. Either method does not alter the staining quality of the solu-
tion and produces negligible amounts of precipitate.

Oil Red O for Fat

FIXATION: 10% formalin or other aqueous general fixatives.
SOLUTIONS:
Oil red O stock solution:
oil red O, C.I. 26125, saturated in 99% alcohol (250-500 mg.
dye/100 ml. alcohol)
Working solution:
stock solution
distilled water

Let stand 5-10 minutes (no longer). Filter. Use immediately.

Hematoxylin, see page 125.
PROCEDURE:
1. Frozen sections, 15 microns.
2. Place in distilled water.
3. If HgCl, is present, treat with Lugol’s and thiosulfate; wash.
4, Stain in oil red O: 10 minutes, in closed container to reduce evap-
oration. If dye begins to settle out before 10 minutes have elapsed,
remove sections to water, or precipitate may settle on sections.
5. Wash in water. Make transfers with glass rod and wipe it clean
after each section.
6. Stain in Mayer’s hematoxylin (or like): 2-3 minutes.
7. Wash in tap water: 3 minutes.
8. Blue in Scott’s solution (page 412): 3 minutes.
9. Wash in tap water: 5 minutes. Mount in gum syrup or glycerol
jelly. For permanency, ring cover glass.
RESULTS:
fat—orange-red or brilliant red
nuclei—blue

Oil Red O with Tween 40 (BELL, 1959)

FIXATION: Baker’s formalin recommended (page 14), or other general
aqueous fixative, no alcohol.
Fat Staining 255

SOLUTIONS:
Alcohol-Tween solution:
a Bary evey a2 est 2 op ekce
een eee 10) mil.
Gustilledswaten tot. + atic. ok 4 ee Aes 70.0 ml.
absolute alcohol to make .. ....920
14 026c445 100.0 ml. total

Oil red O solution:

Add to above, 250 mg. Oil red O, C.I. 26125. Incubate at 60°C for
not less than 24 hours well sealed to prevent evaporation. Agitate oc-
casionally. Cool, filter under vacuum.
Hematoxylin,
j
see page 125.
D5

PROCEDURE:
1. Frozen sections, 15 microns; place in water.
2. Transfer to alcohol-Tween without dye: 10 minutes, agitate.
3. Stain at least 4 hours: may be overnight.
4. Differentiate in alcohol-Tween: 10-15 minutes.
5. Wash in distilled water.
6. Stain in Mayer’s hematoxylin (or like): 2—3 minutes.
7. Wash in water, and blue in Scott’s solution.
8. Wash in tap water: 5 minutes. Mount in gum syrup, glycerol jelly,
or Farrant’s medium. Ring cover glass for permanency.

RESULTS:
fat—brilliant red
nuclei—blue

COMMENTS:
This method rarely deposits precipitate on the sections. “The author
always uses it in preference to the former method.

Sudan IV or Sudan Black B (CHIFFELLE AND puTT, 1951)

FIXATION: 10% formalin, no alcohol.

SOLUTION:
Dissolve 0.7 gm. Sudan IV, C.I. 26105, or Sudan black B, C.I. 26150,
in 100 ml. propylene or ethylene glycol. Add small amounts at a time,
and heat to 100—110°C, stirring, for a few minutes. Do not exceed
110°C or a gelatinous suspension is formed. Filter hot through What-
man #2 paper, and cool. Filter again through fritted glass filter with
aid of suction, or glass wool with vacuum.
2 Atlas Powder Co., Wilmington, Delaware.
256 _ Cytoplasmic Elements (cHAP. 18)

PROCEDURE:
1. Frozen sections, 15 microns, into water.
2. Place in pure propylene or ethylene glycol: 3-5 minutes, 2 changes.
Agitate.
3. Stain, 2 changes: 5—7 minutes each, agitate occasionally.
4, Differentiate in glycol and water (85:15): 3-5 minutes, agitate.
5. Wash in distilled water: 3—5 minutes.
6. Counterstain in hematoxylin if desired.
~I Mount in glycerol jelly.

RESULTS:
fat—Sudan IV, orange to red; Sudan black B, blue-black to black

COMMENTS:
Chiffelle and Putt recommend glycols as a perfect solvent for a fat
stain because it does not extract lipids.
Zugibe et al. (1958, 1959) suggest Carbowax 400 as a solvent for Oil
red O and Sudan IV.
Gomori (1952) questions the use of glycols because they are sol-
vents for so many water-insoluble substances, and he prefers triethyl
phosphate. It has a low volatility and is harmless to lipids. His method
follows.

PROCEDURE:
1. Frozen sections are rinsed in water and transferred into 50% al-
cohol: few minutes.
2. Stain in a saturated, filtered solution of any of the fat dyes in 60%
triethylphosphate, 5-20 minutes.
. Differentiate in 50% alcohol: 1 minute.
09
He Counterstain in hematoxylin or any preferred stain.
5. Mount in glycerol jelly.

Osmic Acid (MALLory, 1944)

FIXATION: 10% formalin, no alcohol.

SOLUTION:
Osmic acid:
oOsmic acid ((Osmitim sretraoxide) a wae 22. G 1.0 gm. ampule
daisiiied Water. o..ee heen. ae ee 100.0 ml.

With file, score a circle around ampule and drop it into bottle with
the distilled water. Several sharp shakes will break the ampule and
Fat Staining 257

allow the water to dissolve the crystals. This method eliminates pos-
sibility of breathing the fumes.
PROCEDURE:
1. Frozen sections, 15 microns, into water.
2. Transfer to osmic acid solution: 24 hours.
3. Wash in several changes distilled water: 6-12 hours.
4. ‘Treat in absolute alcohol: 4—5 hours, for secondary staining of fat.
5. Wash well in distilled water: 5 minutes or more. .
6. Mount in glycerol jelly.
RESULTS:
fat—black
background—yellowish brown

Naphthalene Yellow (sILLs AND MARSH, 1959)

Naphthalene yellow in 60% acetic acid was used to restore the yellow
color to the fat of gross formalin-fixed specimens. This cannot be used
for sections; the color is too pale.

Fluorescent Method (‘METCALF AND PATTON, 1944; PELTIER, 1954)

FIXATION: 10% formalin (salts of heavy metals—HeCls have a quench-

ing effect on fluorescence), or use fresh tissue.

SOLUTION:
Phosphine 5R:
PROspIIne SRS hes 8.c%.. =< age eens ee 0.1-1.0 gm.
Aistatled watery. me irseyee f ic es ode 1 gE oe 100.0 ml.

PROCEDURE:
1. Cut frozen sections, 10 microns.
2. Wash in distilled water.
3. Stain in phosphine solution: 5 minutes.
4. Rinse quickly in distilled water.
5. Mount in glycerol, or examine as a water mount.
RESULTS:
lipids (except fatty acids, cholesterol and soaps) will fluoresce brilliant
white
COMMENT:
For details concerning fluorescent microscope and equipment, see
page 102.
*George T. Gurr, London, England; or Pfatz and Bauer, Inc., Empire State Bldg., N.Y.
258 Cytoplasmic Elements (cHaAP. 18)

Nile Blue A (Sulfate) (MALLORY, 1938)

This is a useful method for separating neutral fats from other fats.
FIXATION: 10% formalin, no alcohol.
SOLUTION:
Nile blue A:
Nilceiwree teks... 511801... oe Meee eel lee 1.5 gm.
CIS WALET oes. oycosiscs « oyepee: hs Ree A & eur 100.0 ml.

PROCEDURE:
1. Frozen sections, 15 microns, into distilled water.
. Stain in Nile blue sulfate: 20 minutes.
NO
oo. Rinse in tap water.
4. Differentiate in 1% acetic acid (1 ml./99 ml. water), 10-20 min-
utes, or until colors are clear; can happen in 1—2 minutes.
5. Wash thoroughly, distilled water, several changes.
6. Mount in glycerol jelly or A bopon.
RESULTS:
neutral fats—pink to red
fatty acids—blue to violet
nuclei and elastic tissue—dark blue
COMMENTS:
There appears to be some doubt about the specificity of this reaction
—whether it actually differentiates between neutral fats and fatty
acids. Gomort (1952) says that not all structures staining pinks, reds,
or blue are lipid. Use the method with tongue in cheek.

Staining for Phospholipids (ELFrMAN, 1957B)

FIXATION:
mercuric chloride, 59% aqueous! 4... )..5.9heee 100.0 ml.
otassium /dichromate: .4 .. chaos {iene 2.D. em,

Mix fresh. Fix for 3 days; oxidizes phospholipids and makes them in-
soluble in fat solvents such as are used in paraffin embedding.
SOLUTIONS:
Sudan black B:
Dissolve 0.7 gm. Sudan black B, C.1. 26150, in 100.0 ml. ethylene gly-
col with heat, 100-110°C (not above). Stir. Filter while hot through
Whatman #2 filter paper. Cool and filter through fritted glass filter
of medium porosity with suction.
Golgi Apparatus and Mitochondria 259

Ethylene glycol solution (or propylene glycol), 85%:

Cthyleme INCOM g as oy a ee eee ae hha 85.0 ml.

GIStINEC Water sole o-oo o 24,e eta ee oe ek ee wre Oa 100.0 ml.

PROCEDURE:

1. Deparaffinize and transfer slides into absolute alcohol: 2 minutes.

2. ‘Transfer to absolute ethylene glycol.
3. Stain in Sudan black solution: 30 minutes.
4. Differentiate in 85% ethylene glycol: 2-3 minutes.
5. Wash in distilled water.
6. Counterstain, if desired.
7. Wash well, mount in A pathy, or in glycerol jelly.

RESULTS:

phospholipids—black

COMMENT:
Phospholipids are finding increasing popularity in the study of mito-
chondria and Golgi apparatus, and are an important constituent of
myelin.

Golgi Apparatus and Mitochondria

The Golgi apparatus (Golgi bodies, Golgi substance, Golgi complex)

usually is lost in routine fixation and requires special treatment. Since
the methods are not always predictable under all conditions, it may be
necessary to modify the fixing and/or staining time before attaining
precise results. In osmium and silver methods the Golgi appears either
as a dark net, a granular mass, a cord, or even a more diffuse condition.
These methods seem to indicate it is fatty in nature.
Mitochondria are tiny bodies, granular, rod-like or filamentous in
shape and scattered through the cytoplasm. They are lipo-protein in
composition and are easily lost in routine preparation. It is therefore
imperative that small pieces of tissue be fixed rapidly in order to pre-
serve any mitochondria.

Golgi Apparatus Staining

Osmium Tetroxide: Ludford’s Method (LILLiE, 1954B; cowpry, 1952)

260 Cytoplasmic Elements (CHAP. 18)

FIXATION: Mann’s osmic sublimate: 18 hours.

osmic acid, 1% (1 gm./100 ml. water) ......... 50.0 ml.
mercuric chloride, saturated aqueous, plus 0.37
gm. SOcimerrculonide. - "Ls ceiyen
bee ss - 50.0 ml.

PROCEDURE:
1. Wash blocks of tissue in distilled water: 30 minutes.
2. Impregnate, 2% osmic acid: 3 days, 30°C
2%, osmic acid: | day, 35°C
1% osmic acid: | day, 35°C
0.5% osmic acid: | day, 35°C
Wash in distilled water: | day.
Dehydrate, clear, and embed.
Section 6—7 microns, mount and dry.
CD
Shag
Deparaffinize, clear, and mount.

RESULTS:
golgi apparatus—black
yolk and fat—black (these may be bleached out with turpentine)
COMMENTS:
If it is advantageous to have mitochondria stained on the same slide,
follow deparaffinization (step 6) by hydrating to water (include cau-
tious treatment with 0.125% potassium permanganate), and stain by
Altmann Method (page 263). Mitochondria will be crimson.

DaFano’s Method (CULLING, 1957; Cowpry, 1952)

FIXATION: 3—18 hours in:
Cobalt gintr ates. cooks ads, se metas eax ee ea 1.0 gm.
distilled swater 4. 45 1... neenya. 1 are 100.0 ml.
HOVIMA LIM hy ies oso apy ea RS ig. cone eee 15.0) mal:

SOLUTIONS:
Ramon y Cajal’s developer:
DiviGlGOG(UINOMG Sukie ts ects A har AGERE cas 2.0 gm.
FORMAL, 2 degree ke ata aE ee. Lee 6.0 ml.
cistillenbwidter-o.%. wakes sac a ss oo ae 100.0 ml.
sodiom sulfite, anhydrous ........-1.-...8.4: 0.15 gm.
Gold chloride:
gold chloride stock solution (page 410) ...... 1.0 ml.
Gistilled Wakes, . 54s 4. a 80—90.0 ml.
Golgi Apparatus and Mitochondria 261

PROCEDURE:
1. Rinse blocks of tissue in distilled water.
2. Impregnate in 1.5% silver nitrate (1.5 gm./100 ml. water): 1-2
days. (Can use 1% for very small pieces or embryonic tissues, 2%
for fatty tissues and spinal cord).
3. Rinse briefly in distilled water.
4. Cut blocks into slices thinner than 2 mm. Reduce in developer:
5 hours.
Wash thoroughly in distilled water.
Dehydrate, infiltrate, and embed.
Section, 6—7 microns, mount and dry.
Deparaffinize and hydrate to water.
9. ‘Tone in gold chloride: 2 hours.
10. Rinse in distilled water and fix in 5% sodium thiosulfate: 3 min-
utes.
11. Wash in running water: 5 minutes.
12. Counterstain, if desired, in hematoxylin, thionin, carmalum, etc.
13. Dehydrate, clear, and mount.

RESULTS:
golgi—black
cytoplasm—grey
mitochondria—medium to dark grey or black
COMMENTS:
The silver preparations depend on fixation with salts of a heavy metal
—cobalt in DaFano’s method. Aoyama (Baker, 1945) varies the
method using
cada “chlOniges fran. 4o. 6.45440 < dee a 1.0 gm.
DOHA. 2 2. oc ek Says oo mice 4 nf) 4-8 Be 15,0) anil,
distilled: water: 2... 2244s ood ss oe. 85.0 ml.

The rest of the procedure is the same as DaFano’s.

Direct Silver Method (ELFTMAN, 1952)

PROCEDURE:
1. Immerse small blocks of fresh tissue in silver nitrate, 2% (2 gm./
100 ml. water): 2 hours.
Rinse briefly in distilled water.
oo
bo Develop: 2 hours, in:
hydroquinone ...... ident 2 4 oo 1A 2.0 gm.
formalin 15% (15 ml./85 ml. water)... ..3g304.. 100.0 ml.
262 Cytoplasmic Elements (cHAr. 18)

4. Return to 10% formalin to complete fixation: at least overnight.

5. Wash, dehydrate, and embed.
6. Section, 6—7 microns, mount and dry.
7. Deparaffinize, clear and mount.

RESULTS:
Golgi—black
COMMENTS:
Do not use buffered formalin; it may limit the solubility of the silver
salts.
If the silver is too dense, Elftman suggests bleaching it with 0.1%
iron alum. Check under the microscope and stop the reaction by
washing thoroughly in running water.
The silver is readily oxidized, so gold toning is usually preferable
for a more permanent slide. This can follow deparafhnization; see
other Golgi methods. Also counterstaining may be included before
dehydrating and clearing slides.
Elftman warns that all the black is not necessarily Golgi.

Mitochondria Staining

Fast Green (HARMON, 1950)

FIXATION: Regaud solution (page 20).
SOLUTIONS:
Fast green:
fast pRech MG, Wl A205. Me a Sc uate am 4.0 gm.
agli, sary, A) fs a ns Seal MA 10.0 ml
Guistilledewaten. ocean
ed ee 90.0 ml

Phosphomolybdic acid:
phosphomelypdic acid: 1. 2a...
eee 1.0 gm.
Gustilled: Water: oo ohassccet
oo eee a een Oo 100.0 ml.

Safranin:
Saimanmin ©. Gal: 50240 5 Poe Wee asia....ie 1.0 gm,
bdeA ethyl alcoho! ger. sere © see tat aot b 100.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Warm fast green solution to 62°C just before use. Add slides, re-
move from heat and allow to slowly cool: 6 minutes.
3. Rinse rapidly in distilled water.
Golgt Apparatus and Mitochondria 263

‘Treat with saturated picric acid, aqueous: 10 minutes.

Rinse in distilled water.
Treat with phosphomolybdic acid: 1 minute.
SED
OS Rinse in distilled water: 1 minute.
Stain in safranin: 3—6 minutes.
Dehydrate, clear and mount.

RESULTS:
mitochondria dark bluish green
erythrocytes, nucleoli, plasmosomes—grass green (only other elements
staining green)

Altmann’s Method: (MODIFIED By COWDRY, MCCLUNG, 1939).

FIXATION: Regaud solution (page 20): change every day for 4 days, in
refrigerator.

Mordant in 3% potassium dichromate: 8 days, change every second

day.

Wash in running water: overnight; dehydrate and embed.

SOLUTIONS:
Altmann’s aniline fuchsin:
Make a saturated solution of aniline in distilled water by shaking the
two together. Filter. Add 10 gm. acid fuchsin, C.I. 42685, to 100 ml.
of filtrate. Let stand for 24 hours. Good for only 1 month.
Methyl green:
methyl green Na 200) fac 224 a. eee 1.0 gm.
Pisce Water... sae sive nd dass dos HOSS: 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Treat with potassium permanganate, 1% (1 gm./100 ml. water):
30 seconds (see comments below).
3. Rinse briefly in distilled water and bleach in oxalic acid, 5% (5
gm./100 ml. water): 30 seconds.
4. Rinse in several changes distilled water: 1-2 minutes.
5. Dry slide around sections with filter paper, cover with Altmann’s
fuchsin. Heat gently until fumes smelling of aniline come off.
Cool. Allow stain to remain 6 minutes.
6. Dry off most of stain. Rinse in distilled water until sections only
hold stain. Ifa large amount is left in sections it will form a trou-
264 Cytoplasmic Elements (cHAP. 18)

blesome precipitate with the methyl green. If too much is re-

moved, mitochondria will be lightly colored.
7. Pipette methyl green on slide, watching colors by holding it over
white paper: about 5 seconds.
8. Drain off stain, rinse briefly in 95% alcohol.
9. Dehydrate in absolute alcohol, clear, and mount.

RESULTS:
mitochondria—bright red

COMMENTS:
If the methyl green removes all fuchsin, mordanting by dichromate
was incomplete. Omit steps 2 and 3, or treat sections for a few seconds
with 2% potassium dichromate before staining.
If fuchsin is so intense that the methyl green removes it imper-
fectly, the mordanting was too great. Correct this in steps 2 and 4.
If the methyl green washes out, omit rinsing in 95% alcohol; go di-
rectly to absolute.
A solution of aurantia,* saturated in 70% ethyl alcohol (30 sec-
onds) may be used in place of methyl green. Go directly to absolute
alcohol.

Altmann-Benda’s Method: (CHAMPY-KULL MODIFICATION, MCCLUNG,

1939)
FIXATION: Champy fluid: 24 hours (page 17).
Wash in distilled water: 1 hour.

Treat 24 hours in:

PyNOlOMEOUS TAC. oc shetty
ead sind yee 50.0 ml.
chromic aed; 1% aqueous 2.225 .. .- ! jae 50.0 ml.
Mordant in potassium dichromate, 2% aqueous: 3 days. Wash in
running water: 24 hours, dehydrate and embed. Cut thin sections,
3—4 microns.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Cover slides with Altmann’s fluid (page 263). Heat gently but do
not boil. Cool and allow to stain 6 minutes.
3. Pour off stain, and rinse rapidly in distilled water.
*Michrome stains: Edward Gurr, Ltd., London, England, or Griibler stains: Roboz
Surgical Instrument Co., Washington, D.C.
Golgi Apparatus and Mitochondria 265

4. Flood with toluidine blue O, 0.5% (0.5 gm./100 ml. water): 1-2
minutes (see comments below).
5. Rinse in distilled water.
6. Cover with aurantia, 0.5% (0.5 gm./100 ml. 70% alcohol): 20-40
seconds.
7. Differentiate in 95% alcohol.
8. Dehydrate, absolute alcohol, clear, and mount.

RESULTS:
mitochondria—red
nuclei—blue
cytoplasm—egreenish yellow

COMMENTS:
If sections are too ved, increase the time in toluidine blue. If they
remain too blue, inctease the time in aurantia.
Benda’s staining solution is sometimes used in place of Altmann’s
fuchsin.

SOLUTION:
crystal violet saturated in 70% alcohol ........ 1 part.
absolute alcohol |... ARS sce E1252 . ‘1 part.
snline Saturated an: Waters <..<s .aebasde noes. +4 2 parts.
Dilute with equal parts of distilled water.

Use like Altmann’s fuchsin.

PROCEDURE:
(a) Blot and immerse in dilute acetic acid (30 ml./70 ml. water): |
minute.
(b) Blot, immerse in absolute alcohol until only a little stain comes
out.
(c) Clear and mount.

RESULTS:
mitochondria—deep violet
background—rose
Iron Hematoxylin also can be used for mitochondria. Best fixative
is Regaud, and mordant in 3°% potassium dichromate: 7 days. Use
the long method of staining: overnight in iron alum and overnight
in hematoxylin. The method is not as specific as Altmann or Benda;
other granules also stain.
266 Cytoplasmic Elements (CHAP. 18)

Osmic Method (NEwcomer, 1940)

FIXATION: Zirkle’s solution: 48 hours.
POtassiUM We MEOMALe... .... ... Gein <7. 1.25 gm.
AMMONIUMMCLGNMOMIATC .. cate ery age 1.25 gm.
COPPER ASU it a 5. oo vaSee ema gs 1.0 gm.
distitlcdivatereee,. <2... ... .0, <s AereieeteecSe- 100.0 ml.

PROCEDURE:
1. Wash tissue blocks 8 hours to overnight.
2. Impregnate in 2% osmic acid (2 gm./100 ml. water): 4-6 days.
Change solution on alternate days.
3. Wash for 8 hours or overnight.
4. Dehydrate, clear, and embed. Use benzene, not xylene, for clear-
ine.
5. Cut 5-micron sections, mount on slides.
6. Deparaffinize and hydrate slides to water.
7. Bleach in 1% potassium permanganate (1 gm./100 ml. water): 5
minutes.
8. Rinse in distilled water.
9. Treat with 3% oxalic acid (3 gm./100 ml. water): 2-3 minutes.
10. Wash in running water: 15 minutes.
11. Dehydrate, clear, and mount.
RESULTS:
mitochondria—black

COMMENTS:
Newcomer used his method on plant cells. A counterstain such as
acid fuchsin may be added.

Saccharides (Carbohydrates)
Carbohydrates appear as four types of substances: (1) simple polysac-
charides (includes glycogen); (2) mucoid substances (mucopolysaccha-
rides, mucoproteids, glycoproteins); (3) glycolipids; and (4) nucleic
acids. In addition to the use of certain specific stains, aldehyde reactions
are a common means of demonstrating carbohydrates. The aldehyde
eroups must first be liberated by some chemical agent, either oxidized
(periodic or chromic acid), or hydrolyzed (dilute hydrochloric acid).
Then the specific reagent for aldehydes can be applied. See the Feulgen
and periodic acid-Schiff reactions, page 292.
Saccharides (Carbohydrates) 267

Alcohol or alcoholic mixtures are usually recommended as fixatives

for saccharides (exception: nucleic acids). Gomori (1952) writes that
theoretically, when glycogen is enclosed in a complex mixture of pro-
teins and lipids, it can be true that a good protein precipitant will coat
the glycogen with a protein membrane. This will be impermeable to
the large glycogen molecules and keep them in situ. He considers
Bouin’s good but mercuric chloride is poor as a glycogen fixative. The
fixative should act and harden rapidly. Alcohol or acid fixatives form
glycogen in coarse droplets, while formalin-containing fixatives dis-
tribute the glycogen in fine granules. In some tissues (liver), unless the
tissue and fixative are chilled, enzymes can cause a loss of glycogen.
Although in most tissues glycogen is more stable, Gomori (1952) recom-
mends placing the tissue in fixative in the refrigerator.
After proper fixation paraffin embedding is satisfactory, but after
deparaffinizing the mounted slides, rinse them in absolute ethyl alcohol
and protect the sections with a coat of 1% nitrocellulose to prevent
diffusion of the glycogen during staining. (Gomori, 1952)

Glycogen Staining
Best’s Carmine

FIXATION: avoid aqueous media (McManus and Mowry, 1958)

Absolute alcolto]’ 4.2204; 2s: s5cke+es sys 9 parts.
Tonmalimn! | 22) 4255555..6 Ses dong 1 or <Se ] part.
Start dehydration for embedding with 95% alcohol.

MOUNTING SLIDES: The author has preserved better localization of

elycogen by mounting paraffin sections with 95% alcohol in place of
water. As soon as sections are spread, drain off excess fluid and con-
tinue to dry.

SOLUTIONS:
Best’s carmine, stock solution:
Carmine, GAS TO4I0 vn os is OE Ss an oS 2.0 gm.
Potassium: Carbenate 4... 2. 22.225. xe ogee eels 1.0 gm.
potassium chionidets. i206. 5 6s sai
ne at Aes 4BE 5.0 gm.
aistilled: water tn 262...
.. 2. uae 60.0 ml.
Boil gently: 5 minutes. Cool. Filter. Add:
ammonium hydroxide ede se M Gig a Re 20.0 ml.
Lasts 3 months at 0-4°C.
268 Cytoplasmic Elements (cHAP. 18)

Working solution:
carmine stockrsolution: :.'!.) + . Qa TE OSes 30.0 ml.
ammonium hydroxide’: 4) Ea Bo aye 25.0 ml.
Meth yltaleabow ese) 0S. eA AO ae} 25.0 ml.
Lasts 2—3 weeks.

Differentiating fluid.
absoluteretnylalcohol «. ..1sjser eaten! oe a: 16.0 ml.
Methyl AMCG NOM) 3, £5, wae. See eee es he 8.0 ml.
cistied water yi... .. sjsu ce oka be Cie cee 20.0 ml.

PROCEDURE:
1. Deparaffinize and transfer into absolute alcohol: 3 minutes.
Coat with 1% celloidin; dry slightly in air.
Dip 2 or 3 times in 70% alcohol and then into water.
Stain in Mayer’s hematoxylin: 5 minutes.
Wash, blue in Scott’s, and wash.
Stain, Best’s carmine working solution: 15-30 minutes.
‘Treat with differentiating fluid: 5-15 minutes.
Io
ow
TR
CID
Rinse quickly in 80% alcohol.
9. Dehydrate, clear, and mount.

RESULTS:
elycogen—red
nuclei—blue
COMMENTS:
Pearse (1953) proposes the following method of fixation, claiming
that it shows a localization of glycogen comparing favorably with that
preserved by freeze-drying.
Lison’s “Gendre fluid.”
picric acid, saturated in 96% alcohol ....... . 85 parts.
ROVU aN wis, be a ee: 2 eee 10 parts.
glacial: acetictacid —9a gir). en ote 5 parts.
Cool to —73°C before use in an acetone-CO. snow mixture.

Use small pieces; fix for 18 hours.

See also the Bauer-Feulgen reaction for glycogen, page 301.

Saccharides (Carbohydrates) 269

Acid Mucopolysaccharides

Alcian Blue Method (srepMAN, 1950; MopIFIED BY Mowry, 1956) °

FIXATION: 10% formalin or any general fixative.
SOLUTION:

alcian blue; 3G.X3°GCil. 74280) oe ce. es no ee 0.5-1.0 gm.

distilled water ............... . 1£00,0 ml.
glacial acetic acid |. o0nail.
Filter; add thymol crystal to prevent mold.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Stain in alcian blue: 30 minutes.
3. Rinse in distilled water.
4. Counterstain with hematoxylin, safranin, or other nuclear stain.
5. Dehydrate, clear, and mount.

RESULTS:

acid mucopolysaccharides—blue green

nuclei—depending on stain used
COMMENTS: :
I. Can be followed by PAS (page 298) for demonstration of both
acidic groups and 1,2 glycols, or the Feulgen Reaction (page 293).
2. Lison (1954) counterstains with chlorantine fast red for efficient
differentiation of mucin from collagen (mucin—bluish green; col-
lagen—cherry red): Follow steps 1, 2, and 3 above: Then
a. Treat with phosphomolybdic acid, 1% aqueous: 10 minutes.
b. Rinse in distilled water.
c. Stain in chlorantine fast red: 10-15 minutes.
chilorantine fast red 9B® 2... 2.424.
2 ean 0.5 gm.
distilled water .................6.e
cues -. 100.0 ml.

d. Rinse in distilled water, dehydrate, clear, and mount.

3. Williams and Jackson (1956) observed possible diffusion of acid
mucopolysaccharides under aqueous conditions. The fixative should
form complexes insoluble in water and alcohol. They suggest two
possible solutions containing organic chemicals which form such in-
soluble complexes with acid mucopolysaccharides:
5 Also see Metachromatic Method, p- 280.
* Edward Gurr, 42 Upper Richmond Road West, London SW 14, or Ciba, Ltd., Montreal,
Canada.
270 Cytoplasmic Elements (CHAP. 18)

(a) 0.5% cetylpyridium chloride (CPC) in 4% aqueous formalin.

(b) 0.4% 5-aminoacridine hypochloride in 50%, ethyl alcohol.
4. Spicer and Meyer (1960) precede staining with alcian blue by 5
minutes in aldehyde-fuchsin (page 287); to 70% alcohol, running
water, and then into alcian blue.
5. Mowry (1960) reports that lots of alcian blue have differed in
staining efficiency and fastness. Current stocks of alcian blue SGX
are more soluble, less fast and more stable. He has changed his stain-

chloric acid-sodium citrate buffer: 1—2 hours.

Colloidal Iron Method (RITTER AND OLESON, 1950)

FIXATION: 10% formalin in 90% alcohol; do not use dichromate—it
oxidizes polysaccharides.
SOLUTIONS:
Colloidal iron solution:
dialyzed" iron, (MIGrCks |. eueeee
is ee ee 50.0 ml.
2M acetic acid: (11-5 ml: 7/885 ml water) 20>. 50.0 ml.
Potassium ferrocyanide in HCI:
potassium ferrocyanide, 1% aqueous ......... 86.0 ml.
IN HIGI(page 408) ss st aac eyes eee 14.0 ml.
Periodic acid:
periodietacidh4ss S7RL se ). Ce ere ee 0.4 gm.
distiliedawater 20s settee, steemeee os et Meee 100.0 ml
M/5 sodium acetate (135 mg. hydrated salt/35
Eveth yiealeohol) te Pee
te eee 0 In:
Reducing rinse:
potasstumiodide: 5 ys.) eee 3.)..) ohare 1.0 gm.
SOGIUIM tiiGsUl tate’ an. 5. eee oe eee 1.0 gm.
ensculled) water: =. 7 ho 0) sive |. ah seeeee . 20.0 ml.
Add with stirring:
absolute ethyl -alcohol oF are eee gach 30.0 ml.
DIN EIG!: (pape s408) cs OG ee. watieen:. cit 0.5 ml.
Sulfite rinse:
WEKEGEN E14. 0 RR SEERSIRE) DO)CROORE ARTEL laa | Ee 50.0 ml.
concentrated’ HCO ERCP INTE eas AS Oe 0.5 ml.
potassium imetabisulfite 2... fee tee 0.2 gm.

Schiff reagent (page 294)

Saccharides (Carbohydrates) 271

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
Treat with colloidal iron: 10 minutes.
Wash well in distilled water.
Of
Hm
DO Treat with potassium ferrocyanide: 10 minutes.
Wash well in distilled water.
Flood with 70% alcohol.
Treat with periodic acid: 5 minutes.
Rinse with 70% alcohol.
ID Transfer
Ow to reducing rinse: 5 minutes.
10. Rinse with 70% alcohol.
11. Transfer to Schiff reagent: 1 hour.
12. Use 3 sulfite rinses: 1.5 minutes in each.
13. Wash well in running water: 5 minutes.
14. Stain with hematoxylin if desired.
15. Dehydrate, clear, and mount.

RESULTS:
acid polysaccharides—blue
elycogen—magenta

COMMENTS:
Nito and Stokes (1960) prepare a dialyzed iron as follows: Dissolve
100 em. ferric chloride in 230 ml. distilled water. Add 130 ml.
glycerol and mix well. Divide into 2 portions of { and 3. To the large
volume add concentrated ammonia in small quantities with vigorous
stirring. A precipitate forms and dissolves more and more slowly
until the mixture becomes pasty (approximately 110 ml. ammonia).
Add drop by drop and with constant stirring the smaller portion until
the solution becomes clear dark reddish brown. It now contains a
slight excess of ferric chloride.
Dialyze against distilled water using a cellophane bag—capacity
about 4 times that of the solution. Change the distilled water every
day until the solution in the bag has reverted to its original volume
(about 2 weeks). Store at room temperature. For use dilute with equal
amount of water.
Acid mucopolysaccharides combine with dialyzed iron and then
become blue in potassium ferrocyanide (Prussian blue). Mowry
(1958) considers this method more specific than others, and it stains
more strongly than alcian blue.
272 Cytoplasmic Elements (CHAP. 18)

Colloidal Iron Method (Mowry, 1958)

SOLUTIONS:
Colloidal iron solution:
Stock solution:
Bring 250 ml. distilled water to boil. While boiling pour in 4.4 ml.
of 29% ferric chloride (USP XI) solution. Stir. When solution is
dark red, remove from heat and allow to cool. The color must be dark
red and clear. It is stable for months.
Working solution:
elacial-acetic acid 2.0)... 4 Se 5.0 ml.
Gistililed? -water’’-. : 2°) pat)
See ee 15.0 ml.
stock colloidal iron solution 20.0 ml.
pH should be 1.1—1.3. Nonspecific staining takes place at pH 1.4 or
higher. Effective for | day.
Hydrochloric acid-ferrocyanide.
24% TAG) (Zimis/9semlk water). ..95 9...
.. 4k. 2, 50.0 ml.
2% potassium ferrocyanide (2 gm./100 ml.
Wot iie Mp eA. oat ety. {cena 50.0 mi.
Mix immediately before use.
PROCEDURE:
1. Deparaffinize and hydrate slides to water: remove HgCle.
2. Rinse in 12% acetic acid (12 ml./88 ml. water): 30 seconds (pre-
vents dilution of reagent).
3. ‘Transfer to freshly prepared colloidal iron (working solution): 60
minutes.
4. Rinse in 12% acetic acid, 4 changes: 3 minutes each.
5. Treat with hydrochloric acid-ferrocyanide, room temperature: 20
minutes. (Include a control slide, one not treated with colloidal
iron.)
6. Wash in running water: 5 minutes.
ea Counterstain if desired: Feulgen, PAS, or hematoxylin.
8. Optional: dip in aqueous picric acid to color cytoplasm and
erythrocytes; rinse for few seconds in tap water. (Author's com-
ment: acetic-orange G also may be used.)
9. Dehydrate, clear, and mount.
RESULTS:
acid mucopolysaccharides—bright blue; uncolored in control slide.
mucins of connective tissue, epithelium, mast cell granules, capsules
of some microbial agents, pneumococci—bright blue
Mucin Staining ro ~I

COMMENT:
For staining of weakly acidic polysaccharides, see Wolman
(1961).

Mucin Staining
Mayer’s Mucicarmine
FIXATION: any general fixative, but alcoholic preferred.
SOLUTION:
Stock solution: (Southgate’s solution, 1927)
Cammine sel, 7OAIO) 2. 5... . deuseae o3 1.0 gm.
aluminum hydroxide, powder ............... 1.0 gm.
aluminum chloride, anhydrous .............. 0.5 gm.
50? eunylalconol 2 vd» .2 oi feg heya nis tac n LOO Oem:
Heat in boiling water bath with frequent shaking: 2.5 minutes. Cool
under tap and filter.
Working solution:
SEO SOUMETON Fe eifek 40 ht bon a:= co Re ee 1.0omi:
GistilleG water tc kines os 5.« . «idee
ee ESA 9.0 ml.

Mix 1-2 days before using.

PROCEDURE:
1. Deparafhinize and hydrate to water; remove HgClbs.
2. Stain in diluted mucicarmine: 15—20 minutes; check under micro-
scope. (Flood the slides with the working solution.)
3. Rinse quickly in distilled water.
4. Dehydrate, clear and mount.
RESULTS:
mucin—red

COMMENTS:
If desirable to stain nuclei, precede staining with mucicarmine by
Weigert’s or Groat’s iron hematoxylin (page 126).
This method has lost its popularity and is not used extensively, but
has been included for technicians who might want to refer to it. For
more popular methods, see the metachromatic procedures (page 279).

Fluorescent Method (HICKS AND MATTHAEI, 1958)

FIXATION: 10% formalin preferred.

274 Cytoplasmic Elements (cHAP. 18)

SOLUTIONS:
Iron alum:
ferric ammenmmeusultate .... > Veesk eee oes. oe 4.0 gm.
Gistulledswatenyae. .. =... J eee eee oo 100.0 ml.

Acridine orange:
acridimelorange, G.1. 46005 4) feeme "te 0.1 gm.
diseiWledwwater: 25)>... 1° 2 Oe Ren See 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate paraffin sections to water, or cut frozen
sections and place in distilled water.
Treat with iron alum solution, 5—10 minutes.
Wash briefly in distilled water.
Stain in acridine orange, 1} minutes.
eneRinse briefly in distilled water, blot and mount in Harleco Fluo-
hae
rescence Oil Mountant.*
RESULTS:
mucin—brilliant reddish orange fluorescence
COMMENTS:
This is not a permanent slide. The iron inhibits the production of
fluorescence with acridine orange in nearly all tissue components
except mucin. Hicks and Matthaei also suggest that the mucins of acid
polysaccharides rather than mucoproteins fluoresce.
For details concerning fluorescent microscope and equipment, see
page 102.

Amyloid Staining
Higman’s Congo Red: (MODIFIED By LILLIE, 1954)
FIXATION: 10% formalin or an alcoholic fixative.
SOLUTIONS:
Congo red:
Congo ned, Ccl, '22120N iy ahaaaacr,
(eet fice 0.5 gm.
BO, ethyl alcohol passes as eeeeeGe
rt cao: ee 100.0 ml.
Potassium hydroxide:
potassium” hydrOxideres seine amet ens fees ae 0.2 gm.
DUP eny alCOla Peres tre near etree, eames he teen 100.0 ml.
Hematoxylin, page 125.
7 Hartman Leddon Co., Philadelphia.
Amyloid Staining no~ICr

PROCEDURE:
1. Deparaffinize slides, transfer through absolute, 95%, and into 50%
alcohol.
Stain with Congo red: 1—5 minutes.
Differentiate in potassium hydroxide: 1-3 minutes.
Rinse in tap water.
Stain in Mayer’s hematoxylin (or the like): 2-3 minutes.
Wash in tap water: 5 minutes.
G0
OR
SID
Ky Dehydrate, clear, and mount.

RESULTS:
amyloid—red
nuclei—blue

COMMENTS:
Amyloid is a hyaline material that appears in connective tissue as a
result of disease and methods should be on hand for pathological
study.
See also Metachromatic Method, page 278.

Fluorescent Method (vAssAR AND CULLING, 1959)

FIXATION: 10% formalin or alcohol. The latter gives a crisper differ-
ential picture.
SOLUTIONS:
Thioflavine:
Uuotlavine: Gil. A9005. o... .. ..aiscthteaaena 1.0 gm.
Gastilled Water = cltsie<. 4.25 2550020cenhawe 100.0 ml.

Acetic acid) 1%:

Placial mcetic ACid2 2.325222
re3 so: ene. eee Ohm:
distilled: water 24 oe boon le te Je aeet a4 99.0 ml.

PROCEDURE:
1. Place frozen sections in distilled water.
Stain sections in thioflavine: 3 minutes.
NO Differentiate
oo in 1% acetic acid: 10 minutes.
4. Wash briefly, blot, and mount in Harleco Fluorescence Oil
Mountant,’ or Apathy Mountant (page 119).

RESULTS:
amyloid—yellow fluorescence
® Hartman Leddon Co., Philadelphia,
276 Cytoplasmic Elements (CHAP. 18)

COMMENTS:
Treatment with acetic acid enhances differentiation and reduces
fluorescence of normal tissue without affecting the fluorescence of
amyloid. Vassar and Culling suggest that the nuclear fluorescence
can be quenched by prestaining with hematoxylin (2 minutes), then
proceeding with step 2 above.
For details concerning fluorescent microscope and equipment, see
page 102.

Mast Cell Staining

Mast cells are of common occurrence in connective tissue. Because

of their cytoplasmic granules, however, staining methods for these cells
have been included in this cytoplasmic element section. The specific
staining of these granules is the primary means of identification of
mast cells.

Neutral Red (ALLEN, 1960)

FIXATION: 10% formalin
SOLUTION:
neutral ned G.1. 50040. scr ea seis eee 0.5 gm.
509, ethylvalcohol... Sinn poh d-ak es i ote 100.0 ml.

PROCEDURE:
1. Deparaffinize and run slides down to 70% alcohol.
Stain in alum hematoxylin: 3 minutes.
Wash, blue, and wash.
Stain in neutral red: 10 minutes.
Differentiate in 70% alcohol: 2-10 minutes.
Dehydrate in 95% alcohol: 3 minutes.
Dehydrate in n-butyl alcohol, 2 changes: 5-10 minutes in each.
ow
TP
WIAClear and mount.

RESULTS:
mast cell granules—red
cartilage matrix—red
nuclei—blue

COMMENTS:
Color is paler than with metachromatic methods, page 281.
Mast Cell Staining ‘
I ~I|
a

Bismarck Brown (spAtz, 1960)

FIXATION: 10% formalin.

SOLUTION:
Bismarck brown:
Bismarck brown; C1. 21000. 065k. csneddss 0.5 gm.
absoluterethyl-alcohol ........% ag seme
ne Se ake: 80.0 ml.
1% aqueous hydrochloric acid .............. 20.0 ml.

PROCEDURE:
1. Deparaffinize and transfer sections to absolute alcohol: 3 minutes.
2. Dipin 95%,, then 70%, alcohol.
3. Stain in Bismarck brown: 30 to 90 minutes.
4. Differentiate in 3 changes 70% alcohol: 2 seconds each.
5. Transfer to 95% alcohol: 2 seconds.
6. Dehydrate in absolute alcohol: 30 seconds.
7. Clear in xylene: 1 second; mount.

RESULTS:
mast cell granules—yellow brown

COMMENTS:
If counterstaining is desirable, follow the 70% differentiation by 3
minutes in hematoxylin (Harris, Mayer’s, etc.); dip in 70% alcohol:
2 seconds, and continue into step 5 above.

Chrysoidin (HARADA, 1957)

FIXATION: any general fixative.
SOLUTION:
Chrysordin a, «Us a2 70). na ko wm wa 2 0.5 gm.
qistmlMed syaler © o2 242s. bbb eon dhesee 2a tage 00:0 mil:

PROCEDURE:
1. Deparaffinize and run slides down to 80% alcohol; remove HgCl:
if present.
2. Stain in chrysoidin: 5-10 minutes.
3. Rinse in distilled water.
4. Dehydrate, clear, and mount.

RESULTS:
mast cell granules—deep brown to black
other elements—yellowish
278 Cytoplasmic Elements (CHAP. 18)

COMMENTS:
Staining with chrysoidin can be preceded with hematoxylin or PAS.
The author likes this method.

Metachromasia

A certain few tissue elements are stainable by a particular group of

cationic dyes, changing in the tissues from blue (the usual orthochro-
matic form) to a purplish-red or reddish-purple hue. Such a dye, called
metachromatic (see page 110), is of considerable value in the study of
specific elements of connective tissue. Among the metachromatic dyes
most commonly used are toluidine blue O, thionin, methylene blue,
azures, crystal violet, cresyl violet, methyl violet, safranin O, celestin
blue, gallocyanin, and pinacyanol. Some of the tissues identified by this
means are mast cells, amyloid, cartilage, and mucus materials (Schubert
and Hamerman, 1956).
‘The methods are tricky and a technician must learn to distinguish a
true metachromasia from a false one. The difficulty in preserving meta-
chromasia lies in the dehydration of the tissue after staining. Increasing
strengths of alcohol (ethyl) revert the dye back to the orthochromatic
form. Sections can be examined in an aqueous condition, but this
produces at best a semipermanent preparation. Some workers use
acetone or tertiary butyl alcohol for dehydration, then follow with one
of the clearing agents. Padawer (1959) uses Ether 181 (page 282) and
Levine (1928) used oil of cloves for dehydration (page 281).

Amyloid ®
Crystal Violet (LIEB, 1947)
FIXATION: 10°% formalin or alcoholic fixative.
SOLUTION:
Crystal violet stock solution:
crystal violet, C.I. 42555 (14.0-15.0 gm.); satu-
rated in 95°, ethyl alcoliolyyn My Ste 100.0 ml.
Working solution:
Stocks solution | 02.0 Aeron Bane se ae 10.0 ml.
dismileds water °:/5a0s ae ae tee ween as oe . 300.0 ml.
hydrochloric acid, concenthated sare ee. 1.0 ml.
*See Amyloid Staining, page 274.
Metachromasia 279

PROCEDURE:
] Run frozen sections or deparaffinized paraffin sections down to
water.
Nd Stain in working solution: 5 minutes to 24 hours.
Rinse in water.
4 Mount in Abopon, page 122
RESULTS:
amyloid—purple
other tissue elements including hyalin—blue
COMMENTS:

Acid in the staining solution makes it self-differentiating and staining

time is flexible. Lieb suggests that if there is only a small amount of
amyloid in the tissue, thicker sections should be cut to make the color
reaction more clearly visible. Slides mounted in Abopon have re-
tained their color for 2 years. dbopon mounts must be sealed with
ringing cement for permanency.
Gomori (1953) outlines a simple method for permanent mounts:
Float paraffin sections on dye solution: 15—20 minutes.
Float sections on distilled water to remove excess dye.
Float on 1-2% acetic acid, aqueous, to differentiate.
Mount on slides in usual fashion and dry.
FO
HR
or
NO
= Remove paraffin with xylene and mount.
This preserves the metachromasia of the crystal violet.

Mucin'?

Toluidine Blue (LILLIE, 1929)

FIXATION: any general fixative, but alcohol preferred.
SOLUTION:
tolumdme 0) Gl 52030)... 5. 21. as eg. 2.0 gm.
ise WALES cae Fe oe ee oO ese a eRe 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Stain in toluidine blue: | minute.
3. Wash in water: 2—3 minutes.
4. Dehydrate in acetone, 2 changes: 3—5 minutes each.
5. Clear in xylene and mount.
See Mucin Staining, page 273.
280 Cytoplasmic Elements (CHAP. 18)

RESULTS:
mucin—reddish violet
nuclei and bacteria—deep blue
cytoplasm, fibrous tissue—bluish green
bone—bluish green
cartilage matrix—bluish violet
muscle—light blue
cell granules—blue violet
hyalin and amyloid—bluish green

Thionin (MALLorRY, 1938)

FIXATION: any general fixative, but alcoholic preferred.
SOLUTION:
eo mins Gls SL0O0 ten re ne eae ee 1.0 gm.
BO REUIY) AlCOMO cet aes otra ge ee te ee 100.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCle.
2. Stain in thionin: 15 minutes to | hour.
3. Differentiate in 95% alcohol.
4. Dehydrate in absolute alcohol, clear, and mount.

RESULTS:
mucin—light to dark red or purple

Acid Mucopolysaccharides™
Thionin (curr, 1958)
FIXATION: 10% formalin or other general fixative.
SOLUTIONS:
Thionin:
thionin, C.I. 52000, saturated aqueous ........ 0.5 ml.
distilled wateric: 42154-2529
es). Eee 100.0 ml.
Molybdate-ferricyanide solution:
ammonium molybdate, 5% aqueous .......... 50.0 ml.
potassium ferricyanide, 1% aqueous 2) 0%). hoe 50.0 ml.

Make up solutions fresh each time.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
™ See Acid Mucopolysaccharides under Saccharides, page 269.
Mast Cells 281

Stain in thionin: 5—15 minutes.

Rinse in distilled water.
Treat with molybdate-ferricyanide: 2 minutes.
Wash in distilled water: 2—3 minutes.
1DR
oo Dehydrate, clear, and mount.

RESULTS:
acid mucopolysaccharides—purple
other cell elements—bluish

COMMENTS:
The molybdate-ferricyanide solution prevents loss of metachromasia.
See Kuwyer (1957) for suggestions concerning fluorescent methods
for mucopolysaccharides.

Mast Cells”

Thionin Method (LEvINE, 1928)

FIXATION: any general fixative.

SOLUTIONS:
Thionin:
thioniy Gob, 52000) oo. Le. 2 seo pees Peele 1.0-2.0 gm.
distilled: water oh.<3 04's = 4s h<.40 14540 R ee Ouse eS 100.0 ml.
Orange G:
orange G, C.I. 16230, saturated solution in oil of cloves. It goes into
solution slowly; stir frequently. Allow to stand 24 hours before using.
Good until it becomes discolored by thionin.

PROCEDURE:
I. Deparaffinize and hydrate slides to water; remove HgClb.
2. Stain in thionin: 2—3 minutes for light stain; 10-15 minutes for
an intense stain.
3. Rinse briefly in water for light stain; blot around sections for in-
tense stain.
4. Dehydrate by dropping a few drops of absolute alcohol on sections.
5. Stain with orange G-clove oil: 1-2 minutes. Repeat orange G until
thionin disappears from cytoplasm, leaving it orange. Mast cells
remain unchanged.
6. Clear thoroughly in xylene, and mount.
See Mast Cell Staining, page 276.
282 Cytoplasmic Elements (CHAP. 18)

RESULTS:
mast cells—deep blue or purple blue
cytoplasm—light gold-orange
nuceli—faint blue

Quick Toluidine Blue Method

FIXATION: any general fixative.
SOLUTION:
toluidmevbine'O;C.1 520407 2. ee eee ee... 0.2 gm.
OOo rethmibalcohol, .....5 4. 2. geeeeeee fear 100.0 ml.
PROCEDURE:
1. Deparaffinize and run slides down to 60% alcohol. Remove HeClz
if present.
2. Stain in toluidine blue: 1-2 minutes.
3. Rinse quickly in tap water.
4. Dehydrate in acetone, 2 changes: 2—3 minutes in each.
5. Clear in xylene and mount.
RESULTS:
mast cells—deep reddish purple
background—faint blue

Toluidine Blue (paDAWER, 1959)

FIXATION: formalin-alcohol preferred, 10% formalin satisfactory.
POLAT a hyena cs. 6 een pl kt eee 10.0 ml.
HEF Ae C50)20) eee me ee pcan ss “os AMR Reero ee 90.0 ml

SOLUTIONS:
toluidine bine;O, CT -5A 04 Oe tte heey: 0.05 gm.
rel Uc)GaN!Kon | CivPyROAM pe Laue lee EMRE OR! 2B 50.0 ml.
Gistilledtiwater aq. Grasse eae One 50.0 ml.
Dissolve dye in water, add ether 181. Filter. Use within 24 hours.
PROCEDURE:
1. Deparaffinize and transfer into absolute methyl alcohol, 2 changes:
10 minutes each.
. Transfer to ether 181, 50% aqueous: 10 minutes.
. Stain in toluidine blue: 20-60 minutes.
. Dehydrate in ether 181, 50% aqueous: 10-15 minutes.
. Dehydrate in ether 181, 100%, 2 changes: 20 minutes each.
NO.
OO
BP
Ct
DM Clear in xylene and mount.
18 Personal communication from Miss Marlies Natzler, U.C.L.A.
* Tetra methylene glycol ether, Ansul Chemical Co., Marinette, Wis.
Pituitary and Pancreas 283

RESULTS:
mast cell granules—reddish purple
background—faintly blue
cartilage—reddish purple (will be the only other tissue staining meta-
chromatically)
COMMENTS:
The toluidine blue in many methods reverts to its orthochromatic
(blue) color during dehydration; therefore it is the usual practice to
prepare an aqueous or glycerine mount to produce only semi-perma-
nent results. Padawer suggests that partial hydration is required for
metachromasia and ether 181 does not completely hydrate the tissue.
A specific concentration is used in which both mast cell polysac-
charides and toluidine blue are poorly soluble.
Belanger and Hartnett (1960) use the following solution:
potassium acid phthalate-tartaric acid... 1.0 gm.
distilled water ......: hs. cxg} sehpata anaes ol 100.0 ml.
toluidine blue: O;C 1752040". cats ase es 0.5 gm.
The dye requires 24 hours to dissolve. Filter before use. Good for one
week.
PROCEDURE: Stain for 10 minutes; rinse in fresh phthalate-tartaric acid
buffer (1 gm./100 ml. water): 2 minutes; dehydrate in tertiary butyl
alcohol, 4 changes: 2 minutes each; clear and mount.

Pituitary and Pancreas

The staining of the cytoplasm and its elements is used to characterize
and differentiate the cells of the anterior prrurrary. The glandular
cells are classed as either chromophils or chromophobes. Seventy-five
per cent of the chromophils are normally acidophilic (acidophils, some-
times called alpha cells) and twenty-five per cent are basophilic (baso-
phils). The latter group is made up of gonadotrophs (delta cells) and
thyrotrophs (beta cells). Every few months a new method seems to
appear in the literature, suggesting that the ideal procedure is being
elusive. Certain reactions seem to be specific for certain cells. Thyro-
trophs and gonadotrophs have an affinity for Schiff’s reagent, thyro-
trophs also stain with Gomori’s aldehyde fuchsin or aldehyde thionin.
Acidophil granules differentiate sharply in Elftman’s (1960) method.
Orange G also stains them. Paget and Eccleston (1959, 1960) use luxol
fast blue to distinguish them.
284 Cytoplasmic Elements (cHAP. 18)

Five methods have been included here. For additional information

some of the following authors may be of assistance: Elftman (1957 A;
1959 A and B; 1960); Gomori (1951); Kerenyt (1959); Landing (1954);
Landing and Hall (1955); Lazarus (1958); Paget and Eccleston (1959,
1960);Pearse (1949, 1950); Shanklin, Nassar, and Issidorides (1959).
Characteristic staining of cells types is also used to differentiate the
various cells of the islets of Langerhans of the PANCREAS. Gomori’s
(1941 B), method is recommended, but the Heidenhain Azan method
(page 149) also is excellent and brilliant.
Trichrome PAS! (PEARSE, 1950)
FIXATION: Helly’s is best.
SOLUTIONS:
Periodic acid:
PERIOUI GyACI. i te SRNR A dca ee ate, 3 = Ree 0.4 gm.
VE />esOdiuIMKACekate) Ah ct ieee se ea eee 5.0 ml.
9527; cethy lmaleohol timertst.. oe oe ae eee ee 30.0 ime,
GaStilled! Water. 2 ei ucnie cet, a eR ee = ere 10.0 ml.
Reducing solution:
POLASsI@MMNGMIGE I <5. fia. ee hae ad se eee 1.0 gm.
SOd TUM ;EMTOS tate ee nate ee Se ee a ee ee 1.0 gm.
507 -cenylralcOne leach ne cae oer. eee . 80:0) mi.
distilledhwatert tt: Bee. tak eer eee 20.0 ml.
ZIN PERGI pa gera08)!: a5 zak. itt knatare ote 0.5 ml.
A precipitate of sulfur forms; do not filter out. Make fresh just before
use.
Schiff’s reagent, see page 294.
Sulfite baths:
sodium, metabisullite, sNa5S,O- 85. 65 aus soe 0.5 gm.
Gastililedeawater ken ye eee en ae 100.0 ml.

Celestin blue solution:

eclestin‘ bluesBMCA. G1OS0M. WR eee. nT 0.5 gm.
ferric ammonium sulfate (iron alum) ........ 5.0 gm.
distilled: watertis. Mi 70s Ge eis. 218)4 Lae 100.0 ml.

Dissolve alum in water, add celestin blue; boil 3 minutes. Cool and
filter. Add:
GIEENUING "iyy 22 4% eae SgBars oad ce Te 14:0 mil:
1 Concerning PAS technics, sce page 298.
Pituitary and Pancreas 285

Mayer’s hematoxylin, see page 125.

Orange G Solution:
OraneerG, MOM G290) exten yee
alas ae ss 2.0 gm.
phosphotungstic acid, 5% (5 gm./100 ml. water) 100.0 ml.
Allow to stand 48 hours. Use supernatant fluid.

PROCEDURE:
Ne Deparaffinize and hydrate slides to water; remove HgCle.
no‘Transfer to 70% alcohol: 2 minutes.
59
Oxidize in periodic acid: 5 minutes.
Rinse in 70% alcohol.
Place in reducing solution: | minute.
Rinse in 70% alcohol.
Treat with Schiff’s reagent: 10-30 minutes.
Treat with sulfite solutions, 3 changes: 1 minute, 2 minutes, and
og)
2 minutes.
. Wash in running water: 10 minutes.
. Stain in celestin blue: 2 minutes.
. Stain in Mayer’s hematoxylin: 2 minutes.
. Wash in running water: 5 minutes.
Stain in orange G: 10 seconds.
. Wash in running water until orange color is differentiated: about
10-15 seconds. Check under microscope.
. Dehydrate, clear, and mount.

RESULTS:

colloid of stalk and parenchyma, and vesicles of vesiculate chromo-

phobes—magenta
basophil (beta) granules—dark red
acidophil (alpha) granules—orange
erythrocytes—orange
nuclei—dark blue

Trichrome PAS (LAzARus, 1958)

FIXATION: Zenker-formol (Helly’s). Bouin’s unsatisfactory.

SOLUTIONS:
Schiff’s reagent, see page 294.
Sulfurous acid:
sodium metabisulfite, NasSoO; .... 04. .c46546s 0:5 em:
distilled water (ioc...as: Oene ee ee 100.0 ml.
286 Cytoplasmic Elements (CHAP. 18)

Weigert’s hematoxylin, see page 126.

Ponceau-orange G:
ponceau ZA GME WOLDO |... Le ered. sh 0.2 gm.
orange KG) GUeEbZ SON. |.) Neaires cLAe: 0.1 gm.
distilled Wallet tyre. tis sc. tate Bee cetasens, -c 100.0 ml.
elaciqlaGeeIeRaelel) 2... ..4, ds SaRemewe eee 2. 1.0 ml.
Light green:
lightereen’ SF yellowish; C.1)42095 0) pot2s 52 1.0 gm.
GhistrleGGwatel: fi... een Aa tee ee eee eh 100.0 ml.
elaciaimacetic. acid. ~ .. . | 52 +2 F eae St ee 1.0 ml.
Phosphomolybdic acid solutions:
phosphomolybdic:acid .225... 2... See 1.0 gm.
distilled! water-22 0/0, Poh |. RES ie SEE 100.0 ml.
To make 0.5% solution, use above 1%
Phosphomolybdtciacid: 75g ee 50.0 ml.
lastilled (Waters 72.06 845104) a eee
oe ae 50.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water: remove HgClo.
. Treat with periodic acid, 0.6% (0.6 gm./100 ml. water): 20
minutes.
. Wash in running water: 5 minutes.
. Treat with Schiff’s reagent: 20 minutes.
. Transfer through 3 sulfurous acid rinses: 0.5 minute each.
Wash in running water: 5—10 minutes.
. Rinse in distilled water.
. Stain in Weigert’s ferric chloride hematoxylin (24 hours old): 10
minutes.
. Rinse in 95% alcohol.
. Differentiate in acidic alcohol (0.5 ml. HC1/100 ml. water).
. Wash in running water: 10 minutes.
. Stain in ponceau-orange G: 2 hours.
. Rinse in distilled water.
. Differentiate in phosphomolybdic acid, 1% until delta cells and
collagen are colorless.
. Rinse briefly in distilled water.
. Stain in light green: 5—30 minutes.
. Wash well in acetic acid, 1% (1 ml./99 ml. water): 1-2 minutes.
. Differentiate in phosphomolybdic acid, 0.59%: 0.5—5 minutes.
19: Wash in acetic acid, 1%: 20 minutes.
Pituitary and Pancreas 287

20. Dehydrate in absolute alcohol, 2 changes: 3—5 minutes each.

21. Clear and mount.

RESULTS:
beta cell granules—pale yellow orange
beta cell cytoplasm—pale yellowish green
alpha cells—deep orange
delta cells—translucent green
glycogen, mucus, glycoproteins—dark red to purplish
nuclei—black
collagen—green
PAS-positive elements—magenta
erythrocytes—yellow

Aldehyde fuchsin-PAS Method (ELFrMAN, 1959 A and B)

FIXATION: overnight or week end in:
mercuric chloride, 5% AIGUCOUIS Hanr et athens9 100.0 ml.
potassium, chromium’ sulfate <0. (44, 62 Jt 5.0 gm.
formalin, {enc tee oe ae She’, cs « Me ee, wae 5.0 ml.
Prepare fresh. Wash in 70% alcohol.
SOLUTIONS:
Aldehyde fuchsin:
basic fuchsin, G.1.,42500: . ...0. 62 e6 sn ge Ae 0.5 gm.
70°, ethyl! alcohol © ...... Se er en ee 100.0 ml.
paraldehydé 227.2252: fet 2 ae ets 0:75: mil.
hydrochloric acid, concentrated ............. 125ml;
Put in 37°C oven early in morning, and will be ready by noon of
succeeding day.
es 8
Schiff’s reagent, see page 9294.
2 5

Orange G:
orange ne AOS WG2 SON segs 2 6 pon sea Mee 3.0 gm.
distilled water brought to pH 2.0 with few drops
of glacial acetic acid or hydrochloric acid . . 100.0 ml.

PROCEDURE:
1. Deparaffinize and run slides down to 70% alcohol; remove HeCls
in iodine-70% alcohol.
. Stain in aldehyde fuchsin: 30 minutes.
HO. Rinse in 95% alcohol.
oo

4. Oxidize in periodic acid, 1% (1 gm./100 ml. water); 15 minutes.

288 Cytoplasmic Elements (CHAP. 18)

5. Rinse in distilled water, 3 changes: 2 minutes each.

6. Treat with Schiff’s reagent: 20 minutes.
7. Rinse in tap water.
8. Stain acidophils in orange G: 10 minutes.
9. Rinse rapidly in water.
10. Dehydrate, clear, and mount.

RESULTS:
thyrotrophs (beta)—deep purple
gonadotrophs (delta)—red
acidophils, erythrocytes—orange
nuclei—unstained
COMMENTS:
If aldehyde is overstained, treat for about 4 minutes with 0.1%
dilution of Clorox. Check under microscope. Wash thoroughly 10 or
more minutes.
If oxidation of tissues is desired before staining, treat with equal
parts of 0.2 potassium permanganate and 1% sulfuric acid: 5 minutes.
Bleach in 5% oxalic acid. Wash.

Rhodacyan Method (GLENNER AND LILLIE, 1957)

FIXATION: Formalin or Zenker-formalin, not acetic formalin or Bouin’s.

SOLUTION:
eosin, C.I. 45400, 1% (1 gm./100 ml. water) .... 8.0 ml.
antliny blue, \Gl, 42780; 10% (seme/l00" mi:
Wider): ge fybe ctet heey onl 32h AE: fcc 2.0 ml.
pH 4.5 buffer composed of
OSUIMSClinicacat. | ater oa nae eo eee oll samt
O'2Mi disodium, phosphate, 2.2... ..5be. yaeee 0.9 ml.
distilledwater-- "geOR Se : 28.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Stain, start staining at room temperature, and place at once in
oven at 60°C: | hour.
3. Wash in running water: 5 minutes.
4. Dehydrate and clear through 50%, 80°% and anhydrous acetone,
acetone-xylene (1:1), xylene, 2 changes, and mount.
RESULTS:
beta cell granules—blue-black
acidophil granules—dark red
Pituitary and Pancreas 289

chromophobe granules—slate grey to pale pink

colloid—red to blue-violet
red cells—orange
collagen—blue

Cameron and Steele (1959) Method

FIXATION: any good fixative.
SOLUTIONS:
Potassium permanganate, 0.5%:
potassium ‘permanganate. 2: ../1f.l
fie. 2h ies te 0.3 gm.
distilled water ....... Ke A ELS 100.0 ml.
sulfuric acid, concentrated 5 Sa Pee asta Me te 0.3 ml.

Sodium bisulfite, 2.5%:

Soaitun isUihiters m5 4:2. 3s eeeaeaes Seas 2.5 gm.
MIStlled) WAC eco as on bo ae eae ele ee 4 100.0 ml.
Aldehyde-fuchsin, page 168 and 287.
Halmi’s mixture (1952):
distilled water ...... seep, yrs ere Tyee 100.0 ml.
lightgreen SF,-yellowish, C.1..42095 . 2... 0...+4. 0.2 gm.
orange G, C.I. 16230 . a i an Mpcael 1.0 gm.
chromotrope 21) GC. 10570) : $hs sien de as hele 0.5 gm.
phosphotungsnc acid: 5... 22.2.2 sesat eee 0.5 gm.
placial aACelic AClG) sen. 5.47 4h ddeomameee
ieee 1.0 ml.

Keeps indefinitely.
PROCEDURE:
Deparaffinize and hydrate slides to water; remove HgCl,.
Oxidize in potassium permanganate: | minute.
Rinse in distilled water.
Bleach in sodium bisulfite until permanganate color is removed.
Wash in running water: 5 minutes.
Transfer to 70% alcohol: 2 minutes.
Stain in aldehyde-fuchsin: 2-10 minutes.
Wipe off back of slide and rinse in 95% alcohol.
. Differentiate in 95% alcohol until no more aldehyde-fuchsin
comes out of sections.
Transfer to 70% alcohol: 2 minutes.
Rinse in distilled water: 1 minute.
Stain in Halmi’s mixture: 45 seconds.
290 Cytoplasmic Elements (cHAP. 18)

13. Wipe off back of slide, differentiate in 95% alcohol plus 0.2% of
acetic acid: 2-3 minutes.
14. Rinse in fresh 95% alcohol.
15. Dehydrate in absolute alcohol, clear, and mount.

RESULTS:
eranulation of beta cells dark purple
delta cells—green
acidophilic granules—orange
nucleoli—bright red
coagulated contents of cytoplasmic granules—orange

Pancreas

Chromium-Hematoxylin-Phloxine (Gomori, 1941 B)

FIXATION: Bouin’s preferred. Stieve’s satisfactory. Zenker’s, Carnoy, and
formalin unsatisfactory.

SOLUTIONS:
Bouin’s solution, see page 13.
Potassium dichromate-sulfuric acid:
Can be made up as separate 0.3% solutions and mixed or:
potassium idichromater,.4..9 |) eee, yo See Ee 0.15 gm.
distalledswatery:.9. 2247 ah Bes aden Seas 1-00 Re 100.0 ml.
sulfuric acta. concentrated ....,65....2-56-0.58 0.15 ml.

Hematoxylin solution:
Nem ato ying tao wha oad? 9s ates Sey yd Bene 0.5 gm.
istilled Waiters a ak Meas tle es iiacne 50.0 ml.
When dissolved, add:
potassium chromium sulfate (chrome alum) 3%,
(1.5-¢in: /50\mill. water) ceoaee.. ster ae or 50.0 ml
Mix well and add:
potassium dichromate, 5% (5 gm./100 ml.
SEEN). -. xu myaeg ieieee. bedceses > Retina: Re Ee gate 2.0 ml
N/2 sulfuric acid (about 2.5 ml./100 ml. water) 2.0 ml.

Ripen for 48 hours. Can be used as long as a film with a metallic

luster forms on its surface after 1 day’s standing. Filter before use.
Pituitary and Pancreas 29]

Phloxine:
phloxiness,.CF 45410). . sucess ee 0.5 gm.
Gistilleditwaterettcs os exc sac oo Reet
1 eae 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Refix in Bouin’s solution: 12—24 hours.
3. Wash in running water: 5 minutes.
4. Treat with potassium dichromate-sulfuric acid: 5 minutes.
5. Decolorize in sodium bisulfite, 5% (5 gm./100 ml. water): 3-5
minutes.
6. Wash in running water: 5 minutes.
7. Stain in hematoxylin solution until beta cells are deep blue.
Check under microscope. 10—15 minutes.
8. Differentiate in hydrochloric acid, 1% (1 ml./99 ml. water):
about | minute.
9. Wash in running water until clear blue: 5 minutes.
10. Stain in phloxine: 5 minutes.
11. Rinse briefly in distilled water.
12. Treat with phosphotungstic acid, 5% (5 gm./100 ml. water): 1
minute.
13. Wash in running water: 5 minutes. Sections turn red again.
14. Differentiate in 95% alcohol. If the sections are too red and the
alpha cells do not stand out clearly, rinse 15-20 seconds in 80%
alcohol.
15. Dehydrate in absolute alcohol, clear, and mount.
RESULTS:
beta cells—blue
alpha cells—red
delta cells (not present in all animals)—pink to red, actually indis-
tinguishable from alphas
COMMENTS:
Also see Heidenhain’s Azan method, page 149.
If the zymogen granules (acidophilic) are to be preserved in the
acinar cells of the pancreatic lobules avoid fixatives containing acetic
acid.
For fluorescent differentiation of the alpha and beta cells, see
Hartroft (1951).
 Chapter 19

Feulgen and PAS

Technics, and
Related Reactions

Feulgen and PAS technics involve two chemical reactions: (1) the
oxidation of 1,2 glycols and/or a-amino alcohol groups to aldehydes,
and (2) the reaction of resulting aldehydes with Schiff reagent (page
294) to form a purple-red color. Among the polysaccharides are gly-
cogen, starch, and cellulose having 1,2 glycol groups which develop a
positive Schiff reaction. Cartilage has a polysaccharide compound mak-
ing this tissue react positively; and among the mucoproteins, the
mucins are carbohydrates and thus react positively. Other structures of
unknown chemical composition, but containing polysaccharides, will
show a positive reaction: striated and brush borders and reticulin
fibers, for example. The two oxidizers most commonly used are chromic
and periodic acids. The latter breaks the carbon chains of the poly-
saccharides containing the 1,2 glycol groupings and oxidizes the broken
ends into aldehyde groups. Chromic acid is a weaker oxidizer with its
action limited almost exclusively to glycogen and mucin (the principle
of the Bauer method, page 301). If necessary, glycogen and starch can
be demonstrated to the exclusion of other reactants, by iodine or Best’s
Carmine (page 267), and mucin by Mayor’s mucicarmine (page 273) or
metachromatic methods (page 278). If it is desirable to prevent the
reaction of glycogen or starch, the saliva or diastase treatment is simple
and effective.

292
Feulgen Reaction 293

Among the nucleic acids, oxidation will not form aldehydes and acid
hydrolysis is required. The two kinds of nucleic acid are (1) thymo-
nucleic acid (desoxyribose or deoxyribose nucleic acid, desoxypentose
or deoxypentose ribonucleic acid, or DNA), the principle component of
nuclei and containing desoxyribose (desoxypentose) sugar; and (2)
ribose nucleic acid (pentose nucleic or plasmo nucleic acid, or RNA),
found in cytoplasmic structures, with ribose (pentose) as the sugar com-
ponent. When nucleic acids are treated with warm HCl, aldehyde
groups are released from the desoxypentose sugars, but not from the
pentose sugars. Thus, when treated with Schiff reagent, DNA reacts
positively, but RNA reacts negatively. Both, however, can be demon-
strated at the same time by selective dyeing with pyronin and methyl
green (page 297).
Schiff’s reagent. In an acid solution and with excess of SO, present,
basic fuchsin (a mixture of several related phenyl methane dyes, ro-
sanilins, and pararosanilins built on the quinoid ring) is reduced to
form a colorless N-sulfinic acid (fuchsin sulfurous acid). This reagent,
with the addition of aldehydes, forms a new phenyl methane dye,
slightly different from basic fuchsin since the color is more purple-red
than pure red. The chemical reaction is not wholly understood. ‘Thus,
areas rich in DNA, after hydrolysis, show deep coloration. Schiff’s is
stable so long as an excess of SOz is present and high acidity. Anything
removing these conditions restores the original dye and produces a
pseudo-reaction. But when regenerated by an aldehyde, the dye be-
comes extremely resistant to such agents. Schiff’s reagent is not a dye; it
lacks a chromophore and is, therefore, colorless. Baker (1958) considers
this an example of localized synthesis of a dye; when the Schiff’s (color-
less) comes in contact with an aldehyde, the chromophore of the triaryl-
methane (quinoid ring structure) dye is reconstituted. The additive
compound of the aldehyde with the Schiff could be called a dye.
Schiff’s deteriorates rapidly at temperatures over 40°C, but at 0°
and —5°C, if kept tightly stoppered, the deterioration is slow. Under
these conditions it will keep as long as six months.
References: Atkinson (1952); Baker (1958); Bensley (1959); Glegg
oo’

Clermont and Leblond (1952); Gomori (1952); Lhotka and Davenport

(1949); Lillie (1951A, 1953, 1954A and B); and Stowell (1945).

Feulgen Reaction
FIXATION: one containing HgCl. is preferred.
204 Feulgen and PAS Technics (cHap. 19)

SOLUTIONS:
N hydrochloric acid, see page 408.
Schiff’s reagent (Lillie, 1951 B):
basic fuchsini@ee 42500! 27.) eee es 0.5-1.0 gm.
distilled weer a... fee tee 85.0 ml.
sodium metabrsuliite, NasSsO- 92s ee 6 ae ee ce 1.9 gm
ING CL ae. a ert ee ee ee 15.0 ml.
Place in bottle with approximately 50-60 ml. of free air space. Shake
at intervals for at least 2 hours or overnight. Add 200 mg. activated
charcoal: 2 minutes; shake occasionally. Filter. If solution is not
water-white the charcoal is old. Try a fresh batch and refilter. Store
Schiff reagent in a bottle with a minimum of air space above the solu-
tion and keep in refrigerator. This will decrease loss of SO2 (Elftman,
oe)
Bleaching solution (sulfurous acid):
ING EIC "(page 208) s i" Ae eared ee ot REL 5.0 ml.
potassium or sodium bisulfite, 10% aqueous
K3550;, ar (NasssOe 215 eee ee 5.0’ ml.
distilled watery. ee eee eae 100.0 ml.
or:
FIG) FCON Centr
ate esc a oei raeee
@enatee tte 1.0 ml.
potassium, orsodium )isul hte: sske<
yon doc Sans 0.4 gm.
distilledpwatetst: OA cic Oe ek aoe eee 100.0 ml.

For best results, make up bleach fresh each time.

Fast Green:
fastiereen diGFy GT, 42058 ly 8s « beve exs pete eee 0.05 gm.
95°, ethyl alcohol i.4:thepteetndis dts ae 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCle. (Leav-
ing slides in 95% alcohol overnight will remove lipids which
might cause a plasmal reaction.)
2. Rinse at room temperature in N HCl: 2 minutes.
3. Hydrolyze at 50°C in N HCI: 20 minutes or 5 minutes at 60°C.
4. Rinse at room temperature in N HCl; rinse in distilled water.
5. Stain in Schiff’s reagent: 2 hours in dark.
6. Drain and transfer quickly into bleaching solution, 3 changes:
1.5-2 minutes in each.
. Wash in running water: 10-15 minutes,
Feulgen Reaction 295

8. Rinse in distilled water.

9. Counterstain in fast green: 10 seconds.
10. Dehydrate, clear, and mount.
RESULTS:
thymonucleic-acid-containing substances—red-violet
other tissue elements—shades of green
COMMENTS:
1. Kasten and Burton (1959) make the following Schiff reagent. It is
quickly prepared, colorless, does not stain the hands, and does not
require refrigeration. It can be made more sensitive by boiling it
for 1 minute.
basretuehsin, C.142500 2... ccc
oe om on 0.05 gm.
Mistmlede Water Oe ai5-d4 oa0s «5c neers eed be _ 300.0 ml.
sodium hyposulfite, Na,S,O,4 ............ rT 6.0 gm.
Solution should decolorize immediately. Filter if necessary. Ready
for immediate use.
2. Pink solutions of Schiff reagent may have lost their potency.
Test by pouring a few drops into 10 ml. of 40% formalin. A good
solution changes rapidly to reddish purple, but if the color changes
slowly and becomes blue-purple, the solution is breaking down.
3. Chen (1943) uses the following weak Flemming’s fixative for
Avian parasites. It gives beautiful results on any kind of smear prepa-
ration and very small pieces of tissue.
FIXATION: 1-4 hours.
chromic acid; 1°, in normal saline’... 0.44042.. 25.0 ml.
acetic ‘acid, 1°, an-normal saline |.<.. $223.08 . 10.0 ml.
osmic acid, 2%, in normalsaline: «=...2. 9374
4.+. 5.0 ml.
Morinall Saline: ou scAet Daed & sack ce fo a oe 60.0 ml.

WASHING: smears—l hour in running water and proceed to step 2, hy-

drolysis: pieces of tisswe—overnight before proceeding to embed
for sectioning.
4. The first of the series of three bleaching solutions (step 6) will
begin to accumulate Schiff’s reagent and turn pink. Then it is ad-
visable to remove that solution, shift number 2 and number 3 along
into positions 1 and 2, and add a new number 3 solution.

Desoxyribonucleic Acid (DNA) Fluorescent Technique

(CULLING AND VASSER, 1961)
FIXATION: 10% formalin for sections; methyl alcohol for smears. (Other
fixatives may require a different time for hydrolysis.)
296 Feulgen and PAS Technics (cHapP. 19)

SOLUTIONS:
Fluorescent Schiff’s reagent:
acriflayvine \dihyedrochloride, \.: 8a te. 1.0 gm.
potassiumemetawisulhite .. .. 5 eee ee: 2.0 gm.
distilled. waitenpad srs ...cn. 11k ee ese eee 200.0 ml.
20.0 ml.

Dissolve acriflavine and potassium metabisulfite in distilled water;

add hydrochloric acid. Keep overnight before using.
Periodic acid:
PCKIOMIeRACICs: fF et Ce co pene ee eee eae 1.0 gm.
Ni esadium. acetate) Soot ae 10.0 ml.
absolute ethyl alcohol 74...2e. fae oe eae 90.0 ml.
Acid alcohol:
TOC et Ligh calle
OlNON Sg eat re etA aad Nes coenl 99) 0small:
hydrochlorc acid; comeentrated #2"... 2. aw 1.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate sections to water. Smears can be carried
directly into next step.
2. Transfer to pre-heated N hydrochloric acid, 60°C: sections, 10
minutes; smears, 3-4 minutes (or 1% periodic acid: 10 minutes
for PAS).
iS) Wash briefly in distilled water.
Transfer to fluorescent Schiff reagent: 20 minutes.
Wash in acid alcohol: 5 minutes.
Wash in fresh acid alcohol: 10 minutes.
Dehydrate in absolute alcohol: 1 minute.
Se
osloClear in xylene: | minute, and mount.
RESULTS:
DNA—bright golden fluorescence
other elements—dark green to black

COMMENTS:
The advantage of this method over the conventional one is that
smaller amounts of dye molecules are more easily observed.
Culling and Vasser warn that previously heated hydrochloric acid
is important; cold hydrochloric acid can produce negative results.

Also timing is important; the reaction may weaken if slides are left
in hydrolysis too long.
Feulgen Reaction 297

For details concerning fluorescent microscope and equipment, see

pages 102.9

Pyronin-Methyl Green for Nucleic Acids (KURNICK, 1952)

FIXATION: absolute alcohol, Carnoy or cold acetone. If formalin is used,
it must be adjusted to pH 7 and used only briefly before the solution
turns acid and produces a faint green staining of cytoplasm. Picric
acid depolymerizes DNA and shows no green chromatin.
SOLUTIONS’
Methyl green:
methy oreensG.1) 42000) .. ... .co-tee
eae ys wes 0.2 gm.
0.1M acetate buffer (pH 4.2) or distilled water 100.0 ml.
Before making solution, methyl green should be purified by extrac-
tion with chloroform. Add approximately 10 gm. methyl green to
200 ml. of chloroform in 500 ml. Erlenmeyer flask and shake. Filter
with suction and repeat with smaller amounts of chloroform until
the solution comes off blue-green instead of lavender (usually re-
quires at least 3 extractions). Dry and store in stoppered bottle. Puri-
fied dye is stable.
Pyronin:
pyronin Y saturated in acetone, or for lighter color in 10% acetone.
The British Pyronin Y for RNA (G. T. Gurr or Edward Gurr) 1s
recommended.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
Stain in methyl green solution: 6 minutes.
NO
©9 Blot and immerse in n-butyl alcohol: several minutes in each of 2
changes.
4. Stain in pyronin: 30—90 seconds. Shorten to a few dips if stain is
too dark.
5. Clear in cedar oil, xylene, and mount.
RESULTS:
RNA containing cytoplasm—red
nucleoli—red
chromatin—bright green
erythrocytes—brown
eosinophilic granules—red
cartilage matrix—ereen
osseous matrix—pink with trace of violet
298 Feulgen and PAS Technics (cHar. 19)

COMMENTS:
These two dyes distinguish between states of polymerization of
nucleic acids, not between the acids themselves. Highly polymerized
nucleic acid stains with methyl green; low polymers of both DNA
and RNA stain with the pyronin. The usual technics resulted in too
pale a methyl green, or if excess staining was tried, water rinses re-
moved the methyl green and acetone removed the pyronin. Dehy-
drating with ethyl alcohol removes most of the color; isopropyl and
tertiary butyl are no improvement. So Kurnick developed the above
method, when he found that n-butyl alcohol differentiated the methyl
green, but it removed the pyronin. He, therefore, decided to leave
the pyronin out of the first solution (it was lost anyway) and tried
following the methyl green with pyronin saturated in acetone. The
acetone must be free of water, so always use a fresh solution.
Later Kurnick (1955) published a method in which he did combine
the two stains:

SOLUTION:
pyronin, ¥,.29, (2 em./100mmi water)... .22 p25. 125 smile
methyl green, 2% (2 gm./100 ml. water) ...... ie ani:
Chistilledh water 4. Aer sco iabeen rece oo es? eee 30.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Stain in methyl] green-pyronin: 6 minutes.
3. Blot carefully with filter paper.
4. Dehydrate, n-butyl alcohol, 2 changes: 5 minutes each.
5. Clear in xylene: 5 minutes; transfer to cedarwood oil: 5 minutes.
6. Mount.

Flax and Pollister (1949) used 0.1% aqueous Azure A, and differ-
entiated overnight in absolute alcohol. The stain was specific for
nucleic acids; chromatin, nucleoli, and cytoplasmic regions high in
nucleoprotein concentrations, but it also stained mast cells, mucus
and cartilage matrix. The latter could be checked by applying ribo-
nuclease.

Periodic Acid-Schiff (PAS) Reaction

Two methods are outlined; one (A) with alcoholic solutions, the
other (B) with aqueous solutions.
Periodic Acid-Schiff (PAS) Reaction 299

FIXATION: any general fixative, but if glycogen or other soluble polysac-

charides are to be demonstrated, fixation and washing should be done
in alcoholic fluids of no less than 70% alcoholic content.

A. SoLuTions (alcoholic):
Periodic acid:
Periodic acidy IO og oes. Hoe age Base bye a 0.8 gm.
Gisimlled Wallen cnet sshd: . yee ee yw gees 30.0 ml.
sodium acetate, hydrated .............. eae 0.27 gm.
(or 10 ml. M/5 sodium acetate)
absolute culm! alCONON = % 5:52 Pagosuee
ss oes ~ 400 ami.

Reducing rinse:
POtassiaN: IOGIdEfkdx .G.~ dea, OO Le 28 2.0 gm.
sodium” thiosulfate; NaoScOg. 22-21.
62 sin 0% 2.0 gm.
GistiNied WALEN 3.75 Vacgnce aes. e ci See S Shae 40.0 ml.
add with stirring:
absolute ethyli:alcohol ta...
2 pope Pow sieo's 60.0 ml.
2 EUG Apape 208) 302 ..2i es ttaeeeene aasag 1.0 ml.

If a precipitate forms, allow it to settle and use solution immediately.

Schiff’s reagent, see page 294.
Sodium bisulfite:
sodium metabisulfite, Na,S,Os .............. 0.5 gm.
distilled) water <..4 fas
cc -: nests wate -. 100.0 ml.

PROCEDURE:
1. Deparaffinize and run slides down to 70% alcohol; remove HgCl,
if present, using iodine in 70% alcohol.
Treat with alcoholic periodic acid: 10 minutes.
NO Rinse with 70% alcohol.
OS

sa ‘Treat with reducing rinse: 5 minutes.

Rinse in 70% alcohol.
Treat with Schiff’s reagent: 10 minutes.
©)
~—J
Or ‘Transfer through sulfite solutions, 3 changes: 1.5—2 minutes in
each (see Comment 4, page 295).
8. Wash in running water: 5 minutes.
9. Counterstain if desired (see Comments, number 3).
10. Dehydrate, clear, and mount.
300 Feulgen and PAS Technics (cap. 19)

B. SOLUTIONS (aqueous):
Periodic acid:
periodic ‘acids (EM@4) ). 2 2). Set Soe 0.6 gm.
distilledsagaterse ote 2.27200.) V2 RR a 850) ore 100.0 ml.
NittiG Were ecancentrated \. 4. -.ee Aa ee 0.3 ml.

Schiff’s reagent, see page 294.

Sulfite solution, see above procedure.
PROCEDURE:
. Deparaffinize and hydrate slides to water; remove HgClbe.
. Treat with periodic acid, aqueous: 5 minutes.
. Wash in running water: 5 minutes.
‘Treat with Schiff’s reagent: 10 minutes.
. Transfer through sulfite solutions, 3 changes: 1.5-2 minutes in
each.
Wash in running water: 5 minutes.
peCounterstain, if desired (see Comments, number 3).
ce Dehydrate, clear, and mount.
RESULTS:
Many tissues give positive PAS reactions: glycogen, starch, cellulose,
mucins, colloid of thyroid, cartilage matrix, chitins, reticulum, fibrin,
collagen—rose to purplish-red
fungi—red
nuclei and other tissue elements—colors of counterstain

COMMENTS:

l. When preparing slides, avoid excessive use of egg albumen; it con-

tains sufficient carbohydrate to react with the Schiff.

ihe) Control Slides:

To remove glycogen, run the slides down to water and subject
them to the saliva test. Saliva contains a diastatic enzyme which
dissolves glycogen and starch. Instead of saliva, a 1% solution of
salt or animal diastase can be used for 20 minutes to overnight at
room temperature.
To remove mucin, treat slides with Lysozyme, 0.1 mg. to 10.0
ml. of Sorensen M/15 phosphate buffer (page 417), PH 6.24, room
temperature: 40-60 minutes.
To remove RNA, treat slides with 0.01 mg. RNase in 10.0 ml.
0.2M acetate buffer (page 414), PH 5.0 at room temperature: 10—
15 minutes (or tris-HCl buffer, 0.05M, pH 7.5: 2 hours, 56°C).
Glycogen 301

3. Counterstains:
for nuclei—hematoxylin
for glycogen—fast green FCF as in Feulgen method
for mucin or acid polysaccharides—an acid dye (fast green)
for other polysaccharides—a basic dye (malachite green)
4. If it is necessary to remove the PAS from the tissue, treat it with
potassium permanganate until all the color is removed, followed
by oxalic acid bleaching of the permanganate.

Glycogen’
Bauer-Feulgen Reaction
FIXATION: avoid aqueous media (see McManus and Mowry, 1958).
absolute ethyl alcohol... .2... :¢8/Ukb. edcecte ot 9 parts.
formalin |. eee es rer eee PE. Ose oie 1 part.

Start dehydration for embedding in 95% alcohol. Mount slides with

95% alcohol.
SOLUTIONS:
Chromic acid:
chromic acid, (TO, 2.2.4 36hss vende e eeet 4.0\ 1m.
distilled water. case. 54 . as adhs ds paeeeeee -. 100.0 ml.

Schiff’s reagent, see page 294.

Sodium bisulfite:
sodium bisulfite, meta, NasS.O; ......... 1.0 gm.
distilled water 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCl. if present.
2. Oxidize in chromic acid: | hour.
3. Wash in running water: 5 minutes.
4. ‘Treat with Schiff’s reagent: 10-15 minutes.
5. Transfer through sodium sulfite, 3 changes: 1, 2, and 2 minutes
(see Comment 4, page 295).
6. Wash in running water: 5 minutes.
7. Counterstain in hematoxylin, if desired.
8. Wash and blue in Scott’s, and wash.
9. Dehydrate, clear, and mount.
See also Best's Carmine reaction for glycogen, page 267.
302 Feulgen and PAS Technics (cHar. 19)

RESULTS:
elycogen—red
nuclei—blue

Desoxyribonucleic Acid, Polysaccharides and Proteins

Triple Stain (HIMES AND MORIBER, 1956)
FIXATION: any good general fixative.
SOLUTIONS:
N HCl, see page 408.
Azure A-Schiff reagent:
amirerd (azure 1) JC. 52000 5eee aoe boc ee 0.5 gm.
bledch;solution;(below) ich. a52) sihaat-2eoRe 100.0 ml.
Lasts several weeks, but add a few drops of 10% potassium metabisul-
fite before reusing.
Bleach:
potassium or sodium metabisulfite, 10% aque-
CUS S eB S Mere eat So DARE geet ey ne Eee 5.0 ml.
IN GENE Toi eas ibe, Meenas es oe ms Shoe a Pane 5.0 ml.
GIStILed Wate — Naas oA e. ok See ere 90.0 ml
Make up fresh each time.
Periodic acid:
penodicvacid: (EIIO4) ice. nie m rreae od eee 0.8 gm.
O:2Migsedrinmaacetate? 2. Ais FeVe ee 10.0 ml.
distilledgwater a7 tyre aks Peet Ee ee 90.0 ml.
Make up fresh each time.
Basic fuchsin-Schiff’s reagent, page 294.
Naphthol yellow S:
Stock solution:
naphthol yellows, C:l, [03WGrwe: 3s. eae 1.0 gm.
aECHIC ACI, 17 AGMEOUS) swiymciie®
ou ats ton 100.0 ml.

Working solution:
stock solution) 20 2h eee orciacloge 2.0 ml.
acenc acid, 1 aqueous: sheave
2 acide: 100.0 ml.

Keeps indefinitely.
Desoxyribonucleic Acid, Polysaccharides and Proteins 303

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCly.
2. Hydrolyze in N HCl, 60°C: 12 minutes, no more.
3. Rinse in distilled water.
4. ‘Treat with Azure A-Schiff: 5 minutes.
5. Rinse in distilled water.
6. Bleach, 2 changes: 2 minutes each.
7. Rinse in distilled water.
8. Oxidize in periodic acid: 2 minutes.
9. Rinse in distilled water.
10. ‘Treat with basic fuchsin-Schiff: 2 minutes.
11. Rinse in distilled water.
12. Bleach, 2 changes: 2 minutes each.
13. Rinse in distilled water.
14. Stain in Naphthol yellow: 2 minutes.
15. Rinse briefly in distilled water.
16. Dehydrate in tertiary or isopropyl alcohol, 2 changes.
17. Clear and mount.
RESULTS:
nuclei—blue to green (DNA)
polysaccharides—red
proteins—yellow
 Chapter 2()

Microorganisms

In this chapter, to simplify the specificity of staining methods, the

microorganisms will be broken down into three groups: bacteria, vi-
ruses, and fungi.
BacrTeriA. Bacteria are customarily studied by direct microscopic ob-
servation and differentiated by shape, grouping of cells, presence or
absence of certain structures, and the reaction of their cells to differen-
tial stains. Bacteria may be stained with aniline dyes; in a single dye,
in mixed dyes, in polychromed dyes, or by differential methods. One of
the most universally used stains was developed by the histologist Gram
while he was trying to differentiate the bacteria in tissue. His method
separates bacteria into two groups, (1) those that retain crystal violet
and are said to be Gram positive, and (2) those that decolorize to be
stained by a counterstain and are said to be Gram negative.
Some bacteria of high lipid content cannot be stained by the usual
methods but require heat or long exposure to the stain. They are also
difficult to decolorize and resist acid alcohol. ‘These have been given the
name acid fast. The spirochete forms will stain only faintly if at all and
must be colored by silver methods.
Many bacteria form a capsule from the outer layer of the cell mem-
brane, and the capsule appears like a halo around the organism, or over
a chain of cells. This capsule will not stain in the customary stains;
Hiss’s stain (page 317), however, is simple and usually effective in this
situation. Some bacteria are able to form spores that can be extremely
resistant to injurious conditions (heat, chemicals). Boiling will destroy
some of these, but many spores are more resistant. Some bacteria have
flagella, filamentous appendages for locomotion.
Bacteria can be classified into three groups according to shape.
304
Microorganisms 305

1. Coccus: spherical; may be found singly (micrococci), in pairs (di-

plococci), in clusters (staphylococci) or in chains (streptococci). The
cocci cause food poisoning, infectious sore throat, scarlet fever, rheu-
matic fever, gonorrhea, pneumonia, and meningitis.
2. Bacillus: rod-shaped; elongate but oval, short, and thick (cocco-
bacilli), attached end to end (streptobacilli). Bacilli cause typhoid fever,
cholera, undulant fever, plague, tularemia, whooping cough, anthrax,
tetanus, diphtheria, botulism, tuberculosis, and leprosy. Bartonella, a
Gram-positive bacillus, causes infectious anemia in dogs, cats, mice,
guinea pigs, cattle, sheep, and rats. The organism appears reddish violet
in a Giemsa stain and is found in tissue macrophages and erythrocytes.
3. Spiral forms: curved-rod; a rigid spiral (spirillum) and a flexible
spiral (spirochete). These forms can cause relapsing fever, Vincent’s
disease, syphillis, and yaws.
Rickettsia are very small, Gram-negative coccobacillary microorgan-
isms associated with typhus and spotted fever and related diseases. ‘They
may appear as cocci or short bacilli, and may occur singly, in pairs, or
in dense masses. ‘They usually are found intracellularly, seldom living
outside of body cells.
Some species are found only in the cytoplasm, others prefer the nu-
cleus. Rickettsia stain best in a Giemsa-type stain or by Machiavello’s
method.
FuNGI: ACTINOMYCETES AND YEASTS. The fungus diseases are described
in two groups.
1. Superficial mycoses: dermatophytes (ringworm).
2. Deep-seated mycoses: actinomycosis (cervico-facial, thoracic or ab-
dominal infections, restricted mostly to agricultural workers).
The actinomycetes are characterized by fine branching filaments
(hyphae) that form by intertwining and sometimes anastomosing a
colony called a mycelium. Small oval or rod-shaped spores are formed
by an aggregation of protoplasm of some of the hyphae. Sometimes the
filaments themselves may break up and form bacillary-like bodies which
are morphologically indistinguishable from bacteria. These spores are
Gram positive and are considered by some as possible transition forms
between bacteria and fungi.
There are two relatively well-defined groups of actinomycetes; one
(including all the pathogenic forms) does not form aerial mycelium,
but does show a tendency to segment into bacillary forms. The other
group is characterized by formation of spores in aerial hyphae. Some-
times the actinomycetes are broken down into the Actinomyces—the
anaerobic forms (live best without air), and Nocardia—the aerobic
306 Microorganisms (CHAP. 20)

forms (must grow in air). Burrows (1954) uses the name Actinomyces
for the pathogenic actinomycetes.
The fungi proper (Eumycetes) also form hyphae and mycelia. They
give off two types of spores, sexual spores produced by the fusion of
two cells and asexual spores formed by differentiation of the cells
of the spore-bearing hyphae but without fusion. The so-called Fungi
Imperfecti (part of the Eumycetes) form only asexual spores. These
spores in some forms may be produced by the segmentation of the tips
of the hyphae and are known as conidia (Penicillium).
Open or draining lesions are difficult to examine for fungi because
of heavy bacterial contamination, but dermatophytes are easily demon-
strated. Scrapings from horny layers or nail plate or hair can be mounted
in 10-20% hot sodium hydroxide. This dissolves or makes transparant
the tissue elements and then the preparation can be examined as a wet
mount. Fungi in tissue sections are readily stained.
Yeast and Yeast-like Fungi. These are described as unicellular and
nucleated. Some yeasts reproduce by budding and others by fission.
Some can produce mycelium. Only a few are pathogenic, causing torula
meningitis, European blastomycosis, and superficial infections of skin
and mucus membranes.
Viruses. The viruses are microorganisms too small to be visible under
the microscope and are capable of passing through filters. Viruses are
responsible for many diseases, including yellow fever, poxes, poliomye-
litis, influenza, measles, mumps, shingles, rabies, colds, infectious hepa-
titis, infectious mononucleosis, trachoma, psittacosis, and foot-and-mouth
disease. In tissue sections and smears, viruses are characterized by ele-
mentary and inclusion bodies. Elementary bodies are infectious parti-
cles, and inclusion bodies are composed of numerous elementary bodies.
Both types of bodies vary in size and appearance; some are located in
the cytoplasm of infected cells (rabies, psittacosis, trachoma) and some
are intranuclear (poliomyelitis). Special staining methods can demon-
strate them effectively. (Reference: Burrows, 1954)
ANIMAL ParasiTEs. Protozoan, helminth, and arthropod infections:
see Chapter 23.

Bacteria Staining
Gram Staining

When a gram staining procedure has been applied, a gram-positive

cell or organism retains a particular primary dye. This process includes
Bacteria Staining 307

mordanting with iodine to form with the dye a precipitate which is

insoluble in water and is neither too soluble nor insoluble in alcohol,
the differentiator.
For the mechanism of gram reactions see Bartholomew and Mittwer
(1950, 1951); Bartholomew et al. (1959); Mittwer et al. (1950).

Gram-Weigert Method (KRAJIAN, 1952)

FIXATION: 10% formalin or Zenker’s formol.
SOLUTIONS:
Fosin:
EOSIN NE GOO a2 + 4 on DEW HI EES Os ae ea 1.0 gm.
CIStIEO WaAtGH 5 cos ss 6 8 oS pee ees eee 100.0 ml.

Sterling’s gentian violet:

crystal violet, C.I. 42555 (gentian violet) ...... 5.0 gm.
OD euiyl alconol 20s 3.555 .taae eka Boat 10.0 ml.
GNTAU TNR SON Lone eters rca. 5 +p See een eda 2.0 ml.
PoLTS
ortlikexe UG eyCaen aa a eee ore 88.0 ml.
Mix aniline oil with water and filter. Add the crystal violet dissolved

in alcohol. Keeps for several weeks to months.

Gram’s iodine solution, see page 410.
PROCEDURE:
Deparaffinize and hydrate slides to water. Remove HgCly.
Stain in eosin: 5 minutes.
Rinse in water.
Stain in Sterling’s gentian violet solution: 3 minutes for frozen
sections; 10 minutes for paraffin sections.
Or Wash off with Gram’s iodine and then flood with more of same
solution: 3 minutes.
6. Blot with filter paper.
Flood with equal parts of aniline oil and xylene; reflood until
color ceases to rinse out of sections.
8. Clear in xylene and mount.
RESULTS:
gram-positive bacteria, and fungi—violet
eram-negative crganisms—not usually stained
fibrin—blue-black

Brown and Brenn Method (1931)

FIXATION: 10% formalin preferred.
308 Microorganisms (CHAP. 20)

SOLUTIONS:
Mayer’s hematoxylin, see page 125.
Gentian violet:
crystal violet, C.I. 42555 (gentian violet) ...... 1.0 gm.
distilledsiwaitera +270) =": 3! SE ee eS 100.0 ml.
Sodium bicarbonate:
sodium= bicarbonate, .~.... A e en 5.0 gm.
GS till alee css. «eve Re ees eda 100.0 ml

Gram’s iodine, see page 410.

Basic fuchsin:
basiegtuchsin, ‘C.D. 4 250010. 02, es eee ee 0.25 gm.
arstilled: waters... Meee... 4 ee eee ee 100.0 ml.

Dilute 0.1 ml. to 100 ml. distilled water for use.

Acetone-picric acid solution:
PULCIEGEACLC seyne pel vedeenetss? fees Byphir Pa Sie aee 0.1 gm.
ACEtONCHE SS. Sch ols tis 6 ee es ae ree 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Stain in Mayer’s hematoxylin: 2—3 minutes.
3. Wash in running water: 5 minutes.
4. Mix 26 drops of gentian violet and 5 drops of sodium bicarbon-
ate solution. Flood slide, agitate occasionally: 2 minutes.
5. Rinse in water.
6. ‘Treat with Gram’s iodine: | minute.
7. Rinse in water and blot.
8. Decolorize with 1 part ether and 3 parts acetone, flooding slide
until no more blue color comes off. Blot.
9. Stain in basic fuchsin: 5 minutes.
10. Rinse quickly in water, blot but do not dry.
11. Dip briefly in acetone, and immediately differentiate in acetone-
picric acid until sections turn yellowish pink. Overdifferentiation
may take out gram negative stain.
12. Rinse quickly in acetone, transfer to acetone-xylene (equal parts),
then to xylene and mount.

RESULTS:
gram-positive organisms—deep violet or black
eram-negative organisms—bright red
cell nuclei—reddish brown
Bacteria Staining 309

cytoplasm—yellowish
red blood corpuscles—yellow or reddish yellow
cartilage—pink
basophilic granules—red
COMMENTS:
The author finds it easier to differentiate step 8 by using a cophin jar
of ether-acetone (1:3) and dunking the slide.

Glynn Method (1935)

FIXATION: Zenker’s without acetic acid preferred.

SOLUTIONS:
Carbol-crystal violet:
crystal violet, C.I. 42555 (gentian violet) ...... 1.0 gm.
phenol: (carbolic acid) crystals: 2. waz. eisa2
200 1.0 gm.
Work together in mortar, add
absolutesethyl alcohol: 23,22 .-adagceehies
25,5 10.0 ml.
Gsstled Water ss Alo dy 5: + a cA ree bere on 100.0 ml.
Allow to stand 48 hours. Filter.
Lugol’s solution, see page 410.
Basic fuchsin:
basic fuchsin, C.I. 42500 : 0.05 gm.
HCI .05N or acetic acid .IN (page 408) rae 100.0 ml.
Solution should be pH2
PROCEDURE:
1. Deparaffinize and hydrate to water; remove HgCly.
2. Stain in carbol-crystal violet: 2 minutes.
3. Drain, but do not wash.
4. ‘Treat with Lugol’s solution: 1 minute.
5. Flush with acetone from dropping bottle until no more color is
removed: 10—15 seconds.
6. Rinse in distilled water.
7. Stain in basic fuchsin: 3 minutes.
8. Drain, but do not wash.
9. Stain in saturated aqueous picric acid: 30 seconds to | minute.
10. Wash in running water till sections are light yellow.
11. Differentiate (some red is lost in sections) and dehydrate in ace-
tone: 10-15 seconds.
12. Xylene and mount.
S10 Microorganisms (CHAP. 20)

RESULTS:
gram-positive bacteria—deep violet
gram-negative bacteria—red
erythrocytes—yellow
nuclei—red
cytoplasm—faint yellow
COMMENTS:
Glynn suggests treating sections with .01N HCl before staining with
basic fuchsin.

Lillie’s Quick Method (1928)

FIXATION: any good general fixative.
SOLUTIONS:
Crystal violet solution:
crystallivioletsGana2555) ea eee 2.0 gm.
Qo oF ethyl alcohol «coef 2! he ee on pate od ee 20.0 ml.
ammonium oxalate, 1% (1 gm./100 ml. water) 80.0 ml.
Filter. Keeps well.

Lugol’s solution, see page 410.

Safranin:
COME
NT gO eaeOillamelSU2 40RR aoe ee Se 8 re eg ey Sc 0.5 gm.
Gistilled Gwateig osc ork yo en me er eae 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
Stain in crystal violet solution: 30 seconds.
Wash in running water: 3—5 minutes.
‘Treat with Lugol’s solution: 30 seconds.
Wash in running water: 2—3 minutes.
Flush with acetone from dropping bottle until no more color is
removed: 10-15 seconds.
Wash in water.
. Counterstain in safranin: 30 seconds.
. Rinse in distilled water.
Dehydrate and differentiate in acetone. Some of red differentiates
out of cells leaving nuclei red and cytoplasm pink.
MM. Clear and mount.
RESULTS:
gram-positive bacteria blue-black
gram-negative bacteria—red
Bacteria Staining onal

cell nuclei—red
cytoplasm, fibrin, collagen—pink

COMMENTS:
For tubercle and lepra bacilli it may be necessary to stain for a longer
period or heat for 90 seconds on a 50—52°C hot plate.

MacCallum-Goodpasture Method (MALLorRy, 1944)

FIXATION: any good general fixative.
SOLUTIONS:
Goodpasture’s fuchsin:
basic fuchsin, G:ls42500) .... cis avenue eed Se 0.59 gm.
MMMM HOU er a Beret a a Zo Sp eRe ae 10S:
phenol (carbolic acid). ». . . ....osiaedget
yess 1.0 gm.
oraiclted ~. .0 524 o54). ose =REE ta gthe EO" mil:
SU AICONOl 235 ota 24. 4 oMg eee estes 100.0 ml.
Sterling’s gentian violet:
crystal violet, C.I. 42500 (gentian violet) ...... 5.0 gm.
9577, ethylialcohol . 7.4. 5 ety eae 10.0 ml.
C16) cee ae eeoem ao 5:4 2fut Meee
eee re 20 amit
distilled water’. .2.2:24.. . ae Sere | ysBONNE Ne
Mix aniline and water and filter. Add crystal violet dissolved in alco-
hol. Keeps several weeks to months.

Gram’s iodine, see page 410.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
2. Stain in Goodpasture’s fuchsin: 10-20 minutes.
3. Wash in water.
4. Differentiate in formalin (full strength): few seconds. Red color
changes to rose.
Rinse in distilled water: few seconds.
6. Treat with picric acid, saturated aqueous: 3—5 minutes, Sections
turn purplish yellow.
. Wash in distilled water: few seconds.
~I

8. Differentiate in 95% alcohol. Sections turn red; some of it washes

out, also some of yellow.
9. Rinse in distilled water.
10. Stain in Sterling’s gentian violet: 3—5 minutes.
11. Rinse in distilled water.
12. Treat with Gram’s iodine: | minute.
312 Microorganisms (CHAP. 20)

13. Blot with filter paper. Do not dry.

14. Flood with equal parts of aniline oil and xylene, reflood until no
color washes out of sections.
15. Xylene and mount.
RESULTS:
gram-positive bacteria—red
eram-negative bacteria—blue
fibrin—blue
other tissue elements—reds to purples

Acid-Fast Staining
Ziehl-Neelsen (MODIFIED FROM MALLORY, 1944)

FIXATION: any general fixative.

SOLUTIONS:
Carbol-fuchsin:
basic fuchsin, C.I. 42500, saturated in absolute
ethyl alcohol (approximately 6 gm./100 ml.
ALCOMON) 5 pe aes 8 2 ee ee ee 10.0 ml.
carbolic acid, 5% (5 ml. melted carbolic acid /95
Lin eWay Sr oC So eee
hn eee 90.0 ml.
Store at room temperature. It is advisable to filter each time before
use.
Acid alcohol:
hydrochloric acid, concentrated’ 3 28...
4.5. 1.0 ami:
70°, seul lpalcoholay no yt Bets pte te Gel fs = +99) (99:0 yam.
Methylene blue solution:
methylene blue chloride @: 752015. 2 2a. se 0.5 gm.
placial "Ace hiG aGids \ seme neeene a ee eee ee 0:5 mil:
cistilled water, 34.05.44 5 ere oo eee ee 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCle.
2. Stain in carbol-fuchsin, 55°C: 30 minutes.
3. Wash in running water: 5 minutes.
4. Destain in acid alcohol until sections are pale pink; check under
microscope.
5. Wash in running water: 3—5 minutes.
6. Dip one slide at a time into methylene blue, taking care that sec-
tions remain light blue; rinse briefly in distilled water.
Bacteria Staining 313

7. Dehydrate, isopropyl alcohol, 99%, 2 changes: 3-5 minutes in

each.
8. Clear and mount.

RESULTS:
acid-fast bacteria—red
nuclei—blue

Ziehl-Neelsen (PUTT’S MODIFICATION, 1951)

FIXATION: general fixative, but 10% formalin best.
SOLUTIONS:
New fuchsin:
new fuchsin, C.I. 42520 (magenta III) ........ 1.0 gm.
phenol (carbolic acid) 2... 2:25 ¢2.822 yen: res 5.0 gm.
ethyl or methyl alcohol (absolute) ...... ean * LOO rl:
distalled water to: make: . v...i.268 ats 100.0 ml.

Acetic alcohol:
Pinca aceue MCIdie ists: os os Skea
weno ee 5 .0Mrnll
absolute ethyl alcohol 2... 2.8 236 oe tee ews 95.0 mil:

Counterstain:
methylene blue‘chloride,; C.1. 52005 224.04es 0.5 gm.
absolute ethyl alcool .<4...22.5 5 sagen 5s<288 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCle.
2. Stain in new fuchsin, room temperature: 3 minutes.
3. Treat with lithium carbonate, saturated aqueous: | minute. Agi-
tate gently, discard solution if it becomes blue.
4. Differentiate until pale pink in acetic alcohol.
5. Rinse in absolute alcohol, 2 changes: 1-2 minutes in each.
6. Counterstain.
7. Decolorize in absolute alcohol, 2 changes: 3 minute each.
8. Clear and mount.

RESULTS:
acid-fast bacteria red
red blood corpuscles—pink
mast cell granules—deep blue
other bacteria blue
nuclei—blue
oie Microorganisms (CHAP. 20)

Kinyoun’s Carbol-Fuchsin Method (MARTI AND

JOHNSON’S MODIFICATION, 1951)
FIXATION: any general fixative, 10% formalin preferred.
SOLUTIONS:
Carbol-fuchsin:
basic tuchsuapmertost 2500)... 20 earseees382+ 5's 4.0 gm.
phenol earsolic acid) crystals 272-022... .. ~.- 8.0 gm.
95oe sebhiyteaicohol, ::. ¢:iG4 ) Seepira eens 56.524 20.0 ml.
distillediwater =?. ..'.\. . 2. Pat temo ee aio 100.0 ml.
Add 1 drop Tergitol +7 to every 30 ml. of above.
Acid alcohol:
iicte acid: ‘concentrated, <8. 728 ee 0.5 ml.
Do we ray aleOWOly <x. sco 2 a ee ae 95.0" mi:
Malachite green:
malachite:sreen: oxalate, C.I. 42000 . aayaz.ahie 1.0 gm.
distilled, watery. 2: ete ted ndcpoue tae eee 100.0 ml.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
. Stain in carbol-fuchsin: 30 minutes, room temperature.
. Wash in running water: 5 minutes.
H. Decolorize in acid alcohol: 3 minutes. Mixture should not be yel-
DO
©

low.
Wash in running water: 5 minutes.
. Rinse in 95% alcohol: | minute.
. Wash in running water: 2 minutes.
. Stain in malachite green: 0.5 minute.
. Wash in running water: few minutes.
— . Rinse in 95% alcohol, 10 dips.
—OH
OO
OID
= . Dehydrate, absolute alcohol; clear and mount.
RESULTS:
acid-fast bacteria—red
tissue elements—shades of green
COMMENTS:
Instead of malachite green, Lillie’s (1944) methylene blue can be
used; 1% methylene blue in 0.5% aqueous acetic acid: 3 minutes.

Fite-Formaldehyde Method (WADE’s MODIFICATION, 1957)

FIXATION: Zenker’s preferred; removal of HgClz not necessary, it dis-
appears during processing.
Bacteria Staining 315

SOLUTIONS:
Phenol new fuchsin:
new fuchsin, C.I. 42520 (Magenta III) ........ 0.5 gm.
phenol (Carbolic acid). i. s:24 chesMeta sla te 5.0 gm.
ethylormethy alcohol ,...2sscme. 2) el Sas 10.0 ml.
distiilediwater tocmake, . .. 2 sae see ees as 100.0 ml.
Van Gieson, modified:
acid fuchsin Gal AlO85" .. Oe eases oh oe eee 2 0.01 gm.
PLCC ACI he Ea 01% 0 3 a eRe Meteo SR 0.1 gm.
GIStier Sater Pici2 2 Ss ts 25 weed ered ona tackSe 100.0 ml.
PROCEDURE:
1. Deparaffinize in turpentine-paraffin oil (2:1) 2 changes: 5 min-
utes total.
2. Drain, wipe off excess fluid, blot to opacity, place in water.
3. Stain overnight in phenol-fuchsin, room temperature.
4. Wash in tap water.
5. Treat with formalin (full strength): 5 minutes (turns blue).
6. Wash in running water: 3—5 minutes.
7. Treat with sulfuric acid, 59% (5 ml./95 ml. water): 5 minutes.
8. Wash in running water: 5-10 minutes.
9. Treat with potassium permanganate, 1% (1 gm./100 ml. water):
3 minutes.
10. Wash in running water: 3 minutes.
11. Treat with oxalic acid, 2-5% (2-5 gm./100 ml. water) individ-
ually with agitation: not more than 30 seconds. Slides can remain
in water while others are being treated.
12. Stain with modified Van Gieson: 3 minutes. No wash.
13. Rinse for few seconds in 95% alcohol.
14. Dehydrate, clear, and mount.
RESULTS:
acid-fast bacteria—deep blue or blue black
connective tissue—red
other tissue elements—yellowish
COMMENTS:
Beamer and Firminger (1955) emphasize care in the use of formalin.
Old formalin yields poor results, while a redistilled form produces
the most brilliant staining. The reagent grade in 16 oz. brown bottles,
if kept tightly closed, give a good stain.
Tilden and Tanaka (1945) outline a method for frozen sections,
essentially the same as the one above after mounting the sections with
316 Microorganisms (CHAP. 20)

celloidin protective coat. They also emphasize the need for good for-
malin; if the sections fail to turn blue, try a different batch.

Tubercle Bacilli Staining

Fluorescent Method (ricHarps, et al., 1941; RICHARDS, 1941;
BOGEN, 1941)

FIXATION: 10% formalin for sections; smears by heat.

SOLUTIONS:
Auramin stain:
apna OSC.1. 'ARO00. i tare ae estes er 0.3 gm.
cHstilled: Water! 2. <ten. 5) ye ee one ae eee 97.0 ml
meltediphenoll (carbolic acid)ix. 220) eae 3.0 ml
Shake to dissolve dye or use gentle heat. Solution becomes cloudy on
cooling, but is satisfactory. Shake before using.
Decolorizing solution:
hydrochloric acid;jconcentrated 2 . i a5.t
lle 0.5 ml.
10°, <ethyl aleohols «533 .sep7 ee A coke it era 100.0 ml.
sodium jchlonidé, 292. «125 ppmareeers 2taaete: 0.5 gm.

PROCEDURE:
1. Deparaffinize and hydrate sections to water.
2. Stain in auramin, room temperature: 2—3 minutes.
3. Wash in running water: 2—3 minutes.
4. Decolorize: 1 minute.
5. Transfer to fresh decolorizer: 2—5 minutes.
6. Wash in running water: 2—3 minutes.
7. Dry smear and examine. Mount sections in fluorescent mountant
(page 122) or Harleco fluorescent mountant for examining.

RESULTS:
bacilli—golden yellow

COMMENTS:
Bogen counterstains with Loeffler’s alkaline methylene blue solution
before mounting:
methylene blue Gal.052 1105 22 ee ee 3.0 gm.
absolmte ethyl, alcohol g*..... 2 > ings ees eee 30.0 ml.
potassium hydroxide, 0.01% aqueous ......... 100.0 ml.
Bacteria Staining 317

Capsule Staining
Hiss’ Method (Burrows, 1954)
FIXATION: any good general fixative, 10°% formalin is satisfactory.
SOLUTIONS:
Either of 2 following stain solutions may be used:
A. basic fuchsin, C.I. 42500 .... ae 0.15-0.3 gm.
distilled water < os alae ceaoe ites ves A DO:0" malt
DB, (Crystal wiGlets AZO 0D: we. o ways eta es | phi ie 0.05-0.1 gm.
Gistwledawater) (sors es os oA se eee ea eas 100.0 ml.

Copper sulfate solution:

copper sulfate crystals. 2... 222: 2 ese e ds See ee 20.0 gm.
distilled Water 2222 6 5.2 snc weal nlene an ee 6 LOOIO mil:

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Flood with either staining solution and heat gently until the stain
steams.
3. Wash off the stain with copper sulfate.
4. Blot, but do not dry.
5. Dehydrate in 99% isopropyl alcohol, 2 changes: 1 minute each.
6. Clear and mount.
RESULTS:
capsules—light pink (basic fuchsin) or blue (crystal violet)
bacterial cells—dark purple surrounded by the capsule color

Spirochete Staining

Dieterle’s Method (MODIFIED By BEAMER AND FIRMINGER, 1955)

FIXATION: 10% formalin.
SOLUTIONS:
Dilute gum mastic:
gum mastic, saturated in absolute alcohol ..... 30 drops
95% ethyl alcohol :...... Pena Oo gang ean 40.0 ml.
Developing solution:
distilled water .... . sense.) 20:0 mal.
hydroquinone |... er SER: 0.5 gm.
sodium sulfite ..... - ee e 0.06 gm.
318 Microorganisms (CHAP. 20)

While stirring add:

formalin Gage. Hiss! is. ss i AR se a cles 4.0 ml.
plycerol.\ eaten ant o.oo pS 2 5.0 ml.
When thoroughly mixed add, drop by drop, with constant stirring:
equal parts of gum mastic saturated in absolute
alcohol#and absolute alcohol. ee) oc os: 8.0 ml.
PY BIGUNC Mires i2 2s. ss Seder eee ete 2.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Remove any formalin pigment in 2% ammonium hydroxide (2
ml./98 ml. water): few minutes.
. Transfer to 80% alcohol: 2-3 minutes.
Wash in distilled water: 5 minutes.
G0
we
Gr Treat with uranium nitrate 2-3% (2-3 gm./100 ml. water), pre-
viously warmed to 60°C: 10 minutes.
Wash in distilled water: 1-2 minutes.
Transfer to 95% alcohol: 1-2 minutes.
‘Treat with dilute gum mastic: 5 minutes.
Wash in distilled water, 3-4 changes, until rinse is clear.
lie
So Impregnate with silver nitrate, 2% (2 gm./100 ml. water), pre-
aercede'e
viously warmed to 60°C: 30-40 minutes.
11. Warm developing solution to 60°C. Dip slide up and down in
solution until sections turn light tan or pale brown.
12. Rinse in 95% alcohol, then in distilled water.
13. Treat with silver nitrate, 2% (2 gm./100 ml. water): 1-2 minutes.
14. Wash in distilled water: 1-2 minutes.
15. Dehydrate, clear and mount.
RESULTS:
spirochetes—black
background—yellow
COMMENTS:
Use thin sections, 4—5 microns.
The uranium nitrate prevents the impregnation of nerve fibers
and reticulum.

Warthin-Starry Silver Method (KERR, 1938; FAULKNER AND

LILLIE, 1945A; BRIDGES AND LUNA, 1957)
FIXATION: 10% formalin.
Notes of caution:
All glassware must be cleaned with potassium dichromate-sulfuric
acid.
Bacteria Staining 319

No contamination; coat forceps with paraffin.

Solutions must be fresh (no more than | week old) and made from
triple distilled water.
Carry a known positive control slide with test slide through the
process, or preferably a control section on the same slide.
SOLUTIONS:
Acidulated water:
triple disiiledhwater <i e tide ernie ys oes = 1000.0 ml.
CAtTIGMAGIMO Tee «J hs ns 46s aber ere es sw oe 10.0 gm,
pH 3.8-4.4
2% Silver nitrate (for developer):
SUVeR MMEALC fas aie abs ee eee 2.0 gm.
ACMI ALE. WaALCT mss a4. ass > ie Soe 2 Value eon: 100.0 ml.

1% Silver nitrate:
dilute a portion of above with equal volume of acidulated water.
0.15%, Hydroquinone:
hyG@TOGMINONE 5 5,122.2 hewitt whee
eons s 0.15 gm.
Acigulated’ Water {o24kancus
+ .cekeew eee aes 100.0 ml.

5% gelatine:
sheet gelatine or granulated of high degree of
DUM ier tines ate Po oh eS RM le 10.0 gm.
ACIGUIated: Walkers emt) «ob. 2n sees ee 200.0 ml.

Developer:
Dover mitrates ete 3%) 22s se Sede ee nee lbs
Y/, POMOLIMNC ack 24 seein: Susdvn ae? SNe ; 3: ot
0.15% hydroquinone ..... hd gine an Bee eee 2.0 ml.
Warm solutions to 55-60°C and mix in order given, with stirring.
Use immediately.
PROCEDURE:
1. Deparaffinize and hydrate slides to acidulated water.
Impregnate in 1% silver nitrate, 55-60°C: 30 minutes.
KO Place
oS slides on glass rods, pour on warm developer (55—60°C).
When sections become golden brown or yellow and developer
brownish-black (3—5 minutes) pour off. The known positive can be
checked under microscope for black organisms.
4, Rinse with warm (55-60°C) tap water, then distilled water.
5. Dehydrate, clear, and mount.
RESULTS:
spirochetes—black
320 Microorganisms (CHAP. 20)

background—yellow to light brown

melanin and hematogenous pigments—may darken
underdevelopment will result in pale background, very slender and
pale spirochetes
overdevelopment will result in dense background, heavily impreg-
nated spirochetes, obstructed detail, sometimes precipitate
COMMENTS:
Faulkner and Lillie (1945) use water buffered to pH 3.6-3.8 with
Walpole’s M/5 sodium acetate-M/5 acetic acid buffer (page 415).

Levaditi Method for Block Staining (MALLory, 1944)

FIXATION: 10% formalin.
SOLUTIONS:
Silver nitrate:
SULVeI* STIUEE ACC 6 8 aero ruac he oe eee 1.5-3.0 gm.
distilled: water 30siiOy, LoL. TADS. Oe) Se 100.0 ml.

Reducing solution:
pyrogalllic acids. Gaee ShaSiGt,
ee 4.0 gm.
FOAL SS. ETS RE ye}. EA, A028 Se 5.0 ml.
distilledtwater t) Wives 400 le. ee oe 100.0 ml.

PROCEDURE:
1. Rinse blocks of tissue in tap water.
2. ‘Transfer to 95% ethyl alcohol: 24 hours.
3. Place in distilled water until tissue sinks.
4. Impregnate with silver nitrate, 37°C, in dark: 3-5 days.
5. Wash in distilled water.
6. Reduce at room temperature in dark: 24—72 hours.
7. Wash in distilled water.
8. Dehydrate, clear in cedarwood oil, and infiltrate with paraffin.
9. Embed, section at 5 microns, mount on slides and dry.
10. Remove paraffin with xylene, 2 changes, and mount.
RESULTS:
spirochetes—black
background—brownish yellow

Fungi Staining
Hotchkiss-McManus Method (mcMmANus, 1948)
FIXATION: 10% formalin or any geneal fixative.
Fungi Staining 32]

SOLUTIONS:
Periodic acid:
PCrIOdIE ACI se. ekeT eve 1.0 gm.
distillleclawaleizes ss se, <3. ei) La Lebooe 100.0 ml.

Schiff’s reagent, see page 294.

Differentiator:
potassium metabisulfite, 10% aqueous ........ 5.0m:
INGEI@I(GaGe 20S)e0 ote «io Powe ge ome 5.0 ml.
distelled twaler *7 tas edi. Bo he ae es tak ... 100.0 ml.
Light green:
light green SF yellowish, C.J. 42095 .......... 0.2 gm.
distilled water | var sete 2. 0 »e@ 100.0 mi.
glacial acetic acid ........ De Mods hp tc ot 0.2 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgCly if present,
Oxidize in periodic acid: 5 minutes.
Wash in running water: 15 minutes.
Treat with Schiff’s reagent: 10-15 minutes.
Differentiate, 2 changes: total 5 minutes.
Wash in running water: 10 minutes.
oe
ee Stain in light green:
Lore 3-5 minutes. If too dark, rinse in running
water.
8. Dehydrate, clear, and mount.

RESULTS:
fungi—red. Not specific, however—glycogen, mucin, amyloid, colloid
and others may show rose to purplish red
background—light green
COMMENTS:
To remove glycogen, starch, mucin, or RNA, see page 300.

Gridley Method (1953)

FIXATION: any good general fixative.
SOLUTIONS:
Chromic acid:
CHLOMIC AGI ou oe acu. 2 sc dc. oud « osdanhoeelene 4.0 gm.
distilled “water. 6 oe 26 t 6. ca p's a 4 i hs eRe ee 100.0 ml.

Schiff reagent, see page 294.

322 Microorganisms (CHAP. 20)

Sulfurous rinse:
sodium metabisulfite, 10% (10 gm./100 ml. wa-
GEE De ee eset +o ee 6.0 ml.
Nihydrochlonce acid (page 408). F722... 22 5.0 ml.
aistilled waren. 20)...
1 ee 100.0 ml.

Aldehyde-fuchsin, see pages 168 and 287.

Metanil yellow:
metamilevemOw aes. 5 . swe dae gee cee eR 0.25 gm.
CIS HIME WALEED: cof & o.. 0 «5 ees eee RO Sto 100.0 ml.
Slaciaigaceticracid.:... i. Nein so Pee eE Sg 2 drops
PROCEDURE:
. Deparaffinize and hydrate slides to water; remove HgCle.
. Oxidize in chromic acid: | hour.
. Wash in running water: 5 minutes.
. Place in Schiff’s reagent: 15 minutes.
Rinse in sulfurous acid, 3 changes: 1.5 minutes each.
Wash in running water: 15 minutes.
. Stain in aldehyde-fuchsin: 15-30 minutes.
. Rinse off excess stain in 95% alcohol.
. Rinse in water.
— . Counterstain lightly in metanil yellow: 1 minute.
11. Rinse in water.
12. Dehydrate, clear, and mount.
RESULTS:
mycelia—deep blue
conidia—deep rose to purple
background—yellow
elastic tissue, mucin—deep blue
Gomori’s Methenamine Silver Nitrate Method (GRrocott’s
ADAPTATION, 1955; MOwRyY’S MODIFICATION, 1959)
FIXATION: 10% formalin or any good general fixative.
SOLUTIONS:
Methenamine silver nitrate. Stock solution:
silver nitrate, 5% (5 gm./100 ml. water) ...... 5.0 ml.
methenamine, 3% (3 gm./100 ml. water) ...... 100.0 ml.
Working solution:
borax,.o, (5 gm.7100 ml, water): jacurs.
~ 24. 2.0 ml.
distilled: water °:| Sy) a) 2 See), ee ee eae 25.0 ml.
methenamine silver nitrate stock solution ..... 25.0 ml.
Fungi Staining 323

Light green, Stock solution:

light green SF, yellowish, C.I. 42095 .......... 0.2 gm.
Gishiiled: wate e654. 4% sve
cowed lk 100.0 ml.
placiallsrCetnG aGid. 5.2% 5.6. 7 cee ee oo Re.6a ait QHZ) mill.

Working solution:
light Creem stock SOMULION . 5. da 5 one). cae dws 10.0 ml.
distiledsawater Arid. «Suber Means 2ateteet, 100.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HeCle.
2. Oxidize in periodic acid, 0.5% (0.5 gm./100 ml. water): 10 min-
utes.
3. Wash in running water: 3 minutes.
4. Oxidize in chromic acid, 5% (5 gm./100 ml. water): 45 minutes.
5. Wash in running water: 2 minutes.
6. Treat with sodium bisulfite, 2% (2 gm./100 ml. water): 1 minute
to remove chromic acid.
7. Wash in running water: 5 minutes.
8. Rinse in distilled water, 2-3 changes: 5 minutes total.
9. Place in methenamine silver nitrate, 58°C: 30 minutes. Do not
use metal forceps. Sections appear yellowish brown.
10. Wash thoroughly, several changes distilled water.
11. ‘Tone in gold chloride (10 ml. stock solution /90 ml. water) until
sections turn purplish grey, fungi are black.
12. Rinse in distilled water.
13. Fix in sodium thiosulfate, 5°% (5 gm./100 ml. water): 3 minutes.
14. Wash in running water: 5 minutes.
15. Counterstain in light green: 30 seconds.
16. Dehydrate, clear, and mount.

RESULTS:
fungi—black
background—light green

COMMENTS:
Mowry’s method uses oxidation with both periodic and chromic acid
(former methods use only the latter) with the result that the final
staining is stronger and mere consistent than that of the original
Gomori method.

Fluorescent Method (pickerr et al., 1960)

FIXATION: 10°% formalin, Zenker’s, alcohol and others.
324 Microorganisms (CHAP. 20)

SOLUTIONS:
Acridine orange:
ACHIGICNOTANR CRE oe nl. a5 .s Shoe eee et 0.1 gm.
cistilledhyatenwarest: ci. ic Sine CP ees Bees 100.0 ml.
Weigert’s hematoxylin, see page 126.
PROCEDURE:
1. Deparaffinize and hydrate sections to water.
ND Stain in Weigert’s hematoxylin: 5 minutes.
Wash in running water: 5 minutes.
059 .

Stain in acridine orange: 2 minutes.

Rinse in tap or distilled water: 30 seconds.
Dehydrate in 95% alcohol: 1 minute.
Dehydrate in absolute alcohol, 2 changes: 2—3 minutes.
Clear in xylene, 2 changes: 2—3 minutes.
oA
OODMount in a nonfluorescing medium.
RESULTS:
all fungi fluoresce except Nocardia and Rhizopus; colors of the fluo-
rescing genuses appear as follows:
Coccidioides, Rhinosporidum—red
Aspergillus—green
Actinomyces, Histoplasma—red to yellow
Candida, Blastomyces dermatitids, Monosporium—yellow-green
Blastomyces brasiliensts—yellow
COMMENTS:
Old hematoxylin and eosin slides may be decolorized and restained
as above.
The Weigert’s hematoxylin staining is necessary as a quenching
agent, because some fungi are difficult to see against a background
which also fluoresces. With the hematoxylin, the fungi fluoresce
brightly against a dark setting.

Rickettsia and Inclusion Bodies

Modified Pappenheim Stain (cASTANEDA, 1939)
FIXATION: any general fixative, Regaud recommended.
SOLUTIONS:
Stock Jenner stain:
Vemmenisustaim:\)': 50%, ach Sern hao mie, erie 1.0 gm.
imethylealcohol, absolutest2: 4 (2.2 ue. ee 400.0 ml.
Rickettsia and Virus Staining 329

Stock Giemsa stain:

Giemsarstainwaner 2 lhe A ae | te aie 1.0 gm.
PIVCEVOMN wrens
SS lee eee io ston a /00,06 mil:
Mix and place in oven 2 hours, 60°C. Add
methyl alcohol, absolute ..05 222.0064 iets. 66.0 ml.
Working solution A:
CUS taeda aber a2 0). ah dog 212k Oia oe are Ueno 100.0 ml.
Slacialaceuic acid) 25)! nc Se dels panty fae ects 1 drop
JENNER SLOGK SOMO dis fea heen a Sana 3% 20.0 ml.
Working solution B:
distilledewater .os.cccec85
25. Be eet es 100.0 ml.
placinleacenc ace & 425-2 «...:seweddue
ts See lary: 1 drop
Giemsa stock Solution: . ...«« .s¢2 sais
eeed sos do 5.0 ml.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
. Stain in solution A, 37°C: 15 minutes.
. Transfer directly to solution B, 37°C: 30-60 minutes.
. Dehydrate quickly, 2 changes absolute alcohol.
DO.
oo
Be
Or Clear and mount.
RESULTS:
rickettsiae—blue to purplish blue

Castaneda’s Method (GRADWOHL, 1956)

FIXATION: a general fixative, but Regaud recommended.
SOLUTIONS:
Buffer solutions:
Solution A:
di-basic sodium phosphate, Na,HPO,-12H,O.. 23.86 gm.
Gistiledswater its 1280S 82% Pac odo Ale Hes 1000.0 ml.

Solution B:
monobasic sodium phosphate, anhydrous, NaH,
POG oR eee ees as ce ies Bae hee 11.34. em,
Gastilléd Waters. 73.00.46 06 « nce 0 Seas aeck _.. 1000.0 ml.

Working solution:
SOUT Pe ele
oe a ct oho 88.0 ml.
solution B 12.0 ml.
FOVIMALT cs sents ald beds de ary | 0.2 ml.
326 Microorganisms (CHAP. 20)

Methylene blue solution:

Dissolvesmethylene blue, G.I. 52015 (02........: 21.0 gm.
T9597, \ethveCONOl. |... 2 oc. de ens 300.0 ml.
Dissolve potassium hydroxide .............. 0.1 gm.
In distilled@ewarer 2. 2, . .-¢pc eee 1000.0 ml.
Mix the two solutions and let stand 24 hours.
Staining solution:
Mix buffer working solution with methylene blue solution.

PROCEDURE:
1. Deparaffinize and hydrate slides as far as 50% alcohol.
. Stain in methylene blue: 2-3 minutes.
. Wash in running water: 30 seconds.
. Counterstain in 1% safranin (1 gm./100 ml. water): 1-2 minutes.
. Dip briefly in 95% alcohol.
P . Dehydrate in 2 changes absolute alcohol, clear, and mount.
OF
DM
PO
OO

RESULTS:
rickettsiae—light blue

COMMENTS:
Burrows (1954) recommends this as one of the best methods for
rickettsia.

Ordway-Machiavello Method (GRADWOHL, 1956)

FIXATION: Regaud recommended.

SOLUTION:
Staining solution:
Poriers blue (C:Gs) ol°Aeaqueous eee 10.0 ml.
eosin) bluish, C.1. 45400, 0.459%, aqueous, .......,... 15.0 ml.
Mix just before use. Add slowly with constant shaking:
distilled: water? <. 12.52 4 en eit. enetee a 25-0) mil.
Use within 24 hours.

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Stain: 6-8 minutes.
3. Decolorize in 95% ethyl alcohol until slides appear pale bluish
pink.
4. Dehydrate in absolute alcohol: 1 minute.
5, Clear and mount.
Rickettsia and Virus Staining 327

RESULTS:
rickettsiae, also inclusion bodies—bright red
nuclei and cytoplasm—sky blue

Giemsa Method (AMERICAN PUBLIC HEALTH ASSOCIATION, 1956)

FIXATION: general fixative, Regaud recommended.

SOLUTIONS:
Giemsa stock solution, see page 225.
Buffer solutions:
A. dibasic sodium phosphate, anhydrous, Nas
Oe again 348 as y pe catueeeaie Béla 9.5 gm.
Gisqiledoayater 4% 5.4. .-5 snk oe ea Seces 1000.0 ml.
B. monobasic sodium phosphate, NaH PQO,-
TE i Sat AMA hacdiatie ack 2 a ene enna aa 9.2 gm
distilled water... <4 .656.<9%
ees 1000.0 ml

Working solution, pH 7.2:

SOMO oO 5 cpus oon a pe ees ae 72.0 ml.
SOMO TE! So, 4 4 2 nade sem bated womans BREE ae Bee 28:0) mil.
distilled water ....4........% SEaC eee vowa> 900.0 al,

Giemsa working solution:

Dilute 1 drop Giemsa stock solution with 5 ml. of buffer working
solution.
PROCEDURE:
1. Deparaffinize and hydrate slides to water. (Smears can be carried
directly to water: wash well in running water: 5-10 minutes.)
Leave slides in Giemsa working solution overnight, 37°C.
Rinse thoroughly in distilled water. Dry between blotters.
DO Dip rapidly in absolute alcohol. If overstained,
©9
Hm use 95% alcohol
to decolorize them, dip in absolute alcohol.
(con Clear and mount. (Smears, after treatment with absolute alcohol,
can be washed in distilled water: 1-2 seconds, and blotted dry.
Examine with oil or mount with a cover glass.)
RESULTS:
rickettsiae and inclusion bodies (psittacosis)—blue to purplish blue

Modification for trachoma.

SOLUTION:
Giemsa working solution:
Dilute 1 drop of stock Giemsa with 2 ml. of buffer solution above.
328 Microorganisms (CHAP. 20)

PROCEDURE:
1. Hydrate slides to water (these will be smears).
2. Stain in working solution, 37°C: | hour.
3. Rinse rapidly, 2 changes 95% ethyl alcohol.
4. Dehydrate in absolute alcohol, clear, and mount.
RESULTS:
inclusion bodies—blue to purplish blue

Negri Bodies (Rabies)

Schleifstein (1937) Method

FIXATION: Zenker’s preferred.

SOLUTIONS:
Staining solution:
basic: fuchsin CG. e472 500) 20) cy) aot co) 1.8 gm.
methylene blue, Gil s2015>. 2 aa 2 aan 1.0 gm.
PIV CORON, 1.2 eel aaah: oe Geet 2k, Se fotos: eee 100.0 ml.
MEL yVarCONOIANS ) a5) e Gk een etme eet 100.0 ml.
Keeps indefinitely.
For use add about 10 drops to 15—20 ml. of dilute potassium hydrox-
ide (1 gm./40,000 ml. water). Alkaline tap water may be used.
PROCEDURE:
1. Deparaffinize and hydrate slides to water; remove HgClo.
. Rinse in distilled water and place slides on warm electric hot plate.
NO. Flood
©9 amply with stain and steam for 5 minutes. Do not allow
stain to boil.
4. Cool and rinse in tap water.
5. Decolorize and differentiate each slide by agitating in 90% ethyl
alcohol until the sections assume a pale violet color. This is im-
portant.
6. Dehydrate, clear, and mount.
RESULTS:
Negri bodies—deep magenta red
eranular inclusions—dark blue
nucleoli—bluish black
cytoplasm—blue-violet
erythrocytes—copper
COMMENTS:
Schleifstein outlines a rapid method for fixing and embedding so the
Negri Bodies (Rabies) o29

entire process can be handled in 8 hours, including fixing embedding,

and staining.

Massignani and Malferrari (1961) Method

FIXATION: 10% formalin or saturated aqueous mercuric chloride-abso-
lute alcohol (1:2).
EMBEDDING and SECTIONING: paraffin method, 4 microns.
SOLUTIONS:
Harris hematoxylin, see page 125.
Dilute hydrochloric acid:
hydrochloric acid;,concentrated:. cs dci6 nig i: 1.0 ml.
distilled water ..... sop SA SOR Se ota 200.0 ml.
Dilute lithium carbonate:
lithium carbonate, saturated aqueous oh ok fete 1.0 ml.
distilled water ........ ‘4 200.0 ml.

Phosphotungstic acid-eosin stain:

Grind together | gm. eosin Y (C.I. 45380) and 0.7 gm. phospho-
tungstic acid. Mix thoroughly into 10.0 ml. of distilled water and
then bring volume up to 200.0 ml. with distilled water. Centrifuge at
1,500 rpm: 40 minutes. Pour off supernatant solution but do not
throw it away. Dissolve the precipitate in 50 ml. of absolute alcohol.
When dissolved, add to the supernatant solution. The solution is
ready to use. If the eosin and phosphotungstic acid are not ground
together and then dissolved it requires 24 hours for the dye-mordant
combination to form.
PROCEDURE:
1. Deparaffinize and hydrate sections to water, remove HgCls.
2. Stain in hematoxylin: 2 minutes.
3. Wash in running water: 5 minutes.
4. Dip 8 times in dilute hydrochloric acid.
5. Wash in running water: 5 minutes.
6. Blue in dilute lithium carbonate: | minute.
7. Wash in running water: 5 minutes.
8. Dehydrate to absolute alcohol.
9. Stain in phosphotungstic acid-eosin: 8 minutes.
10. Rinse in distilled water.
11. Dehydrate by quick dips in 50%, 70%, 80%, and 90% alcohol,
then follow with 95% alcohol: 1 second.
12. Complete dehydration in absolute alcohol: 2—3 changes: 4 min-
utes each.
13. Clear and mount.
330 Microorganisms (CHAP. 20)

RESULTS:
Negri bodies—deep red
COMMENTS:
Massignani and Refinetti in their paper (1958) adapted the Papani-
colaou stain for Negri bodies. Further study, however, led to the
discovery that eosin combined with phosphotungstic acid is respon-
sible for Negri body staining and the above stain was developed
specifically for these bodies.

Antigen-Antibodies
The subject of immunity is complex and has filled several large text-
books. It is mentioned briefly here to bring to mind certain principles
used in this field, and to highlight one of the most important technics
developed for locating antigens and antibodies in tissues.
If foreign materials, living or nonliving—bacteria and viruses, for
instance—invade the body, certain substances are formed in body fluids
to combat these foreign materials and “‘neutralize” them. The defense
substances are proteins called antibodies that have the power to com-
bine specifically with the invading foreign materials (antigens) which
induced the formation of the antibodies. Eventual immunity to an
infection can be brought about either by entrance of the antigen natu-
rally or artificially—by “‘shots,” for instance. In the latter case, serum,
called antiserum (immune serum) from an artificially immunized ani-
mal can be injected into a nonimmune animal to induce immunity.
Antiserum can be used in this way to combat an infection already
present (mumps, measles, anthrax, tetanus) or it can be used to prevent
infection (measles, poliomyelitis, tetanus, diphtheria).
If the fluid portion of blood plasma is allowed to clot (fibrinogen
precipitates out) a relatively stable serum remains. This is made up of
two protein fractions: albumin and globulin. The globulins consist of
two alpha globulins, one beta globulin, and one gamma globulin. It is
the latter globulin which is associated with immunity. Antibodies have
been classically identified with gamma globulin (to a lesser extent with
the other globulins, also) and are considered modified serum globulin.
It has become a common practice to use for immune serum the gamma
globulin fraction, which can be isolated from the other serum proteins
and used in a concentrated form. The difference, therefore, between
immune and normal serum globulin lies in the ability of the former to
combine with an antigen.
Antigen-A ntibodies 331

Coons’ Fluorescent Technic

If antigens can be labeled, sources of antibody production in tissues

can be seen microscopically. Coons and his associates have coupled anti-
bodies with fluorescein isocyanate and isothiocyanate to form fluores-
cent carbamide-proteins. The conjugates thus formed from dye and
antibodies still retain the specific reaction of the antibody for the
antigen that causes its formation. Labeled antibodies are poured over
the tissues or cells and will leave a labeled protein in the tissues which
can be observed under a fluorescent-adapted microscope.
Some of the solutions, antisera (immune serum), and conjugates have
to be prepared in the laboratory; some can be purchased, but it is
recommended that all conjugates be purified by absorption against
tissue powders. For reasons and means of doing this, see Coons et al.
(1955); Coons (1958); and Mellors (1959). The method is complicated
and should not be undertaken without a thorough study of the source
material.
With a few exceptions (some polysaccharides) chemical fixatives must
be avoided to retain specific activity of the antigen. Smears, touch
preparations, tissue cultures, and cell suspensions can be used. If sec-
tions are preferred, freeze-dried or unfixed frozen sections cut in a
cryostat (page 337) are mandatory. Tissues may be quick-frozen in
petroleum ether previously chilled to —65°C with dry ice-alcohol mix-
ture in a Dewar flask. When completely frozen, blot and store in
tightly stoppered test tube at —20°C to —70°C until used. Long storage
is to be avoided; the tissues dehydrate (Tobie, 1958). Coons et al. (1955)
place small pieces of tissue against the wall of a small test tube and
plunge it into alcohol cooled to —70°C with solid carbon dioxide. Store
at —20°C until used.
After sectioning by either method, the tissue sections must be fixed
before applying the antigen-antibody solution. Fixation depends on the
antigen.
FIXATION: proteins: 95% ethyl alcohol, sometimes absolute methyl alco-
hol; polysaccharides: can be fixed in the block by picric acid-alcohol-
formalin (Rossman fluid, page 20) and _ paraffin-embedded; lipids:
10% formalin; viruses: acetone.
DIRECT METHOD (staining directly with fluorescein-labeled antibody
against specific antigen).
Slides are dried after fixation, rinsed in buffered saline (0.8% sodium
* Arnel Products Co., N.Y. (Sylvania Chemicals) Baltimore Biological Laboratory, Balti-
more, Maryland.
332 Microorganisms (CHAP. 20)

chloride with 0.01M phosphate, pH 7.0) and the excess saline wiped off
except over the sections. Labeled antibody is pipetted over the section;
the slides, covered with a petri dish with moist cotton or filter paper
attached to its undersurface, are allowed to stand, room temperature:
20-30 minutes. The slide is wiped dry except for the section and
mounted in buffered glycerol (anhydrous glycerol, 9 parts, buffered
saline, | part), then covered with a cover glass. Examination is made
under the fluorescent microscope.
INDIRECT METHOD (layering).
A tissue section containing antigen is covered with unlabeled specific
antiserum to allow the antibody molecules to react with it and be fixed
in situ (humid environment): 20 minutes. The slide is rinsed off in
buffered saline: 10 minutes, then wiped dry except for the section, and
fluorescein-labeled antiglobulin serum (prepared against the species
which furnished the specific serum) added: 20 minutes. The slide is
washed in buffered saline and mounted in buffered glycerol. Cells that
reacted with the antiserum will be covered with antibody (globulin)
and will have reacted with the labeled antiglobulin. This has been
termed layering because the bottom layer is antibody, the middle layer
the antigen, and the labeled antibody hes on top.
Coons (1958) recommends control slides and uses a blocking tech-
nique. Unlabeled specific antiserum is added to a slide and allowed to
react for 20 minutes. Then, if washed with buffer solution and treated
with a drop of labeled antibody, only a faint fluorescence, if any,
should be exhibited.
Silverstein (1957) described a means of applying contrasting fluores-
cent labels for two antibodies using rhodamin B with fluorescein.
The Coons methods have been applied for the detection of viruses,
rickettsiae, epidemic typhus, mumps, influenza, canine hepatitis,
chicken pox, canine distemper, measles, psittacosis, and poliomyelitis.

This description is intended as only a brief résumé of Coons’ technics

in order to acquaint the technician with their potentialities. The fol-
lowing references contain extensive and pertinent discussions of the
procedures: Coons (1956 and 1958) and Mellors (1959).
A tremendous bibliography is developing in this field; the following
references will lead to many others: Buckley et al. (1955); Cohen et al.
(1955); Coons et al. (1942, 1950, 1951, 1955); Coons and Kaplan (1950);
Kaplan et al. (1950); Lacey and Davis (1959); Leduc et al. (1955); Mel-
lors et al. (1955); Noyes (1955); Noyes and Watson (1955); ‘Tobie (1958);
Watson (1952); and White (1955).
 HISTOCHEMISTRY
ce jeeD
III MISCELLANEOUS
SPECIAL
PROCEDURES
 Chapter 2]

Histochemistry

By definition the field of histochemistry is concerned with the locali-

zation and identification of a chemical substance in a tissue. In a broad
sense this might include staining, combining chemical and_ physical
reactions, instances where acidic and basic methods demonstrate basic
and acidic properties of the tissue. But strictly speaking, histochemistry
is being applied only to chemical methods immobilizing a chemical at
the site it occupies in living tissue. These methods can apply to in-
organic substances: calcium, iron, barium, copper, zinc, lead, mercury,
and others; they also apply to organic substances: saccharides, lipids,
proteins, amino acids, nucleic acid, enterochromaffin substance, and
some pigments. Some substances are soluble and react directly, others
are insoluble and must be converted into soluble substances before a
reaction takes place. Occult or masked materials are part of a complex
organic molecule. This has to be destroyed by an unmasking agent be-
fore the chemical can react. Some chemicals may be fixed in place, others
which are soluble or diffusible have to be frozen quickly and prepared
by the freeze-drying method involving no liquid phase. Sometimes it is
advisable to make control slides, thereby preventing confusion between
a genuine reaction and a nonspecific one giving a similar effect.
A sharp distinction between staining and histochemical methods has
proved difficult and not wholly practical. Some will disagree with the
present arrangement of methods, but a sequence according to similar
tissue or cell types seemed adaptable to general laboratory application,
the primary intent of this book. The section “Histochemistry,” there-

230
336 Histochemistry (CHAP. 21)

fore, will cover mainly procedures used to identify enzymatic activity.

The field is a tremendous and puzzling one of increasingly extensive
activity. It is impossible and impractical to include all histochemical
methods and their variations in this type of manual. There are avail-
able many excellent books in the field. Also there are journals that
describe the newest methods and modifications; therefore, only the
most familiar (at least to the author) methods are being incorporated in
this text.
For greater detail start with: Cassellman (1959); Danielli (1953);
Davenport (1960); Glick (1949); Gomori (1952); Gurr (1958); Lillie
(1954); and Pearse (1960).

Freezing-Drying Method for Embedding

Sometimes enzymes, proteins or other substances can be lost during
fixation, dehydration and embedding, and a freezing-drying method
must be used. Small pieces (approximately 1 mm.) are frozen solid
instantly with isopentane cooled by liquid nitrogen to a temperature of
—150°C. The tissue must be frozen rapidly to prevent large crystal
formation which would disrupt the cells. The initial freezing is
commonly called quenching; it stops all chemical reactions that have
been going on in the tissues. Immediately after freezing (it must not
thaw) the tissue is dehydrated in a drying apparatus in vacuo at a
temperature of —30 to —40°C and the ice is sublimed into water vapor
and removed.' There is no liquid phase present at any time, and there-
fore no diffusion of enzymes.
When the material is dry, it is allowed to rise to room temperature,
is infiltrated with degassed paraffin or carbowax and embedded. The
advantages of this method are that shrinkage and diffusion artifacts are
reduced to a minimum, and enzymes and chemical structures are pre-
served. ‘There are some disadvantages; the equipment is bulky and
expensive, and only small pieces can be used in order to prevent distor-
tion by ice crystals. Because of sensitivity to water, the sections cannot
be floated on water, but must be applied directly to warm albumenized
slides (Mendelow and Hamilton, 1950). Embedded blocks have to be
kept stored in a desiccator.
Carbowax or paraffin can be cut at room temperature, but many
enzymes and other tissue constituents necessitate sectioning at low
temperatures of —25 to —20°C or they are lost. Since embedding is
1See new ‘‘Unitrap,”’
Pp Fisher Scientific Co.
Freezing-Drying Method for Embedding “rey

impractical at these temperatures, tissue is frozen in isopentane

(—160°C) or in dry ice-ethyl alcohol mixtures (—70°C). The tissue must
be maintained at least at dry ice temperature (—20°C) and sectioned
and mounted at this temperature. This is performed inside a cold
chamber cryostat? (—25 to —20°C). The object holder is precooled and
the block frozen to it with a few drops of water. The knife must be
sharp and kept as dry as possible. After the section is cut, dip a clean
cooled slide in ethyl alcohol or isopentane (—20°C) and pick up section
by touching slide against it (Louis, 1957). A cover glass can be used in
the same manner. (Slides sometimes must be albumenized.) Dry in a
cold room at 0 to —2°C: 1 hour with fan. Mellors (1959) thaws them
quickly at room temperature for 15 minutes, aided by a warm finger
against the undersurface of the slide. If slides must be stored (but only
for a short time) place them over desiccant (CaSQ,) in refrigerator.
Fitz-William et al. (1960) recommend that immediately before each
section is cut the tissue block be coated with 20% polystyrene dissolved
in methylene chloride (volatile at cryostat temperature). Wait until a
highly reflecting surface, which forms, disappears and then cut section.
Curling of sections is minimal and they can be picked up with fine
forceps. The polystyrene solution should be stored in the cryostat.
These authors also include complete directions on the care of the
microtome used in the cryostat.
See also Greenbaum (1956) and Coons et al. (1951).

Ice-Solvent (freeze-substitution) Method

Freeze-dry methods can be troublesome and costly. A simpler and
cheaper method with excellent results is the ice-solvent procedure
wherein the tissue is rapidly frozen and then the ice formed within the
tissue is slowly dissolved in a fluid solvent such as ethyl or methyl
alcohol. (Other polar solvents also have been tried. See Patten and
Brown, 1958.) When the tissue is free of ice and completely permeated
by the cold solvent, it is brought to room temperature and embedded.
In addition to being simpler than freeze-drying, other advantages are:
no disruptive streaming movements in the tissue—the ice simply dis-
solves—and the substitution solution can sometimes be chosen to con-
tain a specific precipitant for a specific substance. The method can be
used for radioautography, fluorescent preparations, many enzymes,
and other cyto-histochemical methods. There can, however, be cases in
which the freeze-drying procedure is preferred—for instance an enzyme
*Linderstrom-Lang Cryostat, manufactured by Harris Refrigeration Co., Cambridge,
Mass.; also, Fisher & Lipshaw advertise Cryostats.
338 Histochemistry (CHAP. 21)

might be damaged by the denaturing action of the alcohol as it warms

up.
PROCEDURE: (Feder and Sidman, 1958; Hancox, 1957; Patton and Brown,
1958).
1. Cool a beaker of liquid propane-isopentane (3:1) to —170°C-—
175°C with liquid nitrogen.
no. Place tissue specimen (small pieces, 1.5—3 mm.) on thin strip of
aluminum foil and plunge into the cold propane-isopentane
(quenching). Stir vigorously with a 12” ruler: 30 seconds.
3. Transfer frozen specimen to ice-solvent (see Comments) (already
chilled to —60 to —70°C). After a few minutes the tissue can be
shaken loose from the foil. Store in dry ice chest for 1 to 2 weeks.
(52 days did no harm; Patten and Brown)
4. Remove fluid and tissue to refrigerator, wash 12 hours or longer
in 3 changes of absolute alcohol.
5. Transfer to chloroform: 6-12 hours in refrigerator, to remove
any traces of water. (This step may be omitted in most cases.)
6. ‘Transfer to cold xylene (or similar agent), —20°C; and remove
to room temperature for 10 minutes.
Transfer to fresh xylene, room temperature: 10 minutes.
‘Transfer to xylene: tissuemat, 56°C, vacuum: 15 minutes.
Infiltrate, tissuemat #1, vacuum: 15 minutes.
Transfer to tissuemat #¢2, vacuum: 15 minutes.
FS
“I
Oo) Transfer
Socom to tissuemat #£3, vacuum: 15 minutes.
12. Embed.

COMMENTS:
Ice-solvents used with best results by Patten and Brown (1958) were
absolute methyl and absolute ethyl alcohol. Hancox (1957) used
n-butyl alcohol and could infiltrate directly from the ice-solvent,
thereby bypassing the xylene step. Feder and Sidman (1958) used 1%
solution of mercuric chloride in ethyl alcohol, and osmic acid in
acetone, trying to combine a chemical and physical fixing action. The
latter authors include complete directions concerning obtaining,
storing and disposing of liquid nitrogen, isopentane and propane,
also details about equipment.
Hancox (1957) obtained his best sections in a cryostat. He tried
dry mounting, 85% alcohol flotation and water flotation of sections.
The latter gave inferior results; certain cell inclusions had disap-
peared, and the cytoplasm resembled a vacuolated reticulum. Patten
and Brown (1958) found that mounting on water gave the most dis-
Acetone Fixation and Embedding 339

tinct and intense sections, but they were chiefly interested in fluores-
cence. So perhaps the best suggestion is to try all methods and dis-
cover which serves the particular investigation being undertaken.
Blank et al. (1951) use glycols if fixation is to be avoided, alcohol
or acetone if fixation is permissible. This method is as follows:
PROCEDURE:
1. Place slices (2-3 mm.) of tissue in cold propylene glycol in deep
freeze below —20°C: I-2 hours.
2. Transfer to mixture of carbowax, 55°C: 2-3 hours.
Carbowax .000\ oho. . 5 a. scutes os aee.4 EATS 9 parts
CAMO NWaS WOOD) ny ce ost a.m,oc et EA ee ee ] part
3. Prepare blocks, page 81.
4. Cut sections and float on slides according to Blank and McCarthy,
page 81.
Or Stain or use for autoradiographs. Water-soluble substances have
not leached out. For autoradiographs, before each cut coat the
block face with thin molten wax. Press wax-reinforced sections
against emulsion for exposure. See also: Freed (1955); Meryman
(1959, 1960); Woods and Pollister (1955).
Rejerences: Blank er al. (1951); “Feder (1958); Mreed (1955);* Gersh
(1932); Glick and Malstrom (1952); Hancox (1957); Hoerr (1936);
Ibanez et al. (1960); Jennings (1951); Louis (1957); 'Mellors: (1959);
Meryman (1959, 1960); Packer and Scott (1942); Patten and Brown
(1958); Simpson (1941); and Woods and Pollister (1955).

Acetone Fixation and Embedding

Gomori (1952) Method
Chilled alcohol (95% or absolute) and acetone are used for preserva-
tion of tissues to be used for many kinds of enzyme localization. Ace-
tone is generally preferred, being applicable to more enzyme techniques.
Keep a tightly stoppered bottle of it stored in the refrigerator, ready for
any occasion (see also Burstone, 1958).
PROCEDURE:
1. If possible chill tissue for a short time before immersing in ace-
tone. It may prevent some shrinkage artifacts. But fix within 10
minutes of death for best results.
ihe) Fix in cold acetone, 3 changes: 12 hours each.
3. Transfer to ether-absolute alcohol (1:1): 2 hours, refrigerator.
340 Histochemistry (CHAP. 21)

4. Infiltrate with dilute celloidin or nitrocellulose (approximately

10%): overnight, refrigerator.
Or. Drain on cleansing tissue. Place in benzene or chloroform: 0.5—1
hour. Stirring on a mag-mix aids penetration of fluid.
6. Infiltrate with paraffin, 56°C (52°C is better): 15-20 minutes in
oven without vacuum.
. Infiltrate with paraffin, 56°C (or 52°C): 30 minutes with vacuum.
. Infiltrate with paraffin, 56°C (or 52°C): 10-15 minutes without
vacuum.
Embed. Store in refrigerator.
. Section. Float on lukewarm water, briefly (10 minutes maximum).
Drain off water and dry. If kept for some time, put in oven 5-10
minutes to melt paraffin forming protective coat against atmos-
pheric influence. Store in cold room or refrigerator. Safest way
to store tissues is in uncut paraffin block in refrigerator.
COMMENTS:
Novtikoff et al. (1960) find acetone fixation is good for some cryostat
preparations.
Control Slides: Most enzyme reactions should be accompanied by a
control slide in order to help detect false positives. An enzyme can be
inactivated by one of the following methods:
le Place slide in distilled water and bring to boil: 10 minutes.
Immerse in IN HCl: 15 minutes; wash in tap water: 0.5 hour.
ho Incubate
OS slide in substrate medium containing an inhibitor.
NaCN or KCN.
Incubate untreated slide in a medium backing substrate.
Preservation of incubation media: Klionsky and Marcoux (1960)
prepared twelve different media, froze them in dry ice-acetone mix-
ture, and stored them in a dry ice chest. Within at least 6 months, the
solutions remained as good as freshly prepared solutions. The types
of media tested were:
i azo dye and metal precipitate methods for acid and alkaline phos-
photases.
alpha-naphthyl and indoxyl acetate for esterase.
5-nucleotidase.
nitro-BT for succinic dehydrogenase.
post-coupling technic for beta-glucuronidase.
D triphosphopyridine nucleotide and diphosphopyridine nucleotide
oo
Or
1D
diaphorase.
Alkaline Phosphatase 341

Alkaline Phosphatase

Sections containing active phosphatase are incubated in a mixture of

calcium salt and phosphate ester, usually sodium glycerophosphate, so
calcium phosphate is precipitated at the sites. The enzyme liberates the
phosphate ions, which are held on the spot by salts of metals whose
phosphates are insoluble. (Magnesium ions are often required as an
activator and are added in the form of magnesium sulfate or chloride.
‘The mechanism of activation is unknown.) The insoluble phosphates
are made visible either by (1) silver nitrate, producing silver phosphate
and metallic silver; or (2) cobalt nitrate and ammonium sulfide, pro-
ducing cobalt phosphate and finally black cobalt sulfide.
Phosphatase is dissolved or destroyed by ordinary fixatives, but alco-
hol or acetone will preserve it. Celloidin or paraffin embedding can be
used safely, but paraffin sections must be covered with a film of celloidin
to protect against dissolution of enzyme. See Burstone (1960) for fluores-
cent demonstration of alkaline phosphatase.
FIXATION: Thin slices (1-2 mm) are fixed at once in chilled acetone, 3
changes: 24-48 hours. Other fixatives may be 80%, 95%, and abso-
lute alcohol, or cold formalin (4°C) followed by freezing method.
INFILTRATION AND EMBEDDING: For paraffin embedding use chloroform
and paraffin at no higher than 58°C melting point, preferably 52°C.
Cut sections 6-10 microns. Do not dry slides at higher than 37°C.
Store all blocks at 6°C until needed.

SOLUTION:
Incubating Solution:
SOGUms Danbital. wasn. .ccoednles
ose eeotcs ie knife-point full
(regulates alkalinity, only a bit raises it)
3% sodium glycerophosphate (3 gm./100 ml.
water) Keep inreiriperator.. 32. 3. 22.6564: 5-10 ml.
2% calcium chloride (2 gm./100 ml. water) .... 20-25 ml.
1% magnesium sulfate (1 gm./100 ml. water) .. 10 drops
distilled water to.make ........«2¢.+-440;5008 50.0 ml.
PH should be 9.4; if solution is turbid, filter out free phosphate.
PROCEDURE:
1. Deparafhnize (light petroleum can be used), pass through acetone
and coat with 0.25-0.5% celloidin; continue hydration.
342 Histochemistry (CHAP. 21)

INe) Place in incubating solution, 37°C: 0.5—3 hours. (Warm solutien

up to 39°C before starting incubation.)
05 . Rinse in distilled water.
4. Change to colored form by either of following methods:
A. Immerse in 1—2% cobalt salt (chloride, nitrate, acetate or sul-
fate) (1-2 gm./100 ml. water): 3—5 minutes.
Rinse thoroughly, distilled water, 3 changes.
Immerse in fresh dilute solution of yellow ammonium sulfide (5-6
drops in coplin jar of distilled water).
Rinse in distilled water, 3-4 changes.
Counterstain if desired, dehydrate, clear, and mount.
B. Immerse in 5% silver nitrate (5 gm./100 ml. water) under ul-
traviolet lamp: 30 minutes. Or use 0.5% silver nitrate (0.5 gm./
100 ml. water) in direct sunlight, depending on light intensity.
Rinse in distilled water.
Fix in 1—2% sodium thiosulfate: 2 minutes.
Rinse, counterstain, dehydrate, clear, and mount.

Counterstains may be phloxine-methylene blue, PAS, eosin, safranine,

or the like.

RESULTS:
cobalt method—brownish black
silver method—golden brown

COMMENTS:
False positives can be due to hemosiderin or melanin and these pig-
ments should be checked for presence.

References: Ackerman (1958); Gomori (1941A, 1946); Kabot and

Furth (1941); and Moffat (1958)

Azo-Coupling Method
The sections are incubated in a solution of sodium alpha naphthyl
phosphate and a diazonium salt. ‘The phosphatase activity liberates
alpha naphthol to couple with the diazonium salt and form an insoluble
pigment.

FIXATION: cold 10°% formalin (4°C): 8-16 hours.

Frozen sections, 10-15 microns: mount on clean slides with no ad-
hesive, air-dry 1—3 hours.
Acid Phosphatase 343

SOLUTION:
Incubating solution:
sodium a-naphthyl phosphate ............... 10-20 mg.
Ow Mrveronall acetate buffer, pl. 9:2) 0.22.55. 20) smi:
stable diazotate of 4-benzoyl amino-2:5-dimeth-
oxyaniline (fast blue RR) ..... Re Rae 20.0 gm.

PROCEDURE:
1. Filter enough incubating solution onto slides to cover sections
adequately. Incubate, room temperature: 15-60 minutes.
Wash in running water: 1—3 minutes.
Counterstain, Mayer’s hematoxylin: 4—6 minutes.
Wash in running water: 15-30 minutes.
be
ye
ormMount in glycerol jelly.

RESULTS:
sites—black

COMMENTS:
Alcohol fixation can be used, but is of no great advantage for frozen
sections.

References: Gomori (1951), Manheimer and Seligman (1949), and

Pearse (1960)

Acid Phosphatase

Most methods lack uniformity and some loss occurs during fixation
and preparation for sectioning. Gomori (1956) considered the lead sul-
fide method “capricious,” producing artifacts, and that the benzoyl-
naphthol phosphate post-coupling method of Rutenburg and Seligman
(1955) was best. Since then Burstone’s (1959B) method has appeared.
Both of the latter are included.

FIXATION:
Rutenburg and Seligman Method:
1. Cold neutral 10% formalin: 24—48 hours (neutralize with NaCl).
Wash in 0.85% NaCl 2 changes: 30 minutes.
Cut frozen sections.
2. Fresh frozen sections (6-10 microns) cut in cryostat, mounted on
uncoated slides, air-dried.
344 Histochemistry (CHAP. 21)

Immerse 3 minutes in 3 solutions of NaCl (0.85%, 1% and 2%)

before incubation.
Burstone Method:
Fix in cold acetone, 8—24 hours. Pieces 3 mm. thick. Double-embed
(page 339).

SOLUTIONS:
Rutenburg and Seligman Method:
Solution A:
sodium 6-benzoyl-2-naphthyl phosphate? ...... 25.0 mg.
Cis tailed awrater:. 2.02... Ae ee eee ee tte 80.0 ml.
acetaresbutker. (0:5) glad 00 Seem ea. aoe a) 20:07:
make hypertonic by adding 2 gm. NaCl to each 100 ml. of solution.
Solution B:
tetrazotized diorthoanisidine. 447... 4)52. ) 95. 50.0 mg
distilled. Water? Ace eee eae en eke ee 50.0 ml
make alkaline with NasCOs.
Burstone Method:
Either of two substrates may be used.*4
a. Naphthol AS-MX phosphate
b. 6-benzoyl-2-naphthyl phosphate
Dissolve 5 mg. in 0.25 ml. DMF (N, N-dimethy1
formamide)° and add distilled water ........ 25.0 ml.
O.2MGacetatespiiter, yptde o.2 tte atone
ee Zo. Ormalll
diazotized 4-amino-2,5-diethoxybenzanilide,
Casi Lo (tast blues BINesaltjye — 0. eases 30.0 mg.
manganese chlonde; NOGA). antes... Sep eee 2 drops

Shake and filter.

PROCEDURE:
Rutenburg and Seligman Method:
1. Transfer frozen sections or cryostat sections to substrate (solution
A) room temperature: 1—2 hours.
2. Wash, 3 changes: 10—5 minutes each, in cold saline (fresh tissues)
or water (fixed tissues).
® Dajac Laboratories.
‘ Dajac Laboratories.
> Matheson, Coleman and Bell.
® General Dyestuff Corporation.
Aminopeptidase 345

3. ‘Transfer to cold (4°C) freshly prepared solution B, agitate: 3—5

-

minutes till blue dye produced.

4. Wash, 3 changes of cold 0.85% NaCl or water (fixed tissues): 10
minutes each.
5. Mount in glycerol jelly.
Burstone Method: (simultaneous dye and substrate procedure)
1. Deparaffinize in petroleum ether, 3 changes: 4 minutes each.
2. Acetone, 2 changes: 4 minutes each.
3. Acetone-water (1:1): 2 minutes.
4. Rinse in distilled water: 30 seconds.
5. Incubate, room temperature: 45-60 minutes.
6. Rinse in distilled water.
7. Mount in glycerol jelly.
RESULTS:
sites—blue

COMMENTS:
If decalcification is necessary, Schajowicz and Cabrini (1959) say
that 2% formic acid and 20% sodium citrate (1:1), pH 5 is satisfac-
tory up to 15 days. There is loss of enzyme if versene is used.

References: Burstone (1959B); Burton (1954); Goetsch and Reynolds

(1951); Gomori (1950A, 1956); Rutenburg and Seligman (1955); and
Tandler (1953).

Amiunopeptidase
Burstone and Folk (1956)
FIXATION and EMBEDDING:
1. Cold acetone fixation and paraffin embedding, page 339.
2. Freeze drying and embedding in vacuum gives better retention of
enzyme.
SOLUTIONS:
Substrate and Dye:
Stock solution of substrate:
[-leucyl-b-naphthylamide ................... 1.0 gm.
distilled mWwateryscas 40 cay hues ced scc Ae 100.0 ml.
346 Histochemistry (CHAP. 21)

‘Tris buffer, puxi.19:

0.2 M tris (hydroxymethyl) aminomethane .... 25.0 ml.
ORTIN (ELC een o.0. ae Re eb 45.0 ml.
dilute withvdiselled water t0 =: Yaaneeeoro.-. « 100.0 ml

Working solution:
StOCK “SUlStrateman 0... -. > eed geen = 1.0 ml.
distilled Swattenien an 8! 5... io Se Sanne 2! 40.0 ml
trisdib wien hc. 5.5" 22e Soe ee ek © 10.0 ml
Garnet GBC? (diazotized o-aminoazotoluene) .. 30.0 mg.

Shake and filter.

For substrate, the alanyl compound may be substituted: 10 mg. in 40
ml. hot distilled water (90°C). Cool and add 10 ml. of tris buffer and
30 mg. garnet GBC.

PROCEDURE:
1. Chloroform or petroleum ether, 2 changes: 4 minutes each.
. Absolute acetone: | minute.
95% acetone: 1 minute.
85% acetone, several dips.
. Distilled water, few dips.
. Substrate: 15 minutes to 1 hour, room temperature.
Tap water: 5 minutes.
. Hematoxylin: 3 minutes.
NY . Tap water:
OF
Or#
Or~AIm
Oo 10 minutes.
_ i=). Glycerol jelly.

RESULTS:
sites—red

COMMENTS:
If necessary to purify GBC salt (not Imperial Chemical Industries
:oduct):
. Dissolve 10 gms. salt in 50 ml. methyl alcohol. Let stand 0.5 hour.
Filter.
ie) Repeat with residue.

3. Dissolve second residue in 500 ml. ether. Let stand 2—3 hours.
4, Filter. Wash residue 2—3 times with ether. Store dry powder in re-
frigerator.
* Imperial Chemical Industries, Manchester, England, recommended.
Esterases and Lipases S47.

Fsterases and Lipases

These terms are used in reference to enzymes which hydrolyze esters

of carboxylic acids. There is an overlapping between enzyme types and
some seem to occupy intermediate positions between lipases and ester-
ases, and react like both forms. (Chessick, 1953; Gomori, 1952) Broadly
speaking, esters of short-chained fatty acids are acted on by esterases,
long-chained esters by lipases.
Esterases or lipases are not destroyed by acetone fixation, are resistant
to heat and can be embedded in paraffin if not used excessively (3 hours,
maximum, 58°C). They can be incubated with the long-chained fatty
acid esters of sorbitan and mannitan, water-soluble commercial prod-
ucts by the name of ‘Tween.’ On hydrolysis, the liberated fatty acids
form insoluble calcium soaps deposited at the sites. These are converted
into lead soaps and then into brown sulfide of lead.
Fsters of naphthols also are used and naphthol is liberated and dem-
onstrated by azo-coupling.

Esterase, Azo-Coupling (Gomori, 1953)

FIXATION: cold acetone, paraffin embedding, page 339.

SOLUTIONS:
Barbital buffer:
sodium diethyl barbiturate (10.3 gm./500 ml.
water) .. eigeewee: , O6beml:
0.1 M HCl . : . oo.5' mall
Shake and filter.
Substrate:
dissolve a or b-naphthylacetate .............. 10.0 mg.
in acetone |. oe ee 1.0 ml.
add to solution of:
a-naphthyl diazonium naphthalene-1,5-disulfo-
nate ; 7 ind ; re 40.0 mg.
OME INAaGIEG eee 42 wae 50.0 ml.
barbital buffer, pH 7.8 _ 20.0 ml.
distilled water ......... wapiaa ve oil: 2 See 29.0 ml.

PROCEDURE:
1. Xylene, 2 changes: 3 minutes each.
2. Absolute acetone: 1 minute.
348 Histochemistry (CHAP. 21)

> 99%, acetone: | minute.

. 85% acetone: 1 minute.
50% acetone, 1 minute.
. Water: 1 minute.
. Incubate, room temperature: 3—20 minutes.
. Wash in tap water: 2 minutes.
. Hematoxylin: 3 minutes.
. Wash, tap water: 5 minutes.
mH
oo
TW. Glycerol jelly.
OID
SO
ped
—

RESULTS:
sites—red

COMMENTS:
Nachlas and Seligman (1949) prefer b over a-naphthylacetate, say-
ing that it gives greater brilliance of reaction.
Wachstein and Wolf (1958) incubate 30-90 minutes in:
1% propylene glycol (in 0.2M phosphate buffer
ait “OEM G PO) her eRe Red oo Re oe Se 40.0 ml.
1% naphthol-AS acetate in acetone ............ 0.4 ml.
fast" blue B salta(G.W S775). ioe oe, «sorter 80.0 mg.

Lipase (Gomori, 1950E, 1952)

FIXATION: chilled acetone: 24 hours (2mm. sections: prechill tissue to
lessen shrinkage; trim thinner when tissues have gained some con-
sistency). Embed by double embedding, page 339. ‘Temperature
should not exceed 58°C. If sections refuse to flatten completely, blot
onto slides, dry at room temperature, then melt in paraffin oven.

SOLUTIONS:
Substrate:
Tween 60 (or product #81, Onyx Oil and
Chemical Co., or G2151, Atlas Powder Co.) . . 5.0 gm.
Gaspilled! WatODRy 3, 275 ig on meets oc ees oh 100.0 ml.
Tris buffer, pH 7.2-7.4:
ine leiveeaCe Reece ee Meme a ae eee toons © aye 29.0 gm.
tris (hydroxymethyl) aminomethane .......... 30.3 gm.
disailled “waters i¢-2.. 4. fitity eeey Braete 2 yes 500.0 ml.
addm@enancoal ... 5, ehtimes See oe 5 pees Yee 2.0 gm.

Shake, let stand 10 minutes, and filter.

Esterases and Lipases 349

Buffer working solution:

STOCKE SOLON 4 a. oss hc ee ee 40.0 ml.
NCINGOM MGA yies oo 5 os. ckwer ees gongs erekn 20.0 ml.
dilute“tom total Of .) .. i tkeene -= *L00%0" mi:

(Preserve substrate and buffer with $% chloretone or crystal of thy-

mol.)
Calcium chloride:
CAlCamaRsCM One® 44.5.4: Tae e gabe Seen 4c 10.0 gm.
Gristle water. css few.a «fetes See WE es ke -. 100.0 ml.

Working solution:
butter working solution «. ...scs0..58e%
dns oes 9.0) nl,
caloimmrchloride® os 3 ons. 08 340 2R8 0 bh et aed 20a,
SUM Grete cs Ss oc. 54g 2a Ae is oe ee Ay 2.0 ml.
distilled water ...... S Srcoateie i can: 40 0a,

If solution becomes turbid, filter through fritted glass or porcelain

filter of medium density.
PROCEDURE:
1. Deparafhnize and transfer slides to absolute alcohol.
2. Flood with 0.5% celloidin to protect against loss of enzyme.
3. Hydrate to water.
4. Incubate: 12—48 hours.
5. Wash in distilled water.
€. Treat with 1% lead nitrate (1 gm./100 ml. water): 15 minutes
(calcium stearate transformed to lead stearate).
7. Wash in several changes distilled water.
8. ‘Treat with dilute ammonium sulfide (1:5): few minutes (brown
sulfide produced).
9. Rinse in tap water.
10. Counterstain lightly, hematoxylin and eosin if desired.
11. Dehydrate.
12. Clear in crude gasoline or tetra-chloroethylene (avoid xylene,
fades stain).
13. Mount, using resin in one of above; not xylene.
RESULTS:
sites—golden brown
COMMENTS:

Sources of error: brownish pigment or calcareous deposits, pig-

ments visible in unincubated section. ‘These may be hemosiderin
350 Histochemistry (CHAP. 21)

(perform Prussian blue reaction) or calctwm deposits (remove before

incubation in citrate buffer of pH 4.5: 10-20 minutes).
George and Thomas (1960) coat sections with thin layer of 5-10%
aqueous solution of pure gelatine (preserve with thymol not phenol).
Place in refrigerator freeze chamber to solidify coating. Place in 6%
cold neutral formalin to harden. Wash and proceed to incubate.
Eapen (1960) proved that lipase is activated by gelatine and coated
sections to give better results.

Succinic Dehydrogenase
The succinic oxidase system is responsible for the oxidation of the
majority of tissue substrates and is a part of the oxidation-reduction
system in tissues. It is widely distributed, particularly in heart muscle
and kidney.
Succinic dehydrogenase is demonstrated by the reduction of tetra-
zolium salts. ‘The tetrazolium (colorless) acts as a hydrogen acceptor and
becomes reduced to a blue water-insoluble pigment, diformazon.

Tetrazolium salts:

TPT—2,3,5-triphenyl tetrazolium chloride: simplest, with limited sen-

sitivity.
BT—2,2’,5,5’-tetraphenyl-3,3’ (3,3’-dimethoxy-4,4’-biphenylene) ditetra-
zolium chloride: better pigment qualities.
Nitro BT—2,2’-di-p-nitrophenyl-5,5’-diphenyl-3,3’-(3,3’-dimethoxy-4,4’-
biphenylene) ditetrazolium chloride.

Most methods require anerobic (absence of oxygen) conditions to pre-

vent atmospheric oxygen from competing with the tetrazolium for the
electrons of the dye and thus decrease reduction. Sodium succinate is
the substrate.
Sections must be fresh frozen, cut at 30 microns (not less than 20 mi-
crons) and are improved by cutting in a cryostat. ‘They may be floated
on a beaker of the incubating fluid inside the cryostat or mounted on
slides and air dried in the cryostat. For anerobic conditions remove air
from the solution with vacuum, or boil the substrate and then cool to
37°C. In either case, keep in full, tightly closed bottles (small weighing
bottle are excellent). Sections are dropped in and the bottle immedi-
Dopa Oxidase Reaction

ately closed. Slides may be placed in fluid in covered coplin jars with
nitrogen bubbling through.

SOLUTIONS:
Substrate:
Stock buffered succinate; equal volumes of:
0.2M phosphate buffer, pH 7.6
0.2M sodium succinate
Incubating solution; equal volumes of:
buffered succinate
nitro BT (10 mg./10 ml. distilled water)

PROCEDURE:
1. Place sections or slides in incubating solution, 37°C: 1—2 hours or
until sufficient color is present.
2. Wash in physiological saline: 1 minute.
3. Fix in 10% formalin: 1-24 hours.
4. Mount loose sections in glycerol jelly, or mount on slides and pro-
ceed to step 5.
5. Dehydrate, clear, and mount.
Sections may be counterstained in safranine or hematoxylin.

RESULTS:
sites—blue

References: Farber and Bueling (1956); Farber and Louviere (1954);

Friede (1958); Nachlas et al. (1957); Novikoff et al. (1960); Pearson
(1958); Rosa and Velarde (1954); and Rutenburg ef al. (1950, 1956)

Dopa Oxidase Reaction

The Dopa reaction is considered specific for melanoblasts and mye-

logenous leucocytes which are supposed to contain an intracellular fer-
ment, dopa-melanase. The latter converts the 3,4 dioxyphenylalanine
into a blackish brown pigment, the black condition of the reacting cell.

Laidlaw’s Method (cLick, 1949)

FIXATION: Only fresh tissue with no fixation (or 5° formalin for no
longer than 3 hours, but results are inferior to those from fresh tis-
sue).
352 Histochemistry (CHAP. 21)

SOLUTIONS:
Dopa stock solution:
DL b (3,4 dioxyphenylalanine) phenyl] tyrosine’. 0.3 gm.
distillediwatengeers 22... 2G 6 A aa eae «© 300.0 ml.
Store in refrigerator. Discard when it turns red.
Buffer solutions:
A. disodium hydrogen phosphate ............ 11.0 gm.
distilledswater :.|. ic .aceeiee eee oe 1000.0 ml.
B. potassium dihydrogen phosphate ........ 9.0 gm.
Gistilled Water pasxin.. hatieeei ieee he ey 1000.0 ml.
Buffered Dopa solution:
Stocks Depa solution... jo<4) alec acresae 25.0 ml.
buiter solution AL.) 0 oe 2.4. .eee ee ee 6.0 ml
butter: SOlntiOn. 235 ease mag eee ee eee 2.0 ml
Filter through fine filter paper. Should be pH 7.4.
Buffered control solution:

buifer‘solution-Aie te
een ee 6.0 ml.
butter sOlutioness ey co 4 ee ee 2.0 ml.

Cresyl violet:
cresyl (wioletracetaten, «is. shee ete ete. ei 0.5 gm.
Gistilledwwaitere. hte se: Get) Y. Waeee eee th Pee 100.0 ml.

PROCEDURE:
1. Cut frozen sections, 10 microns.
2. Wash in distilled water: few seconds.
3. ‘Treat with buffered Dopa solution, 30-37°C. The solution be-
comes red in about 2 hours, and gradually becomes sepia brown
in 3-4 hours. Do not allow sections to remain in a sepia-colored
solution; they may overstain. After first 30 minutes, change to a
new solution.
4. Wash in distilled water.
5. Counterstain in cresyl violet.
6. Dehydrate, clear and mount.
RESULTS:
Dopa oxidase—black
leukocytes, melanoblasts—grey to black
melanin—yellow brown
8 Eastman Kodak Co. #4915.
Arginine Test 353

COMMENT:
Freeze-dried and embedded tissues give a good Dopa reaction.

Arginine Test
The arginine reaction uses the reaction of a-naphthol with guanidine
derivatives in the presence of hypochlorite or hypobromite to produce
an orange-red color. The reaction proceeds with guanidine derivatives
in which one hydrogen atom of one of amino groups is substituted by
alkyl, fatty acid or cyano radical. Proteins containing arginine give the
color, and in tissue fixed by ordinary methods the reaction is specific
for protein-bound arginine and other guanidine derivatives such as
elycocyamine or agmatine. Guanidine, urea, creatine, creatinine, and
amino acids, other than arginine, do not give the color.

Thomas (1950); Liebman (1951)

FIXATION: Bouin’s or Stieve’s.

SOLUTIONS:
a-Naphthol stock solution:
anaphihol (Bastmancse170) ..<122096 6223 aes 1.0 gm.
absolute ctw alconol o. a. 2g ee Aaa 100.0 ml.
Keep in refrigerator.
Working solution:
SHOCKHSOUUITION ts ¢ gegen, i ae cee Panta weeaees 5.0 ml.
GISEIMEd Water: b2eFcreces
awiete.2 22 bus BS EARS 3 100.0 ml.

Hypochlorite and potassium hydroxide:

Nesodiumaiypochlonte: 5 3, x..-.2.<-4 veneer 15.0 ml.
distilled water ....... Fs cane eet ee 80.0 ml.
IN oO tasstam MVATOSAGE: 3.2.5 Js
es ee oe berm.
Urea, potassium hydroxide:
BIGER: axe or eats A oe eee cc Oe. ok Gone eee 2 oe 10.0 gm.
distilled water <<.S.<.4..-. 2s.65-5s nde teem 15.0 ml.
WN spotassium: Iydroxide <... pe: .2.2.62)
+5 21eee 5.0 ml.

Shake until urea is in solution, add:

tertiary butyl alcohol, anhydrous ............ 70.0 ml.

PROCEDURE:
Reagents above are kept in ice bath and allowed to cool well before
354 Histochemistry (cHAP. 21)

use. Keep in bath during procedure. Other reagents at room tempera-

ture.
1. Deparafhnize slides and run down to 70% alcohol.
Place in a-naphthol: 15-30 minutes.
oo
PO Transfer (no wash) into hypochlorite-potassium hydroxide: 1.5
minutes.
4. ‘Transfer quickly into urea-potassium hydroxide, 2 changes.
First change: 10 seconds, agitate slides.
Second change: 2 minutes.
5. Transfer into absolute tertiary butyl alcohol, 2 changes.
First change: 10 seconds, agitate.
Second change: 3.5 minutes.
6. Transfer into xylene, 3 changes.
First change: 10 seconds, agitate.
Second change: 1-2 minutes.
Third change: 2—3 minutes.
7. Drain off xylene, but do not dry. Mount in mineral oil.
RESULTS:
arginine—orange to red. Good for about 6 months.

COMMENTS:
Make all transfers as quickly as possible without allowing slides to
drain. Turn jars so slides do not hit lugs on sides.
The first changes in steps 4, 5, and 6 are intended to wash off the
previous fluid, and the slides should be moved gently back and forth
to facilitate the process. This may be done in the second changes, but
more slowly. Timing must be accurate; a stop watch is advisable.
Change solutions frequently; the reagents must be fresh. Carry not
more than 5 slides through the a-naphthol and hypochlorite-potas-
sium hydroxide and not more than 30 through any of the other solu-
tions.
Solidification of the tertiary butyl alcohol may be caused by the
transfer of cold slides into it, so it is advisable to warm it to 30—40°C
before use. Change the alcohol frequently. Carry about 10 slides
through | change; the first solution may then be discarded and re-
placed by the second change. The latter is then replaced by a fresh
solution.
Before mounting, drain off the xylene thoroughly, but do not dry.
Apply several drops of mineral oil, and add cover glass. Draw off ex-
cess mineral oil with filter paper. (The excess oil is desirable because
Mounting Media 355

it dilutes the xylene to a minimum.) The cover glasses tend to slip,

so seal them with one of the ringing cements (page 123).

Mounting Media
Burstone (1957A) recommends for some azo-dye methods:
I: Mount directly from 75-80% alcohol in 20% solution of polyvinyl
acetate (Gelva 2.5, Shawinigan Resins Corporation, Springfield,
Massachusetts) in 80% alcohol. It is soluble also in glacial acetic
acid, n-butanol (90%), aniline, pyridine, and ethyl cellosolve.
It preserves the following:
esterase, alkaline phosphatase, PAS, hematoxylin, celestin blue,
aldehyde fuchsin, eosin.
Cover glass is immovable after 1 hour. No bubble formation. In-
dex of refraction, 1.3865, but increases after evaporation of alcohol
and water.
 Chapter DP

Special Procedures I

Exfoliative Cytology
Exfoliative cytology (study of cells as opposed to Histology, study of
tissues) concerns the preparation and examination of desquamated
cells. ‘These are cells which have been shed or pulled off from a superfi-
cial epithelium, mucus membranes, renal tubules, or the like. For ex-
ample, the horny layer of epidermis is shed normally, but in disease or
inflammation the process may become exaggerated and form abnormal
sized flakes or scales. These may be placed on slides, stained, and exam-
ined.
Methods in this field have reached high priority among diagnostic
- procedures for Pathology. By screening slides made from smears (vagi-
nal, cervical, prostatic, or the like) or from body fluids (peritoneal,
pleural, gastric, urine, spinal, etc.), rapid diagnosis of malignancies is
made possible.
The cells in question degenerate rapidly and smears should be made
and fixed immediately, in equal parts of 95% alcohol and ether, for a
minimum of 15 minutes. The slides may remain in this fluid as long
as a week before staining.
Add to body fluids an equal volume of 95% alcohol and centrifuge
as soon as possible, medium speed, 2000 rpm., 3 minutes. Remove super-
natant fluid. (If smears cannot be made immediately, cover the sediment
with absolute alcohol and place in refrigerator.) Drop some of the sedi-
ment on an albumen-coated slide and, using another slide, smear the
sediment as evenly as possible over the albumenized area. Allow the
smear to begin to dry around the edge but still remain moist in the
center. Fix in ether-alcohol, | hour.

356
Exfoliative Cytology 357
lod

After fixation, the slides should not be allowed to dry at any time
before or during the staining procedure. In emergencies, they can be
shipped dry, but if possible some protection for the smear is advisable.
Ehrenrich and Kerpe (1959) add 5% of polyethylene glycol to the fixa-
tive. Following complete fixation, allow the slides to dry 5-10 minutes,
and then prepare them for shipping. Papanicolaou (1957) protects
smears for shipping by covering the fixed smears with Diaphane? solu-
tion: 2 parts of Diaphane to 3 parts of 95% alcohol. Allow to dry, 20-30
minutes, and prepare for shipping. The polyethylene glycol or the
Diaphane forms a protective coating over the smear.
Richardson (1960) uses the following solution for shipping purposes;
it is economical, noncombustible, and nonvolatile. Either biopsy pieces
or slides can be shipped in it.
monocthylenc’slycol: ...5..22aS3 Bee 04 ok ed 344.6 ml.
dicihviene P1yGOM Gg6315. 60.83 «2a erases = 2 29 18.1 ml.
LDC) Ga tg ae eae Coe er 3.6 gm.
BYAUER Eayi tet mig Reena ar cis: Sa Paros? oa PRI 577.0: mal,
PIAA ACEC ACIGN 8h oc = oP cue wee ie SE 50.0 ml.

Papanicolaou Method (1942, 1947, 1954, 1957)

Excellent preparations of the following solutions can be purchased
from the Ortho Pharmaceutical Corporation, Raritan, N.J. (Harris He-
matoxylin, EA 65, EA 50 and OF 6). Otherwise the stains can be pre-
pared as follows:
SOLUTIONS:
Harris Hematoxylin, page 125 (omit acetic acid).
Orange G 6 (OG 6):
orange G 6, 0.5% (0.5 gm./100 ml. 95% alco-
OD) fate aca Pace Oe Gite ei Sos Se ninaOR ke 100.0 ml.
phosphotunestic acid’... .. ne.) sa cea 0.015 gm.
Eosin—azure 36 (EA 36 or EA 50, the commercial designation):
light green SF yellowish, C.I. 42095, 0.5% (0.5
pins/ LOOP aml 95%, alconol)\. > chk os ve + aa ace 49.0 mil.
bismarck brown, C.I. 21000, 0.5% (0.5 gm./100
Te O57 AMCONON: <548 pe Ps hs ee 2 ee 10.0 ml.
eosin Y, C.I. 45380, 0.5% (0.5 gm./100 ml. 95%
dlcoMialy\ ey taes Ste 1 hc tes 4 ee ee 45.0 ml.
phosplotungstiemcid:. . . 1.2. .22<24 24 eee 0.2 gm.
lithium carbonate, saturated aqueous .......... 1 drop

1 Obtained from Will Corp., Rochester, N.Y.

358
c
Special Procedures I (CHAP. 22)

Note: EA65 is the same, except for the light green content; 0.259% in
95%, alcohol is used. This gives a lighter, more transparent stain,
which sometimes is desirable in smears containing much mucus. Dif-
ferentiation between acidophilic and basophilic cells is better, how-
ever, with EA 36 (EA 50) and therefore is preferable for vaginal, en-
docervical, and endometrial smears.
PROCEDURE:

I. Out of fixative, hydrate slides to water. Rinse in distilled water.

A few seconds in each solution is adequate, or dip slides up and
down until their surface has a homogeneous appearance.
Stain in Harris hematoxylin, either of following methods:
(a) Papanicolaou dilutes Harris with an equal amount of dis-
tilled water: 8 minutes.
(b) Harris hematoxylin without acetic acid: 4 minutes.
Wash in tap water, running water if flowing only slightly; do not
wash off or loosen parts of smear: 3—5 minutes.
Differentiate nuclei in 0.5°% hydrochloric acid in 70% alcohol
(0.5 ml./100 ml.) until nuclei are sharp against a pale blue cyto-
plasm.
Or Wash in slightly running tap water: 5 minutes or until nuclei are
a clear blue.
Rinse in distilled water.
Transfer through 70%, 80%, and 95% alcohol: few seconds in
each or until surface appears homogeneous.
Stain in OG 6: 1-2 minutes.
Rinse in 95% alcohol, 3 changes: few seconds each.
10. Stain in EA 36 (or EA 50): 2-3 minutes.
Me Rinse in 95% alcohol, 3 changes: few seconds in each.
2: Dehydrate in absolute alcohol, 2 changes: 1 minute each.
1, Clear and mount.
RESULTS:
nuclei—blue
acidophilic cells—red to orange
basophilic cells—green or blue green
cells or fragments of tissue penetrated by blood—orange or orange
ereen
COMMENTS:
Papanicolaou cautions against agitating slides excessively, or crowd-
ing them while staining. If parts should float off from a positive slide
onto a negative one, a false positive can occur,
Exfoliative Cytology

Johnson and Klein (1956) apply the method to paraffin sections.

Carson (1959) substitutes Technicon dehydrant? for ethyl alcohol,
thereby eliminating the need for revenue-taxed ethyl alcohol which
is dificult for some laboratories to obtain.
MacLean (1960) describes processing Papanicolaou smears on the
‘Technicon.

Fluorescent Method

Provided the proper equipment is available, the fluorescent proce-

dure requires less time for preparation and less skill on the part of the
examiner than the Papanicolaou method. The polychrome staining is
so brilliant that cells are seen in sharp contrast against a black back-
ground, rendering mass screening simple and quick. Under low power
atypical cells show increased amounts of fluorescence and suspicious
malignant cells are a brilliant flaming reddish orange (they seem to
glow) in contrast to normal cells, which may be greenish grey with
whitish yellow nuclei. The method is based on the differentiation of
RNA and DNA by acridine orange NO (AO). The RNA in the cyto-
plasm and the nucleolus fluoresces red, while the DNA of the nucleus
fluoresces green.

von Bertalanffy and Bickis (1956); von Bertalanffy,

Masin and Masin (1958)
SOLUTIONS:
Acridine orange (AO):
Stock solution:
acridine orange, GA2a6005 . <2. 0s ceweeees 0.1 gm.
distilled water 100.0 ml.
Keep in dark bottle in refrigerator.
M//15 phosphate buffer, pH 6:
M/15 sodium phosphate, dibasic, Na,H,PO,
(11.876 gm./1000 ml. water) - ml.
M/15 potassium phosphate, KH,PO, (9.078
pity IUD MS Waten ee: bess. xu pane es eae 43.0 ml.
Keep in refrigerator.
Working solution:
AO stock solution ml.
buffer solution |. cn obit en 6 a ml.

* Technicon Chemical Co., Chauncey, N.Y.

360 Special Procedures I (CHAP. 22)

Calcium chloride, M/10:

caletumichiloniders :.., } .':'))) 29 Serena?
| 82%. 11.0 gm.
distilled* watempes 34! 22! o eee te MO00:0 aml:

./ROCEDURE:
1. Smears are fixed by Papanicolaou method, ether-95% alcohol: at
least 15 minutes.
2. Hydrate to water: few seconds in each solution.
3. Rinse briefly in acetic acid, 1% (1 ml./99 ml. water), followed by
wash in distilled water: few minutes.
4. Stain in acridine orange: 3 minutes.
5. Destain in phosphate buffer: 1 minute.
6. Differentiate in calcium chloride: 30 seconds. Wash with buffer.
7. Repeat step 6. If smear is very thick, a longer differentiation may
be necessary. Nuclei should be clearly defined.
8. Wash thoroughly with phosphate buffer. Calcium chloride must
be removed.
9. Blot carefully and mount with a drop of buffer.
Frozen Sections, Method I.
1. Wash out ether-alcohol, freeze, and cut.
2. Omit step 2 and proceed as usual. Use small shallow dishes, mount-
ing sections on slide after staining.
Frozen Sections, Method II.
1. Section immediately after biopsy or wrap tissue in saline-soaked
gauze and wax paper surrounded by ice. Keep in refrigerator, 4°C
until processed. Cut, transfer to slides, and fix in alcohol or Car-
noy’s fluid.
2. Hydrate and stain.

RESULTS:
DNA of chromatin of nucleus—green from whitish to yellowish hues
connective tissue fibrils, cornified epithelia; structures—greenish
mucus—dull green
leukocytes—bright green
RNA of basophilic structures of cytoplasm, main portion of nucleoli
—red and orange fluorescence
sites of strongly acidic groups—deep carmine
malignant cells under low power—brilliant flaming red-orange
proliferating malignant cells under high power—intense fluorescence
cytoplasm—flaming red-orange containing reddish granules,
patches, or fine granules
Exfoliative Cytology 361

nuclei—yellowish hues, yellow-orange to yellow-green

nucleoli—brilliant orange-red
degenerating and necrotic malignant cells—characterized by a grad-
ual loss of cytoplasmic RNA and therefore of the flaming red-
orange
cytoplasm—faint orange or brick red, RNA often concentrated
at periphery in a rim of dark brick red
nuclei—brilliant green, green-yellow or pale yellow-grey
nucleoli—often enlarged and multiple, orange-red
COMMENTS:
Paraffin sections also can be stained by this method.
The buffer mount is a temporary mount, even when ringed. Upon
the author’s suggestion, the laboratory at Los Alamos has tried dis-
solving Elvanol 51-50? in the buffer until the solution is of thin syrup
consistency. This has resulted in a clearer and more lasting mount.
(See also page 122) Seal the cover slip, however, with one of the ring-
ing cements (page 123). Many scientific supply houses are now adver-
tising media for fluorescent mounting.
After the smear has been used for fluorescent examination, it can
be stained by the Papanicolaou technique.
1. Remove cover glass in distilled water.
2. ‘Transfer to 50% alcohol: approximately | minute, 2 changes.
3. Proceed to Papanicolaou method.
Bacteria and parasites will fluoresce and can confuse the picture;
for example: bacteria bright orange; Trichomonas, cytoplasm—
bright orange and nucleus—whitish yellow. These are easily recog-
nized in vaginal smears. See Umiker and Pickle (1960) for lung can-
cer diagnosis.
Dart and Turner (1959) Method
SOLUTIONS:
Mcllvaine’s buffer:
sodium phosphate, Na,gHPO, ............... 10.081 gm.
ciate acid monohydrate... 3. Syeate
sci vas he 13.554 gm.
distilled water pea 3 i ee eee 1000.0 ml.
Keep in refrigerator. Good for | week.
Buffered acridine orange:
Stock solution.
aeridine orange MEN 20005 42: 32251. J. ae 0.1 gm.
Gistilled Wateree ..5554554502015
he dp oS eee eee 100.0 ml.
* PVA, polyvinyl alcohol, E. I. DuPont de Nemours and Co., Wilmington, Del.
362 Special Procedures I (CHAP. 22

Add 2? ml. of ““T'ween 80” to 1000 ml. of stock.

Working solution:
acridine orangeystock solution {: yeas
sn)- 4... 1 part
Mell vaime sitet 5.2... ae RR es ome is 9 parts

PROCEDURE:
1. Hydrate slides to water, 5 dips in each of 80%, 70% and 50% al-
cohol.
2. Dip 4 times in acetic acid, 1% (1 ml./99 ml. water).
3. Rinse in distilled water: 2 minutes.
4. ‘Transfer to MclIlvaine’s buffer: 3 minutes.
5. Stain in buffered acridine orange: 3 minutes.
6. Differentiate in MclIlvaine’s buffer: 4 minutes.
7. Wipe excess material from ends of slides, gently blot with coarse
blotting paper.
8. Apply cover glass with buffer. Let stand 2 minutes before examin-
ing.
Can be restained, as above, with Papanicolaou. After this, can be de-
colorized and restained with acridine orange.

Sex Chromatin
Greenblatt and Manautou (1957)
FIXATION: peripheral blood smears in methyl alcohol: 1-2 minutes.
See Comments below for “buffy coat”’ suggestion.
Oral mucosal and vaginal smears in absolute alcohol-ether (1:1): 2-24
hours.
SOLUTION:

Pinacyanole:
pinacyamolet’ sok \s eb CaN erate. us. Maas eters 0.5 gm.
g0%, ethyl-or methylvalcohole =... -..0 aes 100.0 ml.
Wright's buffer:
monobasic potassium phosphate ............. 6.63 gm.
dibasic sodium phosphate sh «+ bse lacets 3.20 gm.
distilled water tis s..0 MNnnee oo. vi ee eee 1000.0 ml.

PROCEDURES
1. Cover with pinacyanole; blood smears: 30 seconds; oral mucosal
and vaginal smears: 45 seconds.
‘Eastman Kodak Co.
Sex Chromatin 363

2. Dilute with Wright’s buffer (equal to amount of stain); blood

smears: 30 seconds; oral mucosal and vaginal smears: 45 seconds.
3. Wash in running water: 2-3 minutes.
4. Decolorize m_50%,,Yivs 70%, 95%,%, and absolute alcohol: 30 seconds
each.
or (a) Air-dry blood smears and they are ready for use.
(b) Clear oral mucosal and vaginal smears in xylene, 2 changes,
and mount.

RESULTS:
Staining: chromatin—blue
Counting: oral mucosal and vaginal smears—6 cells out of 100
counted must be positive for female sex chromatin to be consid-
ered female. If less than 4 per 100, the specimen is male.
blood smears at least 6 rich-staining “drumsticks” must be
counted in 500 polymorph cells for a female count. No more than
2 drumsticks should appear in a male count. The tiny clubs and
sessile nodules are found in about equal numbers in both sexes,
but cannot be counted as sex chromatin bodies. (See excellent pic-
tures in Greenblatt and Manaotou)

COMMENTS:
For sex chromatin counts, the preparation of a “buffy coat” is
recommended by the present author. It gives the examiner a fighting
chance at a relatively easy cell count. Place the collected blood in a
glass tube plus an anticoagulant and allow it to stand for several hours
or use mild centrifugation. The cellular elements will settle out and
leave a straw-colored solution on top. The leukocytes are not as dense
as the erythrocytes and will settle out last to remain as the so called
“buffy coat” between the red cell layer and the plasma on top. Smear
this “buffy coat” with its concentration of leukocytes on the slide and
fix.

Klinger and Ludwig (1957)

FIXATION: 95% alcohol for smears and thin embryonic membranes: 30
minutes to 4 hours. Tissue blocks in Davidson’s fixative (see below):
3-24 hours and transfer to 95% alcohol. If material was fixed in some
other fixative, transfer immediately to 95% alcohol, but do not leave
in first fixative for longer than 24 hours for good results.
364 Special Procedures I (cHAP. 22)

SOLUTIONS:
Davidson’s fixative:
95%, ethylvalcolol”.... >! {i eae eee. 2}, 30.0 ml.
formalin 7 Ae peepee | Cla a) Sea So ie. oh 20.0 ml.
placial aceticsanid..)..<... .. 0). eeeee 10.0 ml.
distilled#watene 2s (02), LY aR 82) 30.0 ml.
Buffered thionin:
A. thionin, C.J. 52000, saturated solution in 50% alcohol. Filter.
BS SOCMUIMACEEALE ai sass Ree eae 9.714 gm.
SOCHUM Dar DitUcate, 35. a eee ee ae 147 em.
distilled water, CO, tree, 2 yen ee 500.0 ml.
GC ahydrochloric, aciG, spe. 1 Lo ass
ee ae 8.50 ml.
ist lle! WatCieowr oh -how ancy ie ee ee 991.5 ml.

Working solution (pH. 5.7):

Pion solutiony(A) isa e284 eenee 40.0 ml
bufterisokution i) 5.3 Hi. i Se ete ae 28.0 ml
hydrochlome acid (Eire Gee ey ea) eee ee 32.0 ml

PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Hydrolyze in 5N HCl (page 408), 20-25°C: 20 minutes.
3. Rinse thoroughly in distilled water, several changes; no acid
should be carried over into thionin.
4. Stain in buffered thionin: 15-60 minutes.
5. Rinse in distilled water, and 50% alcohol.
6. Rinse in 70% alcohol until clouds of stain cease to appear.
7. Dehydrate, clear, and mount.

RESULTS:
sex chromatin—deep blue-violet
nuclear chromatin—lightly colored
cytoplasm—unstained
fibrin and related structures will show metachromasia

COMMENTS:
The Feulgen technique may be used for sex chromatin body
identification, but the above method is simpler and quicker. The
principle is the same in both methods: the sex chromatin body
differentiates from nonspecific nuclear chromatin because of a higher
content of DNA, which takes longer to extract. If the basophil shell
of the nucleolus is not visible, no attempt at sexing should be made
Sex Chromatin 365

(Klinger and Ludwig). In the Feulgen method, the sex chromatin

stains more deeply than any other Feulgen-positive material present.
Skin biopsies are reliable if the sections are of good quality, but
the simplest specimens are oral scrapings, easy to obtain and equally
easy to stain. Scrape the mucosa of the check with a tongue depressor
and smear scrapings on an albumen-coated slide. Fix immediately in
Davidson’s or alcohol. Papanicolaou fixative (equal parts of ether
and 95% alcohol) also may be used.

Lennox (1956)
FIXATIVE: 10% formalin or mercuric chloride saturated formalin. Avoid
Bouin’s and dichromate fixatives.
SOLUTIONS:
Ribonuclease:
HUPONNCICASC edie nd a.Fap)a.4
spo havdgaahe ade 1.0 gm.
distilled water, glass-distilled ................ 100.0 ml.
Boil 3—5 seconds after dissolving. Keeps about | week in refrigerator.
Gallocyanin:
Chiron aiwInt2 tn,os eeeas.) is ehee greeters Ras Selon 5.0 gm:
dastilledGwater 2h £4@ oes ac 2 Ae eee ane Se 100.0 ml.
Dissolve and add:
PadlOCV AIM: wc.8 ykdses oo a ess a5 see BR eal 0.15 gm.
Shake thoroughly, heat slowly and boil 5 minutes. Cool, filter and
wash through filter with distilled water until filtrate reaches 100 ml.
Usable at once. Keeps about | month.
PROCEDURE:
1. Deparaffinize and hydrate slides to water.
2. Incubate in ribonuclease solution, 37°C: 10-15 minutes.
3. Rinse in distilled water.
4. Stain in gallocyanin, 37°C: overnight.
5. Dip in acid alcohol (few drops HCl in 70°% alcohol) to clean, not
to differentiate.
6. Wash in tap water: 5 minutes or longer.
7. Dehydrate, clear and mount.
RESULTS:
By extracting with ribonuclease, the nuclear picture is sharpened
against a colorless background, making it easier to distinguish the sex
chromatin nodule.
366 Special Procedures I (CHAP. 22)

Guard (1959)
FIXATION: Fix smears immediately in 9% alcohol; do not allow them
to dry: 10 minutes.
SOLUTIONS:
Biebrich scarlet:
biebrich scarlet (water soluble) C.I. 26905 .... 1.0 gm.
phosphotunestic acid ... . ye mepvenre la: 0.3 gm.
elacialeacemieracia. .s... 22. <a. Reesie. 50 emil:
DOP eetiyiealconols :.o. \cs or ee See rene 100.0 ml
Fast green:
fasmoneenue Gs Gil 420550. b2Gre eons
ee ee 0.5 gm.
phasphomolybdic acid...
a. sean eee 0.3 gm.
phosphotungstic acids... 27. a aes ee 0.3 gm.
Placial AceticvaCleie Cece © LEN eae yk, oe eee 5.0 ml.
50eciethylalconol 5 <0 oy. Maal eer tee 100.0 ml
Dilute hematoxylin:
Harris hematoxylin (page 125) 22...
-. eee 0.05 ml.
DOSE vethiviivalconol. 4,6'.7.2.0 see Gans. ae 100.0 ml.

PROCEDURE:
Short Method:
. Transfer into 70% alcohol: 2 minutes.
. Stain in biebrich scarlet: 2 minutes.
. Rinse in 50% alcohol.
ND.
Cfo
Hm Differentiate in fast green: 1-4 hours. Check under microscope at
hourly intervals for green cytoplasm and green vesicular nuclei in
all cells.
. Rinse in 50% alcohol; 5 minutes.
. Dehydrate, clear, and mount.

Long Method:
ite Transfer to 70% alcohol: 2 minutes.
9
a Stain in dilute hematoxylin: 15 seconds, or dip in solution 10
times.
No rinse, stain in Biebrich scarlet: 2 minutes.
. Rinse in 50% alcohol.
. Stain in fast green: 18-24 hours; no microscopic checking neces-
sary.
6. Dehydrate, clear, and mount.
Chromosomes 367

RESULTS:
sex chromatin—red
other nuclei, vesicular and cytoplasm—green
COMMENTS:

In the long method, the hematoxylin mordants the sex chromatin

for biebrich scarlet and makes a firm binding, also slows down the fast
green.
Makowski et al. (1956) identified sex chromatin in the cellular
debris of amniotic fluid, spreading the centrifuged sediment on albu-
menized slides. Vernino and Laskin (1960) identified it in bone de-
calcified in EDTA. Barr et al. (1950) describe it in nerve cells. See
also: Klinger (1958); Marberger et al. (1958); Marwah and Wein-
mann (1955); Moore and Barr (1955); and Moore et al. (1953).

Chromosomes
Squash and Smear Technics with Acetocarmine or
Aceto-orcein Staining

FIXATION: sometimes desirable. Use small pieces, Carnoy’s (page 16),

freshly prepared, few minutes to several days.
SOLUTIONS:
Acetocarmine:
Stock solution.
Boil an excess (approximately 0.5 gm./100 ml.) of carmine in
459, acetic acid, aqueous: 2—4 minutes. Cool and filter.

Working solution:
Dilute stock solution with 45°% acetic acid, aqueous, 1:2. An
iron-mordanted stain often is favored because of darker, bluish-
tinged red. Belling (1926) added a few drops of ferric hydrate in
50%, acetic acid, but only a few drops. ‘Too much iron produces
a precipitate in a short time. Moree (1944) determined quanti-
tatively the amount of ferric chloride to add and includes tables
of various normalities and volumes.
Aceto-orcein:
Add 1-2 em. orcein to 45 ml. of hot acetic acid. When cool, add
55 ml. of distilled water. LaCour (1941) used 2% orcein in 70%
acetic acid.
368 Special Procedures I (cHAP. 22

PROCEDURE:

Temporary Mounts
1. Place material, fixed or fresh, on slide with small mount of either
stain, just enough to reach edge of cover glass. Apply pressure.
Sometimes it is advantageous to allow material to remain in stain
5-10 minutes, then remove to fresh batch before squashing. Frag-
ments of animal tissue (ovaries, testes, biopsy bits) can be put in
a tube with a large excess of stain for 2—7 days. Remove to fresh
solution on slide and squash.
Methods of applying pressure:
(a) Press with ball of thumb.
(b) ‘Tap or press with blunt or flat instrument.
(c) Roll with glass rod or a vial.
All methods are subject to ease and amount of spreading, some
materials spread more easily than others. Avoid actual crushing.
Alternate squashing method: Use a large amount of stain and press out
excess with a blotter, flattening cells at same time. Staining may
be improved by passing slide 3—4 times through a small flame. Do
not allow solution to boil.
2. Seal edges with paraffin or vaseline and allow to stand 1 day.
RESULTS:
acetocarmine—red
aceto-orcein—dark purple
iron acetocarmine—deep bluish red
COMMENTS:
Rothfels and Simonovitch (1958) use an air-drying technique for
spreading chromosomes in cells grown in tissue culture, and recom-
mend the method for material which is lumpy and contains debris
making squashing difficult. The material growing on slides or cover
glasses is fixed and at completion of fixation is placed horizontally to
dry at room temperature with no airstream or humidity control. ‘This
produces uniform flattening of division stages and can be followed
by acetocarmine or aceto-orcein staining.
Puck (1958), Ruddle et al. (1958), Harnden (1959), and others first
treat tissue cultures growing on cover-slip with hypotonic solutions
(page 409), 37°C, 15 minutes to swell the cells, followed by fixation
and then air drying. The combination of swelling and air drying
helps to spread and flatten the chromosomes.
Chromosomes 369

Colchicine pretreatment is efficacious, suppressing cell division,

preventing spindle formation and allowing the chromosomes to
spread. They shorten and thicken and become straight and can be
fixed into almost a single plane when squashed. Use 0.01—0.02%
aqueous solution of colchicine for 2—3 hours, and follow by fixation,
etc. (Burrell, 1939; O’Mara, 1939; Meyer, 1943)

Permanent Mounts
If a slide is to be made into a permanent mount and there is a chance of
losing the specimens, coat the slide with albumen before smearing.

A. Smith (1947) method:

1. Remove paraffin or vaseline seal with razor blade and xylene.
2. Soak in equal parts of acetic acid and 95%, alcohol until cover
glass comes off.
3. Place in equal parts of 95% ethyl alcohol and tertiary butyl
alcohol: 2 minutes.
4. Transfer to tertiary butyl alcohol, 1-2 changes: 2 minutes each.
5. Briefly drain slide and cover glass against absorbent paper or
blotter.
6. Add thin resin mountant to slide and return cover glass to same
position as before.
Step 2 can be followed by 2 changes of 95% ethyl alcohol; mount
in Euparal or Diaphane.
B. Nolts (1948) method:
1. Place slide in covered dish so edge of cover glass dips into 95% O7

alcohol: 6-12 hours.

2. Immerse in 95% alcohol: 1-2 hours.
3. With sharp needle, gently pry cover glass free while immersed.
4. Drain off excess alcohol and add drop of Euparal or Diaphane
and replace cover glass.
C. Other permanent methods:
McClintock (1929); Celarier (1956); and Bradley (194SB). Peary
(1955) uses a frozen method of dehydration, also counterstains with
fast green.
Delamater (1951) describes a freezing method.
Conger and Fairchild (1953) freeze smears while in stain on a block
of dry ice: 30 seconds. Pry off cover glass with razor blade. Place
before thawing in 95% or absolute alcohol, 2 changes: 5 minutes.
Mount in Euparal or Diaphane.
370 Special Procedures I (cHApP. 22

D: Lamination Method, page 394.

For additional Staining Methods, see:
Austin (1959): iron alum modification with acetocarmine.
Bradley (1957): sudan black with acetocarmine.
Griffin and McQuarrie (1942) and Chen (1944): iron hematoxylin.
Melander and Wingstrand (1953): Gomori’s hematoxylin.
Rafalko (1946) and Sachs (1953): Feulgen methods.
Carter et al. (1959) recommend aceto-orcein squashing for cancer
detection; mitotic figures are red and detailed. Sex chromatin is
recognizable.
Sandberg et al. (1960) describe in detail preparation of aspirated
bone marrow for aceto-orcein staining.

Restoration of deteriorated slides (Persidsky, 1954)

If dye has precipitated as dark crystals because the preparation has
dried:
1. Remove sealing material.
2. Place 1 drop of 2N HCl at one edge of cover glass. Apply blotter
to opposite edge to help draw the acid under: 3—5 minutes.
3. Replace HCl with acetocarmine by same method.
4. Heat gently, do not boil. Crystals are redissolved and specimen
restained.
5. Reseal.
References: Austin (1959); Belling (1926); Bradley (1948A & B, 1957);
Carter et al. (1959); Celarier (1956); Chen (1944); Conger and Fair-
child (1953); Griffin and McQuarrie (1942); LaCour (1935, 1941);
McClintock (1929); Melander and Wingstrand (1953); Moree (1944);
Nolte (1948); Peary (1955); Persidsky (1954); Rafalko (1946); Rothfels
and Siminovitch (1956); Sachs (1953); Smith (1947) ; Warmke (1935);
Zirkle (1937); and Zuck (1947).
 Chapter 23

Special Procedures II

Preparation of Invertebrates for Whole Mounts

and Sections

It is impossible to cover specifically all the members of this huge

eroup of organisms, but certain generalizations can be made which
will be applicable to the common forms and perhaps be adaptable to
the less common ones. Fixatives can be applied directly to many of
them, but not to others because of their propensity to contract or ball
up, pulling in their tentacles or other appendages, and thereby making
whole mounts or sections practically worthless. In the latter cases, care-
ful anesthetizing or narcotizing must precede killing and fixation. As
soon as the narcotization is complete, and before death if possible,
fixation can be successful.

Anesthetizing and Narcotizing Agents

]. Magnesium chloride or magnesium sulfate is widely and successfully used
on sea anemones, corals, annelids, tunicates and nudibranchs to name a
few. Crystalline magnesium sulfate can be tied in a bag suspended above
and just touching the water surface, or a 33%, aqueous solution siphoned
slowly in, controlled by a screw clamp. When the organisms are anes-
thetized (no reaction to the touch of a needle), siphon off the water until
the animals are barely covered and carefully add fixative. Disturb the
animals as little as possible. When partially hardened, transfer to fresh
fixative.

ad
al2 Special Procedures II (cHap. 23)

2. Cocaine can be used for ciliates, rotifers, bryozoa, hydra, some worms and
nudibranchs. A 1% aqueous solution is added to the water in proportions
of about 1.0 ml. to 100.0 ml. of the water containing the animals. Eucain
hydrochloride can be used in the same manner. Check for contraction
and fix.
3. Menthol. Sprinkle on the water surface and leave overnight. Good for
difficult to narcotize sessile marine animals, coelenterates, some bryozoa,
hydroids, also flukes. It is more efficient when combined with chloral
hydrate in proportions of 45.0 gm. menthol and 55.0 gm. chloral hydrate.
Grind together in a mortar with a little water. Drop on surface of the
water. Fix animals when they no longer contract. Large marine forms
probably will require overnight treatment. Chloral hydrate can be used
alone, sprinkled on water surface for annelids, molluscs, tunicates, bry-
ozoans and turbellaria.
4. Chloretone. A 0.33 to 1% solution can be used, but it is slow acting.
5. Chloroform can be dropped on the water surface for many aquatic
forms, and is used in special bottles for insects and arachnids (see below).
5. Ether and alcohol can be used by dropping on the water, or alcohol
can be added gradually to the water by a tube controlled with a screw
clamp until the proportion of alcohol to water is approximately 10%.
It is particularly good for fresh water forms and earthworms. Ether
can be used like chloroform for insects.
7. Asphyxiation. Boil water to remove the air and seal it in a jar. Particu-
larly good for gastropods (snails); place them in the boiled water after
it has cooled.
8. Cold. Partially freeze organisms in salt and ice mixture or in freezing
compartment of refrigerator, or in ice water until they are relaxed.
Good for tapeworms. Transfer to lukewarm water and then fix.

Tips on special handling

PORIFERA.
Small forms can be dropped directly into osmic-mercuric chloride
(water, 250.0 ml.; osmic acid, 2.5 gm.; mercuric chloride, 9.0 gm.). Large
forms fix better in alcoholic sublimate (Gilson; Carnoy).
Calcareous sponges can be decalcified in 70-80% alcohol plus 3% of
hydrochloric or nitric acid. Silicious ones can be desilicified in 80°%
alcohol plus 5% hydrofluoric acid added gradually. Perform the latter
in a glass dish coated inside with paraffin. After a few hours, transfer to
80% alcohol. Do not breathe hydrofluoric acid fumes. If sectioning
sponges, small spicules will section easily without disilicification.
Preparation of Invertebrates for Whole Mounts and Sections 379

COELENTERATES.

Hydra. Place in a small amount of water (few drops) in a shallow

dish. When animals are extended, rapidly pipette warm mercuric
chloride-acetic (saturated aqueous mercuric chloride, 95.0 ml.; glacial
acetic acid, 5.0 ml.) at them. Work the fixative from base toward oral
region, thereby preventing tentacles from contracting or curling.
Sea anemones. Anesthetize overnight with menthol; or thirty per
cent magnesium chloride (50 to 100 ml.) can be added gradually over
a period of one hour: leave in the solution until there is no more con-
traction of tentacles. Siphon off the solution until anemones are just
barely covered. Add fixative (Susa, Bouin’s, mercuric-chloride combina-
tions, 10°% formalin sea water) slowly down the side of the container.
Also pipette some directly into the throats of the anemones. When
partially hardened, transfer into fresh undiluted fixative.
Jellyfish. In sea water, while stirring, add fixative (109, formalin)
down side of container until proportions are approximately 10 ml. or
more to 100.0 ml. of sea water. Stir for 3 or 4 minutes. After 2-3 hours,
change to fresh formalin.
Medusae. Must be anesthetized; cocaine is best. Allow them to ex-
pand in a small dish of water, add 3—4 ml. of 1°% cocaine per 100.0 ml.
of water. When they do not contract, add fixative (10°% formalin) with
stirring.
Corals with extended polyps. Narcotize with magnesium sulfate and
fix in hot saturated mercuric chloride plus 5% acetic acid.
Freezing sometimes can be tried on coelenterates.

PLATYHELMINTHES,

Planaria. Starve for a few days before killing. Place in a small amount
of water on a glass plate. When extended, add a drop of 2% aqueous
nitric acid directly on it. Follow by pipetting fixative (Gilson or
saturated mercuric chloride in saline) on it and then transfer into a
dish of fixative.
Trematodes. Anesthetize large trematodes with menthol: } hour.
Then flatten by the following method: saturate filter paper with Gil-
son’s and lay on a plate of glass. Lay worms on paper and quickly cover
with another sheet of saturated paper and a second plate of glass. Add
a weight, but not such a heavy one that the worms are crushed: 8-12
hours. Remove glass and paper and transfer worms to fresh fixative. A
single specimen can be flattened between slides, tied together and
dropped into fixative. After 2-3 hours, remove the string, but do not
SI Special Procedures II (cHaApP. 23)

disturb the slides. Leave overnight. Remove slides and transfer animal
to fresh fixative.
Small trematodes can be shaken in a small amount of 0.5-1.0% salt
solution: 3 minutes. Add saturated mercuric chloride plus 2% acetic
acid. Shake for several minutes. Change to fresh fixative: 6-12 hours.
If they should be flattened, they can be laid on a slide and covered with
another slide or, if very delicate, with a cover glass. Pipette fixative
carefully along the side of the cover and then lower carefully into
fixative.
Cestodes. Place a large tapeworm in ice water until relaxed (over-
night). Then flatten between glass plates for microscope-slide whole
mounts (as for trematodes—see above). For museum mounts, the worm
can be wound on a tall bottle or 100 ml. graduate and fixative poured
over it.
Small cestodes can be stretched from an applicator stick supported
over a tall container and fixative poured down the length until they
hang straight. ‘Then immerse them completely. Then they can be
flattened between microscope slides.
Nemertinea. Drop into saturated mercuric chloride-acetic acid

NEMATHELMINTHES.

Nematodes (hookworms). Shake 3 minutes in physiological saline.

Pour off and drop worms into hot glycerol alcohol (70-80% alcohol plus
10%, of glycerol). A sublimate fixative can be used. (also for ascaris)
Very small nematodes can be relaxed and killed in a depression slide
or watch glass by gentle heat. An incubator regulated to 50—52°C is most
reliable. If using a flame or hot plate, be careful not to boil the worms.
Transfer to fixative.
Formalin-acetic-alcohol is an excellent fixative for small nematodes:
S507 ethivl alcohol Us 5 eee, oS 5d eee eee 12-20.0 ml.
Slacial aceticvactel s-5) h. sere...
Scie dy: 1.0 ml.
OEE eed oe eS 6.0 ml.
Gustilled Watery. 6: suse uae es re 40.0 ml.

If a few drops of saturated aqueous picric acid is added, the worms take
up a little color.
Slide whole mounts are easy to prepare. Place the worms in the al-
cohol glycerol mixture in an incubator (35—37°C) or on top of an oven
to permit slow evaporation of the alcohol. When the solution is almost
Preparation of Invertebrates for Whole Mounts and Sections OFO

pure glycerine, mount in glycerine jelly (page 119). Helminth ova also
can be mounted in this way. For other methods see Whole Mounts,
page 379.
Microfilaria. Blood infected with microfilaria is smeared and dried
as for any blood smear. Then fix in any mercuric chloride fixative and
stain with Delafield’s hematoxylin. If it is preferable to have the slides
dehemoglobinized, after fixation treat them with 2% formalin plus 1°%,
of acetic acid: 5 minutes. Wash and stain. An alternate method is to
smear and dry; dehemoglobinize in 5% acetic acid and air dry; fix in
methyl alcohol and stain with Giemsa (page 225).

BRYOZOA.

Anesthetize with menthol. Fix salt water forms in chromo-acetic

(10% chromic acid, 7-10 ml.; 10% acetic acid, 10 ml.; water to make
100 ml.). Fix fresh-water forms in 10% formalin.

ANNELIDA.

Earthworms. If sections of worms (aquatic or terrestrial) are desired,

the intestines must be freed of grit and other tough particles.
Several methods have been devised for earthworms. Cocke (1938)
feeds them on cornmeal and agar (1:1) and some chopped lettuce for 3
days, changing the food every day. Becker and Roudabush (1935) recom-
mend a container with the bottom covered with agar. Wash off the agar
twice a day for 3—4 days. Moistened blotting paper can be used. When
the animals are free from grit, place them in a flat dish with just enough
water to cover them. Slowly siphon in 50% alcohol until the strength
of the solution is about 10% of alcohol. Chloroform also can be used
for narcotizing. Fix in Bouin’s or mercuric chloride saturated in 80°,
alcohol plus 5% of acetic acid. The worms may be dipped up and down
in the fixative and then supported by wire through a posterior segment,
hanging down in the fixative, or placed in short lengths of glass tubing
in fixative to keep them straight. This is necessary if perfect sagittal
sections are to be cut. Embedding is probably most successful by the
butyl alcohol method. After removal of mercuric chloride in iodized
80%, alcohol, transfer to n or tertiary butyl alcohol: 24 hours (change
once). Transfer to butyl alcohol saturated with paraffin (in 56—60°C
oven): 24 hours. Pure paraffin: 24 hours and embed.
Sea worms can be kept in a container of clean sea water, changed
every day for 2 or 3 days, then anesthetized with chloroform and fixed,
using fast-penetrating fixative (Bouin’s, or a mercuric chloride fixative).
9 I op) Special Procedures IT (cHap. 23)

ARTHROPODA.

Insects and arachnids. Ether, chloroform or potassium cyanide are

used for killing. Simplest method: Place a wad of cotton in the bottom
of a wide mouthed jar and cover the cotton with a piece of wire screen.
Dampen the cotton with ether or chloroform or lay a few lumps of
potassium cyanide on it before adding the screen wire. Keep tightly
closed with a cork or screw cap. A piece of rubber tubing soaked in
chloroform until it swells and placed under the screen wire will hold
chloroform for several days. If the appendages should be spread when
fixed, as soon as the insect is dead place it on a glass slide with another
slide on top of it. Run fixative in between slides.
For whole organisms, rapidly penetrating fixatives should be used:
picro-sulfuric, sublimate fixatives, mixtures containing nitric acid (Car-
noy), alcoholic and ordinary Bouin’s, or Sinha’s (1953) fixative.
For whole mounts, the clearing of the exoskeleton is sometimes difh-
cult. Body contents have to be made transparent, or sometimes have to
be removed. Lactophenol (page 121) mounting will serve the purpose
in the first case, but heavily pigmented arthropods probably will have
to be bleached in hydrogen peroxide for 12 hours or longer. Fleas, ticks
and the like make better demonstrations if not engorged; in any case
they should be treated with 10% potassium hydroxide, 8-12 hours, to
swell and dissolve the soft tissues, thus clearing out the body contents.
Wash well to remove the potassium hydroxide. Acid corrosives are
preferred by some because they do not soften the integument as much
as alkaline corrosives.
Acid corrosive:
glacial acetic acids) o> ieee te eee hae 1.0 ml.
chloral shyduate sy 05 0) Jest deen, be le Se 1.0 gm.
Walter: \-niRa wees ogc: Aes CNR Ls 22a ae 1.0 ml.
Do not use a fixative containing alcohol or formalin if a corrosive is
necessary. The organism will not clear.
For methods of mounting whole mounts, see page 378.
Because of chitin, sectioning of insects can be difficult. Avoid higher
ethyl alcohols. Dioxane methods (page 39), butyl alcohol (above), or
double embedding (page 83) must be used and will provide excellent
results. Soaking the tissue blocks overnight or for several days in water
(page 55) will simplify sectioning.

MOLLUSCA.

Snails. Place in boiled water until limp. Fix.

Mussels. Sections of undecalcified shell can be made by grinding.
Preparation of Chick Embryos ord,

Decalcification can be undertaken with 3-4% nitric acid. If the soft

parts are to be fixed, wedge the valves apart with a small length of glass
rod and place entire animal in fixative. Dissect out after it is fixed.
Gills can be removed and fixed flat in a mercuric chloride fixative.

ECHINDODERMATA.

Inject fixative into the tip of the rays of starfish. This will extend the
feet. Then drop the animal in fixative; mercuric chloride-acetic is good.

Staining Invertebrates
Sections can be stained in any manner, depending on the fixatives and
the desired results.
For beautiful transparent whole mounts, the carmine stains (page
382) are the usual choice for most of the invertebrates: obelia, hydra
and hydroids, Daphnia, bryozoa, medusae, flukes, tapeworms, small
annelids, tunicate and ammocoetes larvae to name a few. But Korn-
hauser’s hematein (page 384) is an excellent substitute, particularly for
flukes and tapeworms.
If the cuticle and muscle layers of flukes and tapeworms tend to
remain opaque and obscure the details of internal anatomy, either of
the two methods can be tried to correct the condition. Dehydrate and
clear in an oil, such as cedar oil. Place in a flat dish under a dissecting
microscope and carefully scrape away some of the tissue from both
surfaces. With care, this can free the animal of some of the dense tissue
material and not harm the internal structures. An alternate method is
to finish staining and then wash in water. Transfer to a potassium
permanganate solution made from a few drops of 0.59% solution added
to water until only a pink color develops. When the worm begins to
show a greenish-brown sheen, remove it immediately to distilled water:
5 minutes. Transfer to 2-3% aqueous oxalic acid until bleached and
the sheen is lost. Wash thoroughly in running water at least one hour.
Dehydrate, clear, and mount.

References: Becker and Roudabush (1935); Galigher (1934); Gatenby

(1950); Gray (1952); Guyer (1953); Hegner, Cort, and Root (1927), and
Pantin (1946).

Preparation of Chick Embryos

The following procedure is a standard and simple one for removing,
fixing, and staining chick embryos of any size, from primitive streak to
age 96 hours:
378 Special Procedures II (cHAP. 23)

With the handle of the scissors, break the shell at the air space end,
turn scissors and cut the shell around the long axis, being careful to
keep the tip of the scissors pressed against the inside of the shell while
cutting. This will prevent penetration of the yolk or the embryo. With
ego submerged in physiological saline at 37°C ° in a finger bowl, re-
move top half of the shell. The chick will be found floating on the
upper surface of the yolk. It can be a waste of time to remove the lower
half of the shell, but many workers prefer to do so. This is a matter of
individual preference and ease of preparation. With small scissors, cut
quickly around the outside of the area vasculosa. Except for large em-
bryos, do not cut through the vascular area. Keep one edge gripped
by forceps, and slip a syracuse watch glass under the chick. Withdraw
the watch glass and embryo with a little of the saline. Bring as little
yolk as possible with the chick.
The vitelline membrane most likely will come with the embryo;
occasionally it will float free. If not, wave the embryo gently back and
forth in the saline until the membrane begins to float loose. Release
grip on embryo and remove the membrane. Make certain that the
embryo is wrong side up, that is, the side which was against the yolk
is now uppermost. Straighten out the chick, with no foids. Sometimes
washing with saline in a pipette across the top helps to clean and flatten
it. Pull off remaining saline and yolk, carefully, not just from one edge
but slowly and with gentle pressure on the pipette bulb around the
area vasculosa. Do not get so close that the embryo is drawn up into the
pipette.
Have a circle of filter paper cut with the inside hole approximately
the size of the vascular area if it is to be retained, or a little larger than
the embryo if it only is to be used. Being careful not to disturb the
embryo, drop the circle of paper around it. Press gently around the
paper to make it adhere to the blastoderm. With a pipette apply fixative
(Gilson or Bouin’s), carefully dropping it first directly on the embryo;
ease it on, do not squirt it on, and then work outward toward and over
the paper circle, finally adding enough fixative to completely immerse
embryo and paper. Leave overnight. The embryo should remain ad-
hering to the paper and can be transported in this fashion from solution
to solution. Fixation is followed by proper washing, depending upon
choice of fixative. Embryo is then stained in a carmine, hematoxylin
or hematein stain (see whole mounts) and dehydrated. Remove the
chick from the circle, clear and mount. The cover glass may have to be

° The author has found that with rapidity developed from experience, warm water serves
just as well as saline, with no ultimate harm to the chick if it is to be fixed immediately.
Whole Mounts 379

supported (page 381) to protect the delicate embryo from destructive

pressure. For sections, after washing the embryos can be dehydrated,
cleared and embedded in paraffin.
Incubation time and determination of age can be puzzling. A per-
fectly aged chick sometimes is difficult to obtain, because the age is
determined not only by the number of hours in, and the temperature
of, the incubator, but the length of time the egg was in the hen and
the temperature at which the egg was stored. The temperature of the
incubator should be 37.5°C and it usually requires about 4 hours to
warm up the egg once it is placed in the incubator. In other words,
a 36 hour chick would require about 40 hours in the incubator for
proper development. The accompanying chart (Figure 27) indicates how
counting the number of pairs of somites in the chick will determine its
age; thus 10 pairs indicates an age of 30 hours. This is the best criterion
of development, rather than the number of hours in the incubator.
Any bird or reptilian embryo can be fixed as outlined for the chick.
Amphibian larvae can be dropped directly into fixative, after first dis-
secting off any surrounding yolk or jelly.
Mammalian embryos should be dissected from the uteri and fixed in
Bouin’s. If the uterine site also is to be preserved, cut through the uterus
on either side of the embryo and fix. A little fixative can be injected into
the uterine cavity to quicken the fixing action.
Ascaris uteri are fixed in alcoholic Bouin’s; sometimes Carnoy will
give desired results.

Whole Mounts
Whole mounts are slide mounts of entire specimens small enough to
be studied in toto and mounted in resins in toluene or water, in gums
or glycerine as aqueous mounts, or as dry mounts. Some examples from
the innumerable methods will follow, with the hope that a technician
can adapt them to almost any whole-mount venture.
FIXATION: 95°% alcohol, followed by bleaching if necessary. From 70%
alcohol into bleaching fluid (3-4 drops of Clorox in 10 ml. 70% al-
cohol, or water plus H,Oz2 (1:1) with a trace of ammonia): 24 hours.
PROCEDURE:
1. Wash with 70% alcohol, 3 changes.
. Dehydrate in 95% and absolute alcohol.
oOo. Clearing
NO is often difficult. Try mixture of zylene and beechwood
 aa oe
ie SN a
Bs SS Cc
ee [lage te
80

i
ee
eS
ee
60

"i
HOURS

ANS "0un

SOMITES
Figure 27. Development of chick embryo. [From figures in Patten (1952).]
380
Whole Mounts 381

creosote (or aniline or phenol) (prevents them from becoming

brittle), 2 changes. If opaque patches persist, return them to abso-
lute alcohol.
4. Impregnating with mountant can be difficult. Specimen may
collapse and turn black or opaque when taken directly from
alcohol or clearer to mountant. Impregnate gradually by adding
each day a drop or two of mountant to the clearer containing the
specimen. Carefully stir the mountant into the solution.
Galigher (1934) uses a cone of filter paper containing the moun-
tant, and allows it to enter the solution gradually and continu-
ously.
When the clearer has obviously thickened, allow concentration to
continue by evaporation.
Or Mounting sometimes requires considerable experience (and pa-
tience) in judging the correct method and amount of mountant.
Some specimens, large or delicate, should have supports under the
cover glass, in the former case to prevent tilting of the cover glass,
in the latter to lessen chances of smashed specimens. Various
materials may be used to support the cover glass; bits of cover
olass or slide, thin glass rods, circles, squares, or strips stamped or
cut out of heavy aluminum foil, bits of glass wool.
Place a drop of mountant on slide and place specimen in it.
Lower cover glass carefully; keep it flat (not tilted); have specimen
in center of mount. If several specimens are to be mounted under
one cover glass, chance of their slipping out of place is lessened by
allowing the mountant to air dry for a few hours. Place a bit more
mountant over them and add cover glass. Warming the cover glass
aids in bubble prevention.
COMMENTS:
Demke (1952) embeds helminths in celloidin to support them. De-
hydrate through absolute alcohol, alcohol-ether and into thin cel-
loidin. Pour celloidin and specimens into flat dish (petri dish); allow
solvent to evaporate slowly. Cut out squares of celloidin containing
specimens, dehydrate, clear and mount. No other support of cover
glass required.
Rubin (1951) uses PVA mounting medium, page 120. See other
mounting media in that section.

Glycerol Jelly Mounts

Many materials, including frozen sections, may be mounted directly

from water into glycerol jelly. If there is danger of an object collapsing,
382 Special Procedures II (cHap. 23)

transfer it from 70% alcohol or water into a mixture of 10-15% glycerol

in alcohol or water. Leave the dish uncovered until most of the alcohol
or water has evaporated. Mount in glycerol jelly. Sections are mounted
without cover glass support. Thicker specimens probably will require
a supported cover glass. With a turntable spin a ring of gold size on the
slide. Allow it to dry. If it is not high enough, add more layers to the
height desired.
Melt glycerol jelly in water bath or in oven. Add a drop or two of
jelly onto specimen inside ring. Use just enough to fill ring. Warm cover
glass and ease horizontally into place. Carefully wipe off any glycerol
jelly that works out from cover glass. After a few tries, it becomes rela-
tively easy to estimate the correct amount of jelly to make a clean
mount. Seal cover glass, supported or unsupported, with gold size or
other cover glass sealer (page 123).

Hydra, Embryos, Flukes (stained whole mounts)

FIXATION: Many fixatives are suitable: Carnoy’s or Gilson for worms,
Bouin's or Zenker’s for embryos, formol acetic or saturated HgCl»
acetic (95/5). But before fixing consult the previous section on in-
vertebrates or an authoritative source concerning the problem at
hand.
Follow by proper washing and extraction of any undesirable pig-
ments or crystals.

STAINING: Choice of stain also depends upon type of material. A few of

wide usage will be included here.

Grenacher’s Borax Carmine (GALIGHER, 1934)

SOLUTION:
Carmine, (Gel4/947.0) .a a ewe.)
a ee 3.0 gm
BOLdX . oe oe ee ee ce eee 4.0 gm.
Gustilled. Water t..:c 13 es.
iyo ear ay 100.0 ml.
Boil approximately 30 minutes or until carmine is dissolved. Mix-
ture may be allowed to stand until this occurs. Add:
WOopecthyl alcoholy:(figtel. .eeemeereaccr.
<A eee puke: 100.0 ml.
Allow to stand 1—2 days. Filter.

PROCEDURE: (Hydra, flukes, tapeworms, small crustacea, etc.)

1. ‘Transfer from 50% alcohol to borax carmine: 3—4 hours or over-
night.
Whole Mounts 383

nd Add concentrated oe wntil Gar

HCl, slowly, drop at a time, stirring,
mine has precipitated and is brick red. Let stand 6-8 hours or
overnight.
3. Add equal volume of 3% HCl in 70% alcohol and thoroughly
mix. Let stand 2—3 minutes until specimens have settled. Draw off
precipitated carmine with pipette. Add more acid alcohol, mix,
allow to settle and draw off fluid. Repeat until most of carmine is
removed.
4. Add fresh acid alcohol and allow to destain, checking at intervals
under microscope: may require 2 hours or more. If exceedingly
slow, increase percentage of acid in alcohol.
Or . When destained, replace with 80% alcohol, several changes to re-
move acid: over a period of | hour.
6. Dehydrate, 90% alcohol: 30 minutes or more, depending on size
of specimen; absolute alcohol: 30 minutes or more.
7. Clear. For delicate objects this probably should be done gradually
through the following concentration of absolute alcohol and cre-
osote (or aniline or carbol) xylene; and into pure creosote-xylene.

absolute alcohol creosote (aniline or carbol) xylene

80. parts 20 parts
60 40
50 50
40) 60
20) 80
0 100

8. Mount. For exceedingly delicate objects and round worms, heed

the warning above concerning slow impregnation with the mount-
ing medium. Judgment concerning its use rests with experience,
but slowness is invariably necessary for round worms.

Mayer’s Carmalum (cownpry, 1952)

SOLUTIONS:
Stock solution:
carminic acid. (C.P.-carmine) C.1. 75470 . 222). 1.0 gm.
ammonium alum 1 ily ee 10.0 gm.
distilled water ........ Se rere 200.0 ml.

When dissolved, filter. Add:

FORMA Ieee a2 ho wa hale es: 1.0 ml.
384 Special Procedures II (cuHap. 23)

Working solution:
cCarmalumbstackes \. (0)2 ..<)h ke ee ee. 5.0 ml.
placial acetiqqaetds. "=... 2°): ee Ee: eee 0.4 ml.
distilledswategeeee.....
6c eee 100.0 ml.

PROCEDURE: stain for 48 hours, no destaining. Dehydrate as above.

Carmalum is good and requires no destaining.

Hematein (KORNHAUSER, 1930)

SOLUTIONS:
Stock solution:
Grind 0.5 gm. hematein with 10 ml. 95% alcohol and add to 5%
aqueous aluminum sulfate.
Working solution:
Dilute above 1:10 with distilled water.
PROCEDURE:
1. Stain overnight.
Transfer to 70% alcohol.
Destain in acid alcohol, 5°% HCl in 70% alcohol.
He
19
o9Blue in alkaline alcohol, ammonia or sodium bicarbonate in 70%
alcohol.
5. Dehydrate and mount as above.
Hematein is good for flatworms.
Alum cochineal used to be popular but has become largely replaced by
carmine stains.
Alum hematoxylin may be used for small organisms if not too dense.
Celestin blue method, Demke (1952) may be used.

Protozoa

FIXATION: hot Schaudinn’s, 50°C: 5-15 minutes. Other fixatives are

formol Bouin’s, acetic, or mercuric chloride acetic.
Organisms such as Paramecium should be concentrated by centri-
fuging. Quickly pour off some of the solution and add fixative.
Amoeba will settle on the bottom of a clean culture dish. Decant
off most of the culture media, leaving the bottom barely covered.
Quickly pour hot Schaudinn’s or Bouin’s (50—60°C) over the organ-
isms. After a few minutes add an equal amount of 85% alcohol. Care-
fully loosen any amoebae clinging to the bottom and collect the solu-
tion in a centrifuge tube. For both Paramecium and Amoeba, allow
Whole Mounts 385

to settle or centrifuge down at low speed. Pour off fixative and wash
several times with 70%, alcohol, centrifuging after each wash. If a
mercuric chloride fixative was used, one of the washes must contain
some iodine.
The easiest method for handling is the cover glass method of Chen
(1944 A).

PROCEDURE:
1. Follow the 70% washing with 80-85%, alcohol: 10 minutes.
2. Smear cover glass with albumen fixative. Place them albumen-side
up on slides with a bit of edge projecting beyond the slide. ‘The
projecting edge can be easily grasped with forceps when it be-
comes necessary to move the cover glass. With a pipette pick up a
few drops of alcohol containing organisms. Drop them in the cen-
ter of the cover glass, where they will spread over its surface. ‘The
alcohol will begin to evaporate and bring the specimens in con-
tact with the albumen. Avoid complete drying; the edges may dry,
but the center will remain slightly moist. Carefully add a couple
of drops of 95% alcohol onto the specimens, and then transfer the
cover glass to a petri dish of 95% alcohol. Slides lying in the bot-
tom of the dish will help to handle the cover glasses, as above.
3. Carefully remove the cover glass from the 95% alcohol, dip it
gently in absolute alcohol and then flood it with 1% celloidin or
nitrocellulose. Drain off excess celloidin against filter paper. Wave
it back and forth a few seconds until it begins to turn dull and
place it in 70-80% alcohol. It can remain in this solution until
ready for staining.
4. Any stain can be applied—hematoxylin, carmine, Feulgen—de-
pending upon study to be undertaken.
5. The cover glasses are finally dehydrated, cleared, and mounted,
albumen-specimen-side down on a drop of mounting resin.

COMMENTS:
The above method is excellent and rarely fails.
Smyth (1944) uses a quicker method and directly on slides. After
fixation he carries the organisms through graded alcohols into abso-
lute alcohol. This is dropped on a film of albumen on a slide. Place
the slide in absolute alcohol and continue from there to the stain.
Agrell (1958) places suspensions of minute embryos on albumen-
coated slide. Allowed to almost, but not quite, dry, they become flat-
tened and in close contact with albumen. Dip into absolute alcohol,
386 Special Procedures IIT (cHaAP. 23)

and then into fixative; place them horizontally in 95% alcohol vapor:
1 minute. Then fix. This coagulates the embryos and attaches them.

Animal Parasites

Animal organisms parasitic in or on man include protozoans, platy-

helminths, nemathelminths, and arthropods. Clinical parasitology is
extensive and therefore only a few methods which can expedite a tissue
technician’s work are incorporated here. Since the Romanowsky-type
stains are used on blood parasites (malaria, trypanosomes, filaria, etc.)
the preparation of smears is included in the section on hematologic ele-
ments (p. 218). Tissue sections can be stained in a similar manner.
Parasitic roundworms (pinworms, Trichuris, Ascaris, hookworms), flat-
worms (lung, intestinal, bile duct and blood flukes, tapeworms) and
arthropods (ticks, lice, mites) are discussed in the sections on inverte-
brates and whole mounts in this chapter. Methods given there can be
adapted in most cases of parasitic helminths and arthropods. Sections
of tissue parasitized by protozoans or helminths are effectively stained
by hematoxylin methods, also by periodic acid-Schiff; protozoans and
worms are strongly PAS positive due to stored glycogen in both forms,
and PAS-positive cuticle in the helminths. ‘The scolices hooks in hydatid
disease (Echinococcus), however, do not show with PAS and are better
demonstrated against a hematoxylin background. Ova and larvae can
be handled according to directions given on page 374. Intestinal proto-
zoa (amoebae, flagellates, ciliates, and coccidia) require the following
special methods, both for smears and tissue sections.
Only permanent slide mounts are described. Consult clinical labora-
tory manuals for temporary and rapid examination methods for imme-
diate diagnosis. Gradwohl (1956) is comprehensive.

Intestinal Protozoa: Smear Technics

Preparing Concentrate Smears (ARENSBURGER AND MARKELL, 1960)

PROCEDURE:
1. Add 1 ml. of feces to 10-15 times its volume of tap water. Mix well
and strain through 2 layers of wet gauze in a funnel. Collect in a
small centrifuge tube. Add 1—2 ml. of ether. Using a cork or thumb
for a stopper, cautiously shake the tube. Fill with water to 1 cm.
from top.
Animal Parasites 387

. Centrifuge 45 seconds, 2500 rpm. Break up any plug at top and

decant supernatant fluid.
. Add 2-3 ml. of normal saline and shake to resuspend the sediment.
Fill tube with normal saline to within | cm. of top and centrifuge.
. Decant supernatant fluid. Take a small quantity of the original
fecal specimen on the end of an applicator stick and mix well with
sediment at bottom of tube.
. With an applicator stick, transfer as much as possible of the ma-
terial to a clean slide. Smear as for a conventionally made slide and
fix immediately in Schaudinn’s.
6. Stain as usual, hematoxylin, Lawless’ method, etc.

Goldman (1949) Smears

FIXATION: Schaudinn’s (page 21), 40°C: 5-15 minutes.
SOLUTIONS:
Stock solution A:
hematoxylin, 10% in 95% alcohol (10 gm./
NOOimll 95%, alcohol)”: . ibe
d sees Ba 1.0 ml.
Goe7,, ctayl* alcohol ¢ we s+.» Geulsy me near me ome 99.0 ml.

Stock solution B:
ferric/ammonium Sulfate <..icsh.
53 oc on ls eae 4.0 gm.
glacial acetic acid e re ee er 1.0 ml.
sulfuric acid, concentrated .................. O2 amt
GIStEd! WWACT ten fo ose» ou oe png. ee Ee eens 100.0 ml.
Working solution:
Equal parts of 4 and B. It turns purple, but within a few hours
changes to dark brown. Then it should be filtered and is ready for
use. If it turns greenish black, it is unsuitable for staining; the he-
matoxylin was too ripe and became overoxidized.
Hanson’s Iron Trioxyhematein (page 127) may be used in same way.
PROCEDURE:
1. Rinse slides in 70% alcohol and treat with iodized alcohol.
2. Wash in several changes of 50°% alcohol until brown color re-
moved.
3. Stain in hematoxylin progressively: 3-5 minutes.
4. Wash in running water: 15-30 minutes.
5. Dehydrate, clear, and mount.

RESULTS:
protozoa black nuclear stain
388 Special Procedures II (cHapP. 23)

Kessel (1925) and Chen (1944A) Smears (modified)

FIXATION: Schaudinn’s (page 21), 40°C: 10-15 minutes or more.

SOLUTIONS:
Iron alum:
ferricammonmin sulfate = ../-¢ gusts os ie: 4.0 gm.
distilled Waternee: bo... sf. 2 ok DR ec ace be 100.0 ml.
Hematoxylin.
Stock solution:
hematoncilins ©. Sle i225) Rav PSae eee ee 1.0 gm.
absoluterethyl’ alcohol: Ae. eee eee oe 10.0 ml.
Allow to ripen several months or hasten process (page 128).
Working solution:
hematoxylin) stocksolution 9) (a. fope 0.5 ml.
Gaistilled Water. geyser Pacy rene Nee oe: Se 99.5 ml.
Add 3 drops of saturated aqueous lithium carbonate. If the hematoxy-
lin working solution looks rusty or muddy brown, it is unsatisfactory
and will not stain efficiently.
PROCEDURE:
lL. Transfer slides into 70°% alcohol (from fixative): 2-3 minutes.
. Treat with Lugol’s solution: 2—3 minutes.
. Wash in running water: 3 minutes.
. Decolorize with 5% sodium thiosulfate: 2 minutes.
. Wash in running water: 3-5 minutes.
. Mordant in iron alum, 40°C: 15 minutes.
. Wash in running water: 5 minutes.
. Stain in hematoxylin, 40°C: 15 minutes.
. Wash in running water: 5 minutes.
— m . Destain in iron alum
~I
CO
oO
So
OO
Ff
Or
HO 2% (dilute stock 4% with distilled water,
1:1) until nuclei and chromatoidal bodies are sharp against color-
less cytoplasm. Check under high dry objective.
11. Wash thoroughly in running water: 15-30 minutes.
12. Dehydrate, clear, and mount.
RESULTS:
nuclei, chromatoidal bodies—sharp blue black

COMMENTS:
Saturated aqueous picric acid may be used for destaining. Follow it
with a rinse in dilute ammonia (2-3 drops/100 ml. water) and thor-
ough washing in water.
Animal Parasites 389

Diamond (1945) added 1 drop of Tergttol #7 (Carbide and Carbon

Chemical Corporation) to the diluted hematoxylin solution just be-
fore use (1 drop per 30—40 ml. solution). This he substituted for heat;
it reduces surface tension and increases cell penetration. Reduced
staining time to 5 minutes.
This method may be used for amoebae in tissue as well as in
smears.

Lawless’ Rapid Method (1953)

The staining method may be used for either Schaudinn’s or Schau-
dinn-PVA fixed material.
SOLUTIONS:
Schaudinn’s fixing fluid, see page 21.
Schaudinn-PVA fixative:
With stirrer going at high speed, sift 25 gm. PVA powder (Elvanol,
erade 71-24%) into the vortex of a cool (20°C) solution of 312 ml.
saturated aqueous mercuric chloride, 7.5 ml. glycerine, 25 ml. glacial
acetic acid and 156 ml. 95%, ethyl alcohol. After 10 minutes and
while still stirring, heat in water bath to 75—-85°C: 5-8 minutes, or
until solution is complete. If solution gels during storage, warm in
56°C water bath. Some protozoa fix better in warm PVA fixative.
Stain:
ehromoirope ZR: GA, 16570) 2.2.22 eeeeaaa muon 0.6 gm.
light green SE, yellowish, G.I. 42095 .2..21.56. 0.15 gm.
faspercen PCH NGI 22008): «5 itd wane epee 0.15 gm.
phosphotinestic addes0) oi. .2.08 5 ieee 0.7 gm.
Placa acetiC acid fate. 2se ake 22 Eisen 1.0 ml.
distilled Water Ae sos cant. 46 ons thee eee re 5 100.0 ml.
Add acetic acid to dyes and phosphotungstic acid, let stand 15-30
minutes. Add water.

PROCEDURE:
A small portion of stool is fixed in PVA fixative (1 part stool to 3
parts fixative): 15 minutes to | hour or more. In this form it can be
shipped in a vial. When ready to make slides, decant off excess PVA
solution. Replace cap of vial and shake the emulsion. Remove cap
and cover vial opening with gauze. Place 3 or 4 drops of strained ma-
terial on cleansing tissue or blotter. Allow absorption of PVA for
about 5 minutes. Scrape up moist residue and smear on slide or cover
6 DuPont de Nemours and Co.
390 Special Procedures II (cuHapP. 23)

glass. Drop immediately into iodine-alcohol (page 12). If smears

wash off, too much PVA was carried over; leave material for longer
time on cleansing tissue.
Smears may be fixed directly on slides. Take 1 drop of fecal material
to 3 drops of PVA fixative. Smear over a large area of the slide and
dry in oven overnight. Immerse in iodine-alcohol.
Leave in iodine-alcohol: 1 minute.
Decolorize in 70% alcohol, 2 changes: 1-2 minutes each.
=
NO
oOo Stain: 5-10 minutes.
4. Differentiate in acidified 90% alcohol (1 drop glacial acetic acid/
10 ml. alcohol): 10—20 seconds or until stain no longer runs from
smear.
4. Dehydrate in absolute alcohol; rinse twice, dipping up and down.
6. Dehydrate in second change of absolute alcohol: 1 minute.
7. Clear and mount.
RESULTS:
background—predominantly green
cysts—bluish-green cytoplasm, purplish-red nuclei; green is more in-
tense than background stain
engulfed red cells—vary, either green, red, or black
chromatoidal bodies—intense green, but may be reddish
COMMENTS:
If cysts do not stain or stain predominantly red, fixation is incom-
plete. Warming the fixative may help, although cold fixation yields
more critical staining. A newly prepared stain is predominantly red
in color and reaction; older stains show more violets and greens.
Warning: Press the cover glass in place very gently; the smear is
somewhat brittle and is easily loosened. ‘Too much pressure on the
cover glass may start parts of the smear to floating around on the
slide.
This stain does fade after a few years.
References: California State Department of Public Health, Bacteriol-
ogy Department, Division of Laboratories, Berkeley, California; Go-
mori (1950) and Wheatley (1951)

Amoebae in Tissue

Meriweather (1934) Method

FIXATION: 10% formalin or a mercuric chloride fixative.
Animal Parasites 39]

SOLUTION:
Carmine stock solution:
Carmine MG ales LO) sce. 22 aces wil tte Gees S-aaees 2.0 gm.
POLASsIMMMACATMOMALC, oi. . 0... 45-5 Bietee ns ae 1.0 gm.
potassium) chloride =: 2. 22.2.5 ek, 5.0 gm.
distillediswater, fau.,f2424s. 5 = 2a. “60:07am,
Boil gently for about 5 minutes, color should deepen.
Working solution:
CarmmestOck solution. 4...) fab4 4 he Baa ee. 20.0 ml.
aMMomuan, VATOKICS: «12. enaa 1 eee eas ae 30.0 ml.
methyl alcoNOley ooo. .0 eee see ee Seas 30.0 ml.

PROCEDURE:
1. Hydrate slides to water, removing HgCle.
2. Stain in any preferred hematoxylin.
3. Wash in running water: 5 minutes.
4, Stain in carmine solution: 5 minutes.
5. Differentiate: 5 minutes, in:
absolute ctlayl “alecomon 2 od - fade an ra eae 80.0 ml.
me vay sa COMOl: § 2a bs <4 saed aee pe cee 40.0 ml.
distilled water ...... ab a See eae 2 100.0° nal.
Change solution if it becomes highly colored.
6. Dehydrate, clear, and mount.

RESULTS:
vegetative amoebae—highly stained with carmine, brilliant red; no
cell in intestinal wall takes up carmine

COMMENTS:
Tomlinson and Grocott (1944) describe a phloxine-toluidine blue
stain for malaria, Leishmania, microfilaria, and intestinal protozoa in
tissue.
 Chapter 24

Special Procedures III

Special Mounts
Sections Mounted on Film. Pickett and Sommer (1960)
Pickett and Sommer use film in place of glass slides for supporting
tissue sections. The flexibility of film and the low cost of material and
time can be advantageous. Many sections mounted on a long strip of
film are stained in one process by using a developing reel and then are
plastic-sprayed as a unit instead of having to be individually cover-
slipped. A length of film can be stored in a small space, and single sec-
tions or groups of sections can be clipped or stapled to records or re-
ports, and filed or shipped in envelopes. Film with section also can be
mounted in film holders. If desired, one section of a series can be
mounted on film and a successive section mounted on glass. Sections
already mounted on film can be cut out of it and mounted on glass
slides in the conventional manner. Section details are clear under low
and medium power. For oil-immersion or high-power examination, a
cover glass should be mounted on the section with a nondrying immer-
sion oil (cedar oil dissolves plastic). Only low-power projection can be
used with this film. Pickett and Sommer include the design of a holder
to keep the film flat while being scanned under the microscope.

REQUIRED MATERIALS:
35 mm. film, Cronar, P-40B leader film, E. I. DuPont
Krylon Plastic Spray, Crystal Clear Spray Coating #1302, Krylon
Inc., Norristown, Pennsylvania
Nikor 35 mm. developing reel, stainless steel

397
Special Mounts 393

PROCEDURE:

1. Cut strips of film of desired length; clean by dipping in acid alco-

hol; dry with soft cloth (not gauze).
2. Coat film with albumen fixative.
3. (a) Place tissue sections on water bath, maneuver film under rib-
bon and pick up sections. Dry in 56° oven.
(b) Film can be cut in 3 X 1 in. strips; lay on glass slide; smear
with albumen. Place section on film, pipette water under it and
dry on slide warmer as usual for slides.
4. After drying, load lengths of film in reel and stain as usual. Short
pieces can be handled individually. To guard against loose sec-
tions, protect with coating of 0.59%, celloidin or nitrocellulose. Pro-
ceed as follows:
(a) Xylene: 1-2 minutes.
(b) Absolute alcohol: 1-2 minutes.
(c) 0.59% celloidin: 5 minutes.
(d) Air dry: 3-4 minutes.
(e) 80% alcohol: 5 minutes.
(f) Wash and stain.
) Dehydrate through 95° alcohol.
h) Mixture of 4 chloroform and 3 absolute alcohol, 2 changes.
(zt) Xylene.
5. Place face up on a blotter; do not allow to dry. The film must lie
flat; buckling results in uneven plastic coat. Use weights along
edges. Apply spray quickly from end to end, 2-3 times until sur-
face appears smooth. Hold can about 6 inches above film. Allow
to harden: 5-10 minutes. Repeat 2—3 times. Allow to dry.
6. Label with India ink if desired.

COMMENTS:

Mylar polyester film (DuPont), 0.25 mm. thick also has been recom-
mended for use (Johnston, 1960).
Burstone and Flemming (1959) recommend film for smears, touch
preparations and sprayed-on suspensions. Adherence is excellent and
the preparations can be floated on incubation media, staining solu-
tions, etc. The film can be mounted in glycerine jelly, or water-solu-
ble plastics.
Berton and Phillips (1961) use film for tissue cultures. The live
cells attach and adhere firmly to film.
394 Special Procedures III (cHAp. 24)

Cellulose Tape Mounting. Palmgren (1954)

This method has been devised for problem sections, such as tissues
containing a large amount of yolk, chitinous material, hard tissue which
breaks away from soft parts and falls out of sections. Cellulose tape
(Scotch Tape) is pressed firmly on the section surface of the paraffin block
or the tissue frozen on the freezing microtome. The sticky surface of the
tape attaches to all parts of the section and prevents its wrinkling or
shattering during sectioning; l-micron to 100-micron cuts will perform
equally well. Using this method Palmgren has cut whole adult mice for
radiography on the freezing microtome without loss of parts. ‘The sticky
material is soluble in xylene so the paraffin cannot be dissolved while on
the tape, making it preferable to transfer the sections to a glass slide or
plate.
PROCEDURE:
1. Mount section and tape with section-side upwards on glass. Hold
it in place with small strips of tape, but do not cover the section.
2. Spread a thin layer of 0.59% nitrocellulose over the section, but
not over the tape. Let dry.
3. Immerse in xylene in a petri dish, and leave until the nitrocellu-
lose film and section can be loosened easily from the tape. Leave
in xylene a little longer to remove all sticky material.
4. Smear clean slide liberally with albumen fixative. Float section on
it and press in place. Blot dry.
5. Transfer immediately to absolute alcohol-ether (1:1) and leave un-
til nitrocellulose is dissolved. Section should be adhering to slide.
6. Transfer to 95% alcohol, hydrate to water, and stain.
COMMENTS:
Frozen section either must be dried (for radioautographs) or car-
ried from water up to absolute alcohol before the nitrocellulose is
applied for histological staining.
Serial sections can be made by adding sections in sequence to con-
tinuous tape, rolled at each end of tissue block.

Laminated Thermoplastic Mounts. LaCroix

and Preiss (1690)
LaCroix and Preiss describe a practical and simple plastic mounting
method for chromosome smears, whole mounts, and cross and sagittal
sections. The cellular detail is good and the plastic mounting makes
Autoradiography 395

these preparations practical for class study and demonstration. No dan-

ger of breakage is involved, and if the plastic gets marred, the scratches
can be polished away.
APPARATUS:
Photo-Seal Kit (Therm Appliance Manufacturing Company, 608 S.
First Street, St. Charles, Illinois): consists of an electric sealing press
of two 4” & 5” aluminum platens with a 300W, 115V heater cast into
the bottom platen, two 4” « 5” nickeloid polished plates and other
necessary accessories.
PROCEDURE:
Root tip preparations:
1. Fix, hydrolyze, dehydrate and stain.
2. Arrange root tips in a drop of stain on a sheet of clear Vinylite
plastic, 1” « 3” and 15 mils thick.
3. Cover with a second sheet of plastic.
4. Place preparation between two sheets of bibulous paper, and ap-
ply pressure.
5. When spread, remove papers and top piece of plastic; add identi-
fication label if desired, and a third clean piece of plastic.
6. Place between plates of electric press, tighten wing bolts, and heat
for 3 minutes.
7. Cool under running water, and remove preparation from between
plates.
Section mounts:
1. Mount on plastic in same manner as On glass slide.
2. Dissolve paraffin in carbon tetrachloride: 20 minutes.
3. Hydrate, stain, dehydrate and clear. A short stay in xylene will not
dissolve the Vinylite.
4. Place second sheet on top, and laminate.

COMMENTS:
The nickeloid plates can be protected from tarnishing with alumi-
num foil sheets.

Autoradiography

The use of radioactive isotopes as tracers, localizing in an organ, a

tissue, or individual cell, has pushed autoradiography into a position
of considerable importance. ‘The rays and particles of the isotope in tis-
396 Special Procedures III (cuap. 24)

sue sections produce an image on photographic emulsion. After photo-

graphic processing, the emulsion can be studied by conventional light
microscopic methods, and a comparison and correlation made with
stained sections.
Only a brief summary of methods will be discussed and some refer-
ences included. All kinds of material can be used. Sections of large bone
can be exposed to roentgen-ray film; sections on slides, smeared and
squashed material, can be exposed to a covering of stripping or fluid
emulsion and tissue cultures can be placed against an emulsion to make
an exposure.
As to fixation, formalin usually is satisfactory, but solutions contain-
ing mercuric chloride should be avoided; they produce artifacts on the
emulsion (Kaminski, 1955). Solubilities of isotopes must be checked
carefully; some leach out in water or paraffin solvents and must be pre-
pared by freeze-drying methods (Holt and Warren, 1953). In some cases
carbowax can be used, but it will dissolve water-soluble isotopes (Holt
et al., 1949, 1952; Holt and Warren, 1950, 1953). Mounting must be
handled with care to prevent leaching (Gallimore et al., 1954).
Witten and Holmstrom (1953) freeze the tissue at the microtome 1m-
mediately after excision. The knife is kept cold with dry ice, keeping
the section frozen after it is cut. Then the section is carried still frozen
to the photographic emulsion. As the section thaws it produces a bit of
moisture and thereby adheres to the emulsion.
Bone must not be decalcified for this process. Some of the best tech-
nics for bone use methacrylate or epoxy embedding before sectioning.
(Arnold and Jee, 1954A, 1954B; Norris and Jenkins, 1960; and Wood-
ruff and Norris, 1955)
There are various methods of assuring contact between specimen and
emulsion.
PROCEDURE:
1. The specimen or slide bearing a specimen can be placed directly
on the emulsion, or it may first be protected by a layer of 1% cel-
loidin in amyl acetate and then be placed on the emulsion (Holt
and Warren, 1950).
2. Sections can be mounted directly on the emulsion on a slide.
(a) “Stripping film” method:
“Stripping film’? emulsion is removed from its glass plate. First the
edges of the emulsion are cut with a razor blade and the emulsion
can be peeled off and floated on distilled water with 1% Dupanol
(wetting agent) added. Then the slide with the section uppermost is
Electron Microscopy 397

slipped under the emulsion and lifted out carrying the emulsion with
it. To hold the emulsion in place without slipping it is folded under
three sides of the slide, dried in place and the exposure made. (Bogo-
roch, 1951; Pelc, 1956; and Simmel, 1957)
(b) Dipping in fluid emulsion:
This is simpler than the above method. Mounted sections protected
by a celloidin coat are dipped in a liquid emulsion, dried and left in
the exposure box for the proper time. (Messier and Leblon, 1957; and
Jojies, 1979)
COMMENTS:
In either (a) or (b) method, the film is developed, fixed, and cov-
ered or sprayed with Krylon.
At all times when working with photographic emulsions darkroom
safety measures must be observed (Wratten Safelight #2).
Staining:
Sections used for autographs can be stained. Pelc (1956) has used
celestin blue and hematoxylin, neutral red and carbol fuchsin, hema-
toxylin and eosin, carmalum, Giemsa, toluidine blue, and methyl
green-pyronin. Some workers find stains confusing because the emul-
sion gelatine absorbs them. Simmel et al. (1951) used metanil yellow
and hematoxylin with clearer results. Feulgen staining can be used.
(Also see Mérei and Gallyas, 1960)
Most autoradiographs should be protected by glycerol jelly mount-
ing, Krylon spraying or dehydration, clearing and mounting in a
resin.
General Reference: Fitzgerald et al. (1953)

Electron Microscopy

Among the methods in use for investigators in biological and medical

fields, one of the newest and fastest growing is electron microscopy. By
1940 several electron microscopes had been developed in different parts
of the world. Knoel and Ruska in Germany had a practical marketable
research scope by 1939. England began work in 1936 and RCA in the
United States started to develop one in 1938, and had one on the market
in 1941. RCA also has developed a desk model. In the last few years the
means of cutting sections down to 1/20 micron have become reliable.
The electron microscope has a higher resolving power than the light
398 Special Procedures IIT (cHap. 24)

microscope and has revealed submicroscopic structures invisible until

the present time. It uses electrons as the illuminating beam, and focuses
the beam on the object by the use of magnets. As the beam passes
through the object under observation, some of the electrons are scat-
tered by the object causing shadows of the scattered electrons on photo-
graphic film.
Electron beams must be handled in vacuum, therefore no wet or liv-
ing tissue ordinarily can be observed. Sections must be thin to prevent
appreciable electron absorption, because the image depends on the dif-
ferential scattering rather than absorption.

Specimen Supports

The specimen is supported on a metal grid and is placed in the column

at the electron-gun side of the object-lens aperture. This mechanical
support is made of electron-opaque metal (copper, nickel or steel) per-
mitting observation of the specimen through holes in the grid. A mesh
of 200 holes per linear inch is satisfactory and provides holes small
enough to support a thin film yet offers a large enough area for observa-
tion. England prepares an excellent grid with a center marker and a
solid periphery (obtainable from Ernest Fullam, Schenectady, N.Y.).
All specimens must be placed on some supporting film, supported in
turn on the grid. This film has to be transparent to the electron beam
but strong enough to not tear when bombarded by the beam. Various
materials have been used, but celloidin and formvar are favored. Car-
bon films are becoming popular, but are difficult to prepare (Watson,
1956; Barton, 1960).

Preparation of Film
Fither celloidin, 1% in amyl acetate, or formvar, 0.2% in ethylene
dichloride (1,2 dichloroethane) is flowed on a clean dry microscope
slide, or the slide is dipped in the solution (slide may be previously
treated with a water repellant). Evaporate film on slide and when dry
float film off on surface of water. Place grids, convex side down, on film.
Lower a clean slide on top of grids and sweep the slide through the
water bringing out with it the grids lying between the film and the slide.
Allow to dry. Grids can be left on the slide until used. The film also
may be cast directly on the surface of the water, the grids placed on it
and removed as above. The former method, however, forms a tougher
film (Mellors, 1959). Formvar makes a stronger and more temperature-
Electron Microscopy 399

stable film than celloidin, but the solutions deteriorate rapidly unless
kept in the refrigerator.
The grids are tiny and are most easily handled with stainless steel eye
forceps. The forceps tips can be bent slightly and sharpened with fine
emery paper for a delicate grip. Forceps with the bottom jaw sharpened
to a knife-edge will help to lift grids from a flat surface (Hall, 1953).

Fixation

In order to increase the scattering of electrons, the density of the speci-

men must be increased—some sort of reagent must be applied to it. For
these so-called “electron stains’ chemicals of high atomic number have
been tried, compounds of heavy metals (tungsten and osmium in par-
ticular) using phosphotungstic acid and osmic acid. Pease and Baker
(1950) concluded that osmic acid was the “outstanding” fixative. Luft
(1956) used potassium permanganate for membrane systems in partic-
ular.

Osmic Fixation

In this method, 1-2% osmic is buffered with either I or IT below, pH

7.4:

SOLUTIONS:
I. Edwards et al. (1956); Palade (1952):
0.14M sodium acetate and sodium veronal .... 5.0 ml.
OWUNGHIGD 2c 225 fede > cnt a ca enemys 5.0 ml.
I-2% osmic acid . . ; Song ate Renda
aan 12.5 mil:
With distilled water make up to ............. 25.0 mill.

II. Sjostrand (1956):

Buffer solution A:
SOG ACCHATE =<. ics 5 bce iv et a ok ot pe 2 9.714 gm.
Sodium veromall...6f.46.50..
0-504 be esas oe: . Sse 14 pm:
distilled water 2.2.6 casacsciiewev
dancers Mt 500.0 ml.

Buffer solution B:
SOU ColOnm@er en. shi pas ce oe ee « Se 40.25 gm.
potassiyim chiomde. ..,...8.
2. 6s. 6.6 +s see 2.) oT,
Galen COlOm@e tae. 6 date shoe eee 3 hee 0.9 gm.
GUSTMIER, WaAtEIn ako dice occ scan cose ee 500.0 ml.
400 Special Procedures III (cHap. 24)

Isotonic buffered osmic acid (working solution):

solution: Ae gee... .<.. |: ee eee ee 10.0 ml.
solution Sie 222). ot) LR eeePe 3.4 ml.
OSUN HIG) eteeree, ©. ee eS 11.0 ml.
distilled. Wweatenee ea... -.. \. eee re 50.0 ml.
Adjust to pH 7.4 with 0.1N HCl
Add OsmmiegaGidss 2.2... 45: oe eee 0.5 gm.
Store in brown-glass stoppered bottle in refrigerator.
METHOD:
1. Cut tissue in small fragments, 5 mm.; fix immediately. In spite of
the small size only the outer cells will be well fixed. Fix 30 minutes
to 4 hours minimum = maximum interval), 0-5°C.
2. Wash with Ringer’s solution: 1 hour, changing every 15 minutes.
3. Dehydrate and embed.

Permanganate Fixation (LUFT, 1956)

SOLUTIONS:
Stock solution:
potassium permanganate 2.) seeese os. 5. Bae 1.2 gm.
distilled swater pao konieie 8 See saree 100.0 ml.
Store in refrigerator in well-filled glass-stoppered bottle.
Working solution:
Equal volumes of potassium permanganate stock and Palade’s veronal
acetate buffer (above), pH 7.4—7.6, produces a final concentration of
0.6%, potassium permanganate.
METHOD:
1. Cool fixative in cracked ice. Fix 1-2 mm. pieces at 0°C: 15 min-
utes to 12 hours.
ho. Rinse several minutes in cold (0—5°C) 25% ethyl alcohol, allow to

warm to room temperature in fresh 25% alcohol (15-20 minutes)

3. Dehydrate and embed.

Chromium Fixation (Low, 1955; Low AND FREEMAN, 1956)

Good for reticulum.
SOLUTION:
Cheegmic, acid So a4 ae eR >. ae . 3.0 gm.
fORmaT . Se, ee eee oe 10.0 ml.
sodium achloride a. o- 42 Se. Sepa . O85. em.
pH about 3.2
Eleciron Microscopy 401

METHOD:
1. Short fixation best: 0.25-—0.5 hour.
2. Wash: 0.5 hour, in distilled water.
3. Dehydrate and embed.

Chromium-Osmium Fixation (DALTON, 1955)

SOLUTION:
chironaresaeid stAO. Oi dais a chess « hse gsate « 10.0 ml.
somimmmecnlonides G49). |x.’ 2 has mabe nace aiendss 10.0 ml.
Add-osmiuic acid; 29, + 2.454 ! , 20.0 ml.

Can be used for longer period than some of others (18-24 hours) and
is good for tissue of high lipid content, but produces lower contrast
and increased difficulty in obtaining thin sections.

Lehmann and Mancuso’s Fixative (1957)

SOLUTIONS:
Solution I:
POG, AOUMeMD,.. doo eeu ogee samaand
+span 80.0 ml.
AGCUOME Be Mas a aS.cd Gah eed eer eA eee ae 16.0 ml.
Glacial ACetGaCld en. :S erotes a wea. a neeee 0.5 ml.
Solution IT:
2.59% osmic acid, aqueous

METHOD:
1. Mix equal parts of solutions I and IT. Fix for 30 minutes. ‘This 1s
recommended for preservation of fibrous cytoplasmic structures;
penetrates rapidly and partially dehydrates.
2. A subsequent treatment with chromic acid improves preservation
of some lipid and fibrous structures. After 20 minutes in original
fixative, add 10% chromic acid (2 ml. for every 5 ml. of above).
Leave for additional 20 minutes.
3. Rinse in tap water: 15 minutes, dehydrate and embed.

Embedding

Only plastic embedding permits sections of 0.01 to 0.05 microns to be

cut. Glauert and Glauert (1958) recommend the epoxy resin Araldite,
saying that there is greater stability of resin, less shrinkage of tissue,
ereater degree of variation possible in viscosity of mixture, less tend-
ency to form bubbles and greater hardness and uniformity of block
402 Special Procedures III (cHap. 24)

than is true of other plastics. ‘The required solutions can be obtained as

a Cargille (Nysem) Epoxy Embedding Kit from Cargille Sons, Little
Falls, N.J. Full directions for use are enclosed with the kit. (Also see
Richardson et al., 1960)
Pease (1960) disagrees with the above, saying that the epoxy resin
forms brittle blocks, causing cutting artifacts and producing poor con-
trast in some specimens. Biological systems should be “stained” when
embedded in it. “Epon” epoxy resins are being developed; at the pres-
ent he prefers methacrylate embedding, although the methacrylate de-
composes under electron bombardment.

Butyl Methacrylate Embedding (NEWMAN, BORYSKO,

AND SWERDLOw, 1949)
METHOD:
1. From fixative, quick rinse in salt solution or veronal buffer such as
fixative ions prepared in.
2. Dehydrate in 50, 75, and 95% alcohol: 3—5 minutes each. Several
changes of absolute alcohol, at least 2 changes: 5-10 minutes; |
change: 15—20 minutes.
3. ‘Transfer to equal volumes of absolute ethyl alcohol and n-butyl
methacrylate monomer‘: about | hour.
4. Transfer to monomer (no catalyst) 3 changes: 1 hour each.
5. Set body of #00 gelatine capsule upright in wooden block or other
base. Fill with monomer plus 1% (by weight) of catalyst (2,4-di-
chlorobenzoyl peroxide’). Orient tissue in it and slip on lid.
6. Place in oven, 45—50°C. Important that capsule be heated evenly
over entire surface. Suspend with cellophane tape from rod or
other support inside oven. Within 6-8 hours a solid matrix of pol-
ymerized monomer has formed. Additional period of several hours
insures complete polymerization.
_7. Soak in water and peel off capsule.

COMMENTS:
The n-butyl methacrylate contains an inhibitor which must be re-
moved by repeated washings in a separatory funnel with dilute aque-
ous sodium carbonate until no color is visible in the washings. Follow
with calcium chloride to remove the water. Keep in the refrigerator
to inhibit polymerization. Do not use rubber or plastic container for
storing.
* Rohm and Haas Co., Philadelphia.
* Lucidol Division, Novadel-Agene Corporation, Buffalo.
Electron Microscopy 403

Pease (1960) makes the following suggestions: make solution

changes, fixation, dehydration, etc. in small wide-mouthed weighing
bottles. He considers the blocks formed by pure butyl methacrylate
as too soft and adds anywhere from 10 to 30% of ethyl methacrylate
to his solution. The catalyst dissolves slowly and must be complete
before the solution is placed in the capsules. He recommends poly-
merization by ultraviolet light, saying that it forms a block superior
to that formed by heat. It is more uniform and has fewer bubbles.
Use a Westinghouse Fluorescent Sun Lamp bulb, #5TT12 about 1
inch from the capsules.

Ultrathin Sectioning

Conventional-type microtomes have been modified for ultrathin sec-

tions. Pease and Baker (1948); Newman, Borysko and Swerdlow (1949);
Newman (1950); Geren and McCullock (1951); Nylen and Holland
(1957). Three good specially designed microtomes are available:

i Porter and Blum (1953) utilizes a cantilever action, is manufactured

and distributed by Jvan Sorvall, Inc., Norwalk, Conn.
. Sjostrand utilizes a thermal expansion feed and is motor driven, is
no

manufactured by LAB Produkter in Stockholm, is handled by Sor-

vall.
. Reichert of Austria also manufactures one.

KNIVES
Steel knives must be exceedingly sharp, but are dulled rapidly by
plastic cutting.
Glass knives are popular and cheap. They can be made from 3/16
inch or + inch plate glass, shaped as rhomboidal equilateral parallelo-
grams with a cutting edge of 45° (Porter and Blum, 1953), and fitted
in a specially designed holder (Cameron, 1956 and Sheldon, 1957).
They dull rapidly but are easily replaceable; they can be made in the
laboratory or purchased from Acme Glass Company, Cambridge, Mas-
sachusetts. Gavin and Lloyd (1959) recommend a vycor brand glass
for less scratching in the sections. The angle between front side of
the knife and the plane of the passing section should not exceed 10°,
preferably 3-50. Sjostrand uses a hand-sharpened Schick razor blade
in a special holder.
Diamond knives (Fernandez-Moran, 1953, 1956; Crandall, 1961)
are more durable but are expensive. Sorvall carries them.
404 Special Procedures III (cHap. 24)

SECTIONING
Recovery of sections was difficult until Hillier and Gettner (1950)
developed a water trough attached to the knife, thereby permitting
the sections to float directly onto a fluid surface as soon as they were
cut. The trough is filled with 10-40% acetone or ethyl alcohol in
water to facilitate the flattening (also see Solelo, 1957). The sections
are picked up from the solution onto the coated grids, or, if some-
what compressed, are transferred first to 50% alcohol and then to
orids.
For serial sections see Gay and Anderson (1954), Williams and Kall-
man (1954), and Westfall (1961).
The thickness of the sections can be determined by the color analy-
sis of Porter and Blum (1953). While the sections float on the bath,
they display interference colors depending on their thickness. Silver
indicates a thickness of 20-50 millimicra; gold, 50-80 millimicra;
purple, 80-130 millimicra. Silver and gold sections are best for elec-
tron microscopy, but as they begin to appear blue or green they are
getting too thick for efficient observation.
Three-dimensional images may be obtained by metal shadowing and
this is finding favor with many researchers. Metals, such as silver, chro-
mium, palladium, platinum and others are evaporated on the specimen
at a small angle. A thin film of the metal covers the specimen except for
a ‘‘shadow” caused by the specimen being in the way of passage of the
metal. From the shape and dimension of the shadow, height and con-
tour of the specimen can be observed. (Williams and Wyckoff, 1944;
Williams and Backus, 1949; de Harven, 1958)
Plastic sections can be used for optical examination. The sections
(1-2 microns) are dried on a slide, immersed (12—24 hours) in xylene
to remove the plastic and then can be stained for light microscopy, or
mounted for phase microscopy. (Borysko and Sapranauskas, 1954;
Farquhar and Rinehart, 1954; and Houcks and Dempsey, 1954)
See also Fernandez-Moron (1960) for the application of low temper-
ature techniques for electron microscopy utilizing HELIUM u. In addi-
tion, this source describes the use of a liquid nitrogen stage for cooling
specimens during observation in the electron microscope.
References: Farquhar (1956), Hall (1953), Mellors (1959), and Pease
(1960).
 SOLUTION
., PREPARATION
IV AND GENERAL
LABORATORY
/AIDs
 Section 1

Solution Preparation

Abbreviations and Terms

Abbreviations

ml. (cc)—milliliter, cubic centimeter

gm.—eram
mg.—milligram
aq. aqueous
M—molecular solution, see below
N—normal solution, see below

Molecular Solutions (cowpry, 1952)

A molecular solution contains the molecular weight in grams of the
substance made up to | liter with distilled water. For example: M oxalic
acid (COOH)::2H2O is 126 grams (molecular weight) in 1 liter of
water.
Molecular weight expressed in grams is called the gram-molecular
weight or mole. A millimole is 1/1000 of a mole.

Normal Solutions
A normal solution contains | gram-molecular weight of dissolved
substance divided by the hydrogen equivalent of the substance (that
is, | gram equivalent) per liter of solution. N oxalic acid is half the
concentration of the M solution above.

407
408 Solution Preparation (src. 1)

Percentage Solutions
In the case of percentage solutions, it should be indicated in some way
as to whether the percentage is determined by weight or volume: either
written out in grams and milliliters or expressed thus:

w/v means weight in grams in a 100 ml. volume of solution

v/v means volume in milliliters in 100 ml. total volume of solution

Although it is erroneous, a long-established habit of technicians ts

maintained. Percentage solutions of liquids are diluted as though the
reagent solution is 100% concentration. That is, a 1% solution of acetic
acid is I ml. of glacial acetic acid in 99 ml. of distilled water. In most
procedures the dilution is indicated in parentheses following the per-
centage desired.

Dilutions for 1N Solutions.*

Molecular Percentage Specific gm per ml per

weight assay gravity liter liter

acetic 60.05 99.7—100F 1.0498 1050 57.2

acid 99.0 1.0524 1042 57.6
CH;COOH 98.0 1.0549 1034 58.0

ammonium 17.03 26 0.904 235 72.4

hydroxide 287 0.8980 251.4 67.7
NH,OH 30 0.8920 267.6 63.63

formic 46.03 96 1 Wi 1180 S975

acid 987 1.2183 1194 38.4
HCOOH 99 1.2202 1208 38.0
100 2202 1221 B76

hydrochloric 36.46 36 1.1789 424.4 85.9

acid 37-38 (SS SP 192 45126 80.4
HCl 40 1.1980 479.2 76.0

nitric 63.02 69 1.4091 S723 64.8

acid 70 1.4134 989.4 63.6
HNO; 71 1.4176 1006 62.6
72 1.4218 1024 61.5

sulfuric 98.075 95 1.8337 1742 Zeal

acid 96T 1.8355 1762 27.8
H»SO,4 97 1.8364 1781 20.5

* Calculated from Norbert Adolph Lange, 1956, Handbook of Chemistry, Handbook

Publishers Inc., Sandusky, Ohio. Jfalicized numbers indicate figures used in calculations.
} Indicate most common form of the reagent. In addition, a calculation has been made
for the nearest lower and higher percentages as well.
Stock Solutions 409

To make a normal solution, add to the ml. in the right-hand column

enough distilled water to make a combined total of I liter. This will be
accurate enough for histological preparations.

Stock Solutions

The solutions included under this title are probably found (with a
few exceptions) on most histology laboratory shelves.
Three physiological solutions are included to offer a choice, depend-
ing on the particular need of the type of research in progress. The use
of physiological solutions can be imperative at times. A normal solution
(isotonic) contains the proper amount of salts to maintain tissues in a
normal condition. For instance, red blood cells will remain unaltered
in form and will not experience a loss of hemoglobin if isotonic fluids
are used with the cells. The osmotic pressure and salt content of the
physiological solution and of the blood fluid is the same. If the solution
is hypotonic, the osmotic pressure and salt content is less than that of
the body fluids and the cells will swell. If the solution is hypertonic,
the osmotic pressure and salt content is greater than that of the body
fluids, and the cells will shrink. Plain distilled water will be hypotonic
until the proper quantity of salt has been added.
Just as physiological solutions are useful for land vertebrates, so are
environmental solutions convenient for some invertebrates. In the case
of marine invertebrates, such solutions can become necessary for success-
ful preparations and sometimes are recommended as the basic fluid for
fixing solutions. Among the physical properties of sea water, the salt
content has to be considered. It varies and is complex, but an arbitrary
definition of salt content (salinity) has been calculated. Salinity (S 0/00)
equals the weight in grams (im vacuo) of solids in 1 kilogram of sea
water. The major constituent, which is easily determined, is silver-
precipitating halides. Chlorinity (Cl 0/00) is defined as the weight in
grams (in vacuo) of chlorides in | kilogram of sea water. Standard sea
water is about 19.4 0/00 chlorinity and 34.3243 0/00 salinity. (Barnes,
1954)
Not all of the adhesive or buffer solutions will be found or required
in every laboratory: however, a listing of the more widely used ones
seems practical. Then if a need should arise, the information is at hand.
Stain solubilities are only occasionally useful, but they are definitely
needed when a saturated solution must be made.
410 Solution Preparation (src. 1)

ACID ALCOHOL.
70975 ethyl ealcohol:... 5 Seeaes 2 100.0 ml.
hydrochloric acid, concentrated ............. 1.0 ml.

ALKALINE ALCOHOL.
7097, ethiyitaimool...:... :.. 2 Saga Hee 3.3 100.0 ml.
ammonia scameentzated :. 0.1. epee pets. 1,0,analy
(or saturated aqueous sodium or lithium bicarbonate)
CARBOL-XYLOL.
phenel (earbolic -acid) melted”: te) se 1 part
VIET A. 5.oC aR MR MAE aera ts euhgrAh ic' ee haart 3 parts
During use, keep covered to reduce evaporation of xylene.
For creosote-xylol use beechwood creosote in place of phenol. For
aniline-xylol use aniline.
GOLD CHLORIDE STOCK SOLUTION.
gold, chlonide tac e.prrareceter seis Sit. 4a ee 1.0 gm.
distilled -water,_ Ap séte:ocht Jcega tpt maby 2c dope eee 100.0 ml.
LUGOL’s SOLUTION. There are various formulas to which this name has
been applied.
a. The strongest concentration ts:
Kore Fn 5,cic 1) CR eae Re ae. NI gre) 1.0 gm.
potassium iodide: 22. Ge.) acces ee eee cog 2.0 gm.
Cistillede Water. fe cere 4 oak Reet eecde ras ae 12.0 ml.
b. Weigert’s variation:
iodine} 40}: 2u-bsbces
weet Stredee rsamaek 1.0 gm.
potassium jOdides |pyecuni 2k Pees... ocls aHae 2.0 gm.
distilled awatein rycioceh beer hae 3? of erebeeae 100.0 ml.
c. Gram’s variation:
TOCING |e ees 0G RCS ec eee 1.0 gm.
POtasSiUMMOCIAEY ste ae ete, 9 2 es 2.0 gm.
distilled “water oc. 45 eee >. eee: 300.0 ml.
In all cases, first dissolve the potassium iodide, then the iodine will
go into solution readily.
PHYSIOLOGICAL SOLUTIONS
Physiological Saline—NaCl in distilled water
mammals, 0.9% (97gm./1000 ml. water)
birds, 0.75% (7.5 gm./1000 ml. water)
Stock Solutions 4il

salamander, 0.8% (8 gm./1000 ml. water)

7
/,

frogs, 0.64% (6.4 gm./1000 ml. water)

Locke’s Solution
SOMMIMCnIOMdes... .s . . +. ie deg eeedswl
ead 0.85 gm.
(for cold-bloods, 0.65 gm.)
potassmuny cilomde i252... sowie eager ek i: = - 0.042 gm.
sodium bicarbonate ........................ 0.02 gm.
CalCuaMnCMMOtide® sf cua.« 4.80. b hours Sapdeotids Aad Ss 0.024 gm.
ISCHME AV AET soe oh. ha Wao Meashee 100.0 ml.
Ringer’s Solution
SOcwmMmMCINOMGe: 651.255 2 ithe d ena en > hale 0.9 gm.
(for cold-bloods, 0.65 gm.)
potassium: chloride. . 3.4. 4.4/be..Shac2 vind ws 0.042 gm.
ealcratmiretlOride: hig cc: 5 icc octsbnge Bes cette 0.025 gm.
GIGtINIED Water H.520 cue 4 iia a 28 2 Here Sealer sae 100.0 ml.
Best prepared fresh.

ARTIFICIAL SEA WATER, HALE (1958).

chlorinity, 19 0/00
salinity, 34.33 0/00
ENG xe Be oe eerste een ees sate 25.991 em.
DN ee Pate smeeen 24 he op pasate ea oe eee 0.742 gm.
COGS. 22 Gh iede ce eee eaire: . Lop eem.
(GaCl-GHSO™ 2.2.2
ois ecetaa 2.240 gm.)
DIGIC 2c. aay gee 2a a hse vie Sass 2 5.102 gm.
(MeGle*bl50) oan. ees: /s.. 10/893 ¢m.)
Na,SO, Dare eae cies ee econ eee gaa 4.012 gm.
(NassO.
10H Oa occ taea on 9.1 gm.)
INGOs abe 5 ences HE oO oh es avs tere. 5 0.197 gm.
IN AUBTG & Spre ass evento os Sans 9 mer . 0.085 gm.
(NaBr- 20 2.2 wa ntakodd-es 0.115 gm.)
2)1 O) Sn ee PO a 0.011 gm.
(SrClo-GHLO® «<0 scl cs. oases 0.018 gm.)
PODS eee eee See elec He s comes pod 0.027 gm.

Digplve in distilled water and make up to | liter.

Note: The weights in the right-hand column are for the anhydrous
form of the salt; those in parentheses have water of crystallization
present, as indicated on the reagent bottle.
412 Solution Preparation (sEc. 1)

ScoTtT’s SOLUTION.
sodium: jbtcaroonate: . 5°00, nee eee 2.0 gm.
MapnestuMssUlbates... So. ee en 20.0 gm.
distilleduwatenge ce. fo: . <5 2p eee BE 100.0 ml.
Add a pinch of thymol to retard molds.
SODIUM THIOSULFATE SOLUTION (hypo), 5%
sodium ‘thigsultate Na,S.O; ....--2)--.....-. 5.0 gm.
distilleclewatetrar 7. %. 2: . 144 «ceenee an xr ae 100.0 ml.

CLEANING SOLUTION FOR GLASSWARE.

Strong:
Potassium sdichromate 2h)».
see eee aoe 20.0 gm.
TRIZIELC Ae Ne epee ieee «ee Se ce Ny 3 200.0 ml.
Dissolve dichromate in water; when cool add very slowly:
suliuric acid, concentrated 2)\.)c. bert: oo. 20.0 ml.
Weak:
potassium dichromate, 2% aqueous ........... 9 parts
SUG MIE: ACI.” RR: 3.2 en Ee |. 1] part
Sufficiently strong for most purposes.

Adhesive Solutions
Mayer’s Albumen Fixative
Beat white of an egg (only until well broken up, but not stiff) with
egg beater and pour into tall cylinder. Let stand until the air brings
suspended material to the top (overnight). Pour off liquid from bottom
and to it add an equal volume of glycerol. A bit of sodium salicylate,
thymol, merthiolate, or formalin (1:100) will prevent growth of molds.
Many technicians filter their solution through glass wool, but the author
has found this of no advantage.
Faulkner and Lillie (1945B) substitute dried egg white. A 5% solu-
tion of dried egg white in 0.59% NaCl is shaken at intervals for one day.
Do not allow it to froth. Filter in a small Buchner funnel with vacuum.
Add an equal amount of glycerol and 0.5 ml. of 1:10,000 merthiolate
to each 100 ml. of solution.

Masson’s Gelatine Fixative

peelasiinmers i... Pa Ot ah ae ees See isl 50.0 gm.
cistilleas water . 724.4. 20 Soe. sae 25.0 ml.
Adhesive Solutions 413

Recommended by many for alkaline silver techniques, when sections

tend to float off during or after impregnation. Float sections on solu-
tion on slide, and place on warm plate. When sections have spread,
drain off excess gelatine and blot dry with filter paper. Place in
formalin vapor, 40—50°C overnight.

Haupt’s Gelatine Fixative (1930)

PCAC E artes, sak) od Stee eRe kee ees was Gs 1.0 gm.
estilledivaten 3.4 « 2.0 ats na Salheees eke Seles we 100.0 ml.

Dissolve at 30°C (not above) in water bath or oven. Add:

phenol (carbolie acid) crystals <.. ..e¢sa. 00 20 2.0 gm.
PAV CEN Ss . Olt Seeks 2 HaeSe s ay aE eee MIE 15.0 ml.
Stir well, and filter.

Use 2% formalin when mounting sections. This hardens the gelatine;

water is not adequate. Some may find the formalin fumes to be irri-
tating to the eyes and nostrils.
Haupt suggests that if sections tend to loosen, place a small uncovered
dish of concentrated formalin in an oven with the slides while drying
them. The formalin tends to make the gelatine insoluble and helps
to hold the sections in place.

Weaver’s Gelatine Fixative (1955)

SOLUTION A:

PCIANING 5 30d eucbiiten44 coh ing Op aes eeeney: 1.0 gm.

calcium propionate! ...... ee ed: 1.0 gm.
Roccal (1°% benzalkonium patonae Nir cate, 1.0 ml.
distilled water .......... e Lt aa ee 100.0 ml.

SOLUTION B:

chrome alum (Cr2,K(SO,4)4-24H,O) .......... 1.0 gm.

distilled water .......... she oe - 90.0 ml.
fOPIMAII ys «ease
aon s4 &As ere voter © 10.0 ml.

Mix in proportions of | part of A to 9 parts of B. Flood slide with

adhesive mixture, add paraffin ribbons, and allow to stretch as usual.
Drain off excess adhesive and blot. Wipe edges close to paraffin. De-
posits of adhesive should be removed because it does pick up stain.
Good for sections which are difficult to affix.
1 Mycoba, manufactured by E. I. DuPont de Nemours and Co., Inc.
414 Solution Preparation (sEc. 1)

Blood Serum Fixative (PRIMAN 1954)

fresh human blood serum (only from negative
IW aSSeriiaimeyess ors.
sla eee 15.0 ml
caistilled™wategem | |’... ees ene 10.0 ml
formaliniyogpeeee: ss RR ee 6.0 ml
Filter through filter paper and use same as albumen fixative. Does
not stain and sections do not loosen.

Butter Solutions

Molecular Weights of Buffer Ingredients

acetic acid, CH; COOH 60.05
borax, NasByO,;- 10H2O 381.43
boric acid, B(OH); 61.84
citric acid (anhydrous), C3;H4(OH) (COOH); 192.02
citric acid crystals, C;Hy (OH)(COOH);:H20 210.14
formic acid, HCOOH 46.03
hydrochloric acid, HCl 36.465
maleic acid, HOOCCH-CHOOH 116.07
potassium acid phosphate, KH2PO,4 136.09
sodium acetate, CH;COONa 82.04
sodium acetate, crystals, CH;COONa:3H20O 136.09
sodium barbital, CgsH,,03N.Na 206.18
sodium citrate, crystals, C;Hy;OH(COONa);-53H2O SD118
sodium citrate, granular, C;Hy,OH(COONAa);-2H20 294.12
sodium hydroxide, NaOH 40.005
sodium phosphate, monobasic, NaH,PO,-H20 138.01
sodium phosphate, dibasic, Na,HPO,-7H2O 141.98
sulfuric acid, H»SO, 98.082
tris (hydroxymethyl) aminomethane CyH1,NO3 121.14

These are a few of the more commonly used chemicals for buffer so-
lutions; for others consult a chemical handbook. Nine buffer tables fol-
low, and will be adequate for most histological preparations.

0.2M Acetate Buffer (Gomori) (pH 3.8-5.6)

STOCK SOLUTIONS:
acetic acid: 12.0 ml. made up to 1000 ml. with distilled water
sodium acetate: 27.0 gm. made up to 1000 ml. with distilled water
Add a few crystals of camphor to both solutions.
Buffer Solutions 415

For desired PH mix correct amounts as indicated below:

pH acetic acid sodium acetate

milliliters milliliters

3.8 87.0 13.0

4.0 80.0 20.0
4.2 73.0 27.0
4.4 62.0 38.0
4.6 51.0 49.0
4.8 40.0 60.0
5.0 30.0 70.0
Bid 21.0 79:0
5.4 14.5 85.5
5.6 11.0 89.0

Acetate-Acetic Acid Buffer (WALPOLE) (pH 2.7-6.5)

STOCK SOLUTIONS: ,
M/5 acetic acid: 11.5 ml. (99% assay) and 988.5 ml. distilled water

M/5 sodium acetate: 2.72 gm. made up to 1000 ml. with distilled
water
For desired pH mix correct amounts as indicated below:

pH M/5 acetic acid M/5 sodium acetate

milliliters milliliters

2.70 200.0 0.0

2.80 199.0 1.0
2.91 198.0 2:0
3.08 196.0 4.0
3.14 195.0 5.0
3.20 194.0 6.0
BPs) | 192.0 8.0
3.41 190.0 10.0
3.59 185.0 15.0
a2 180.0 20.0
3.90 170.0 30.0
4.04 160.0 40.0
4.16 150.0 50.0
* 4.27 140.0 60.0
4.36 130.0 70.0
4.45 120°0 80.0
416 Solution Preparation (src. 1)

pH M/5 acetic acid M/5 sodium acetate

milliliters milliliters
4.53 110.0 90.0
4.62 100.0 100.0
4.71 90.0 110.0
4.80 80.0 120.0
4.90 70,0.) sur 15010
4.99 60.0) 140.0
5.11 50.0 . 150.0
5.22 40.0 _ 160.0
5.38 30.0 170.0
5.57 20.0 ; 180.0
89 10.0 190.0
6.21 5.0 195.0
6.50 0.0 200.0

0.05M Barbital Buffer (Gomori) (pH 8.7-6.9)

5 sade

STOCK SOLUTIONS:
sodium barbital: 1.03 gm. in 50 ml. distilled water
Add 0.1N HCl according to table below and dilute to total of 100 ml1.:

pH 0.1N HCl
milliliters

8.7 5.0
8.5 7.5
8.3 11.0 Ba
8.1 15.0 ee e
7.9 19.0 :
7.65 26.0
7.45 31.0 .
tks 36.0 oe
7.15 41.0 ye
6.9 43.5

Boric Acid-borax Buffer (HOLMES) (PH 7.4—9.0)

STOCK SOLUTIONS:
M/> boric acid: 12.368 gm. made up ta 1000 ml. with distilled water
M/20 borax: 19.071 gm. made up to 3000 ml. with distilled water
For desired pH mix correct amounts as indicated below:

er.
y
Buffer Solutions 417

pH M/5 boric acid M/20 borax

milliliters milliliters

7.4 90.0 10.0

7.6 85.0 15.0
7.8 80.0 20.0
8.0 70.0 30.0
SZ 65.0 35.0
8.4 55.0 45.0
8.7 40.0 60.0
9.0 20.0 80.0

0.2M Phosphate Buffer (Gomori) (pH 5.9-7.7)

STOCK SOLUTIONS: |
monobasic sodium phosphate: 27.6 gm. made up to 1000 ml. with dis-
tilled water
dibasic sodium phosphate: 53.6 gm. made up to 1000 ml. with dis-
tilled water
For desired pH mix correct amounts as indicated below:

fH ~~ monobasic sodium phosphate dibasic sodium phosphate

milliliters milliliters

59 90.0 10.0
6.1 85.0 15.0
6.3 77.0 23.0
6.5 68.0 32:0
6.7 57.0 43.0
6.9 45.0 55.0
| 33.0 67.0
Ws 23:0 77.0
7.4 19.0 81.0
7.5 16.0 84.0
Von 10.0 90.0

Phosphate Buffer (SORENSEN) (PH 5.29-8.04)

STOCK SOLUTIONS:
M/15 dibasic sodium phosphate: 9.465 gm. made up to 1000 ml. with
distilled water
M/15 potassium acid phosphate: 9.07 gm. made up to 1000 ml. with
distilled water
For desired pH mix correct amounts as indicated below: (Some tech-
nics will require dilution up to 1000 ml.)
418 Solution Preparation (sEc. 1)

pH M/15 dibasic sodium phosphate M/15 potassium acid phosphate

milliliters milliliters

5.29 2.5 97.5

5.59 5.0 95.0
5.91 10.0 90.0
6.24 20.0 80.0
6.47 30.0 70.0
6.64 40.0 60.0
6.81 50.0 50.0
6.98 60.0 40.0
1M) 70.0 30.0
7.38 80.0 20.0
Weld 90.0 10.0
8.04 95.0 5.0

Standard Buffer (McILVAINE) (PH 2.2-8.0)

STOCK SOLUTIONS:
0.1M citric acid (anhydrous): 19.212 gm. made up to 1000 ml. with
distilled water
0.2M disodium phosphate: 28.396 gm. made up to 1000 ml. with dis-
tilled water
For desired pH mix correct amounts as indicated below:

pH citric acid disodium phosphate

milliliters milliliters

2:2 19.6 0.4

2.4 17.76 1.24
2.6 17.82 2.18
2.8 16.83 3. 7
3.0 15.89 4.11
3:2 15.06 4.94
3.4 14.3 5i7
3.6 13.56 6.44
3.8 12.9 pel
4.0 12°29 Heil!
4.2 11.72 8.28
4.4 11.18 8.82
4.6 10.65 9.35
4.8 10.14 9.86
5.0 9.7 10.3
5) 9.28 NOM
5.4 8.85 iSled;
5.6 8.4 11.6
Buffer Solutions 419

pH citric acid disodium phosphate

milliliters milliliters

5.8 7.91 12.09

6.0 yey) 12:63
6.2 6.78 13.22
6.4 6.15 13.85
6.6 5.45 14.55
6.8 4.55 15.45
7.0 3.53 16.47
eZ 2.61 17.39
7.4 1383 18.17
16 27 18.73
7.8 0.85 19.15
8.0 0:55 19.45

0.2M Tris Buffer (HALE) (PH 7.19-9.1)

STOCK SOLUTIONS:
0.2M tris (hydroxymethyl) aminomethane: 24.228 gm. made up to
1000 ml. with distilled water
0.IN HCl (38% assay): 8.04 ml. made up to 1000 ml. with distilled
water
To 25 ml. 0.2M tris add 0.1N HCl as indicated in table below and dilute
to 100 ml.

pH 0.1N HCl
milliliters

7.19 45.0
7.36 42.5
7.54 40.6
7.66 GHAR
7.77 35.0
7.87 32.9
7.96 30.0
8.05 Biss
8.14 25.0
8.23 22.5
8.32 20.0
8.41 17.5
8.51 15:0
8.62 25
8.74 10.0
8.92 TS
9.1 50
420 Solution Preparation (sEc. 1)

Tris Maleate Buffer (Gomori) (PH 5.88.2)

STOCK SOLUTIONS:
malcievaerieen SO Net eee 29.0 gm.
tris (hydroxymethyl) aminomethane.... 30.3 gm.
distilledivvatere.s.... «...\.: ss ae 500.0 ml.
Add 2 gm. charcoal, shake, let stand 10 minutes and filter. ‘To 40 ml.
of stock solution add N NaOH (4%) as indicated below and dilute to
100 ml.

pH sodium hydroxide

milliliters

5.8 9.0
6.0 10.5
6.2 13.0
6.4 1520
6.6 16.5
6.8 18.0
7.0 19.0
Gee, 20.0
7.6 22:5
7.8 24.2
8.0 26.0
8.2 29.0

Stain Solubilities
in water in absolute ethyl alcohol
per cent per cent

(from (from (from (from

Conn, 1953) Gurr, 1960) Conn, 1953) Gurr, 1960)

26°C 15°C 26°C 15°C

acid alizarine blue — 1.0 — 0.75

acid fuchsin — 45.0 _— 3.0
acridine orange — 5.0 — O75
acridine yellow = 0.5 — 0.75
acriflavine = 15.0 — 1.0
alcian blue = 95 = 6.0
alizarine red S$ 7.69 (4) O15 0.15
aniline blue ws = 50.0 = 0.0
auramin O 0.74 1.0 4,49 4.0
Stain Solubilities 4?]

in water in absolute ethyl alcohol

per cent per cent

(from (from (from (from

Conn, 1953) Gurr, 1960) Conn, 1953) Gurr, 1960)

26°C 15°C 26°C 15°C

aurantia 0.0 0.1 0.33 0755

azocarmine G = 1.0 — 0.1
basic fuchsin 0.26-0.39 1.0 5.93-8.16 8.0
biebrich scarlet = 5.0 0.5 0.25
bismark brown 1.36 1.5 1.08 3.0
brilliant cresyl blue = 3.0 — 2.0
celestin blue B == 2.0 == 15
chlorantine fast red = 1.0 — 0.45
chlorazol black E = 6.0 = 0.1
chromotrope 2R ibe) 19.0 0.17 O15
chrysoidin Y 0.86 5.5 224 4.75
congo red = 5.0 O19 0.75
coriphosphine O — 4.75 = 0.6
cresyl violet
(cresylecht violet) 0.38 o.5 0.25 6.0
crystal violet
(methyl violet 70B) 1.68 9.0 13.87 8.75
eosin B S912 10.0 0.75 3.0
eosin (S') alc. sol. (ethyl eosin) 0.03 0.0 113 1.0
eosin Y, ws 44.20 44.0 2.18 2.0
erythrosin B 11.10 10.0 1.87 5.0
erythrosin Y _— 8.5 — 4.5
fast green FCF 16.04 4.0 035 9.0
fluorescein (uranin) 50.02 50.0 7.19 7.0
gallocyanin (bisulfite) ==: 3.0 = 0.5
gallocyanin (hypochlorite) = 0.5 — 1:25
hematein — 15 = 79
hematoxylin gs) 10.0 60.0 10.0
janus green B 5.18 5.0 f12 7.3
light green SF yellowish 20.35 20.0 0.82 4.0
luxol fast blue — 0.0 = 3.0
malachite green — 10.0 — 8.5
martius yellow 4.57 1.0 0.16 0.0
metanil yellow 5.36 5.0 1.45 is
methyl violet 2B 2.99 a H5: 21 =
methyl violet 6B — 4.7 — 9:5
methylene blue 3:55 9:5 1.48 6.0
naphthol yellow S$ —= 12.5 — 0.65
422 Solution Preparation (src. 1)

in water in absolute ethyl alcohol

per cent per cent

(from (from (from (from

Conn, 1953) Gurr, 1960) Conn, 1953) — Gurr, 1960)

26°C ts°G 26°C 15°C

neutral red 5.64 4.0 2.45 1.8

new fuchsin 1413 1.0 oae 8.0
nigrosin, alc. sol. — 0.0 = 2.5
nigrosin ws == 10.0 = 0.0
nile blue sulfate = 6.0 = 5.0
oil red O = 0.0 = 0.5
orange G 10.86 8.0 0.22 0.22
orcein = 2.0 = 4.2
phloxine B == 10.5 = 5.0
picric acid 1.18 £2 8.96 9.0
ponceau 2R == 5.0 = 0.1
ponceau S — 135 = 12
ponceau de xylidine = 5.0 — 0.1
primilin = 0.25 = 0.03
pyronin B — 10.0 == 0.5
pyronin Y 8.96 9.0 0.6 0.5
quinoline yellow — 0.5 == 0.0
rhodamin B 0.78 2.0 1.47 1.75
rhodamin 6G = 5 ——— 6.5
safranin O 5.45 4.5 3.41 55
sudan black B = 0.0 —— 0.25
sudan ITI 0.0 0.0 0.15 0.25
sudan IV 0.0 0.0 0.09 0.5
thioflavine S — 1.0 == 0.4
thioflavine T — 2.0 == 1.0
thionin 0.25 1.0 0.25 1.0
titian yellow — 1.0 = 0.02
toluidine blue O 3.82 5:25 0.57 175
trypan blue — 1.0 = 0.02
victoria blue B _ 4.3 = 8.25
victoria blue 4R 3:23 3.0 20.49 20.0
Solubilities of different batches of dyes can vary, but the above table can be consulted
when preparing saturated solutions. Certain dyes whose solubilities were not available are
not included, such as pinacyanole and saffron. For greater detail concerning dyes see:
Conn (1953); Gurr (1960).
 Section ps

General Laboratory Aids

Labeling and Cleaning Slides

If using ordinary slide labels, which must be glued on with moisture,
processed slides should be cleaned; otherwise the labels will eventually
loosen from the glass. Dip the slides (when mountant is thoroughly hard-
ened) in water to which has been added a small amount of ammonia
and cleanser, such as Bon Ami (glass cleaner may be substituted). Wipe
dry, and paste on label.
A simpler method for labeling is provided by the Professional Tape
Company, Inc., 355 Burlington, Riverside, Illinois. Their labels are
called “Time Microscopic Slide Labels’? and are applied by _pres-
sure only. They are manufactured in a standard thickness, size |x {
inch, and a so-called “‘tissue-high” thickness, size § & } and 2 & { inch.
The latter is used as an end label protecting the cover glass from stick-
ing to any object laid upon it.
All slide labels should contain complete information, name or num-
ber of tissue, section thickness, stain and date, and possibly fixative.
Mounting can be untidy, and some of the resin may ooze over the
cover glass. If this happens, when completely dry, scrape off excess resin
with a razor blade, taking care not to chip or break the cover. It is not
practical, however, to clean away the resin too close to the cover glass;
in fact, a small band of it may be left to protect the cover from chipping
or being caught against an object.
423
424 General Laboratory Aides (sEc. 2)

Figure 28. A cabinet of drawers designed for use in microtechnic classes at

the University of California at Los Angeles. The cabinets are not a
part of the working table, but are arranged along a wall of the class-
room. The drawers are designed to hold coplin jars, mountant jars,
and similar items. The student can carry the drawer to the work
table, turn it around with back-side facing forward, remove the back
panel, and carry on the staining right in the drawer. This relieves
work table of staining equipment when the space is required for
other purposes, and the student can carry all the staining jars in one
“basket” rather than trot back and forth several times with only as
many jars as his hands will hold. If locking the drawers is unneces-
sary, the front panel can be removable; this eliminates having to
turn the drawer around. Locking a removable front panel has been
found impractical. Drawers may be left out of the upper compart-
ment, or upper two compartments, to make convenient storage
nooks for student books, lunches, sweaters, etc. These cabinets are
custom made. Standard cabinets can be adapted to this purpose, but
do not have a removable panel. [Kewanee Manufacturing Company,
Adrian, Michigan (Technical Furniture Inc., Statesville, North
Carolina), Unit #P3, drawers: inside dimensions, 201544” length,
514” height and 1214” wide.]
Two Different Stains on One Slide 425

Restaining Faded Slides

McCormick (1959A) Method
Slides on long standing or if exposed to bright light frequently will
fade. If they must be recovered, McCormick offers the following method
to bleach them for restaining.
PROCEDURE:
1. Remove cover glass by soaking slides in xylene until cover glass
slips off. Do not force it off; the sections might get torn.
2. Soak longer in xylene to make certain that all of the resin is re-
moved.
3. Hydrate to water.
4. Treat with 0.59% potassium permanganate (0.5 gm./100 ml. wa-
ter): 5 minutes.
5. Wash in running water: 5 minutes.
6. Bleach with 0.5% oxalic acid (0.5 gm./100 ml. water) until color-
less: 1-2 minutes. If old stain still remains, repeat steps 4, 5 and 6.
7. Wash thoroughly in running water: 5 or more minutes.
8. Restain, using more-dilute stains or for a shorter time. Potassium
permanganate and oxalic acid make tissues especially sensitive to
hematoxylin and aniline nuclear stains.

Two Different Stains on One Slide

Feder and Sidman (1957) Method
This method can be used advantageously when it expedites a quick
checking of a tissue against two stains, side by side.
PROCEDURE:
1. Hydrate sections to water.
2. Blot carefully and while still moist coat alternate sections with
silicone grease. A soft brush may be used, or more efficiently use
a 1 ml. syringe and #15 needle clipped off and flattened.
aa Apply first staining procedure.
4. Dehydrate and leave in xylene 15-30 minutes to remove coating.
(After 5 minutes in xylene, blotting with filter paper helps to re-
move grease)
5. Rehydrate; apply coating to stained sections.
6. Apply second staining procedure.
426 General Laboratory Aids (sEc. 2)

7. Repeat step 4.
8. Rinse briefly in absolute alcohol, and clear in fresh xylene. Mount.

Reclaiming and Storing Specimens

Graves (1943) Method
If biopsy material has dried, do not try to process it before first soften-
ing and rehydrating it. Place it in physiological saline solution for 1]
hour, then fix, dehydrate, clear, and embed as usual.

Dried Gross Specimens!

‘Tissues which have been stored in alcohol or formalin often become
completely or partially desiccated. If a major catastrophe arises and tis-
sue must be reclaimed, partial recovery can be undertaken with fair
returns for microscopic identification. Van Cleave and Ross (1947) re-
stored desiccated helminths and invertebrates to normal size by soaking
them in a 0.5% aqueous solution of trisodium phosphate. Trial runs of
this nature were made on dried formalin-fixed specimens for from 4
hours to 30 days, depending on hardness and size of tissue. Occasional
changing of the fluid seemed to help in cases of exceptionally dry pieces,
also warming in a 40°C oven. Increasing the concentration of the
phosphate did not speed recovery. Finally the tissues were washed for
from 30 minutes to 2-3 hours, again depending on size.
If the specimen was fixed in a mercuric chloride fixative, the triso-
dium phosphate was found to be ineffective until the tissue had been
pretreated as follows: (1) soaked in water with enough Lugol’s solution
added to color the solution a deep brown: 1-2 days (if the solution be-
came colorless, it was renewed); (2) washed in running waters: 2 hours;
(3) treated with 5% sodium thiosulfate: 3-1 hour; (4) washed: 2 hours;
(5) treated with 0.5% trisodium phosphate until soft.
Only fair results were obtained by the above methods. Considerable
shrinkage remained in the cells, and the nuclei stained lightly. The
latter condition improved somewhat after mordanting in mercuric chlo-
ride or potassium dichromate.
Since recovery was based on the detergent action of trisodium phos-
phate, the inevitable question occurred, why not try one of the modern
household detergents? A 1% solution of Trend in water was added to
dried tissues and kept overnight in a paraffin oven at approximately
1 Special acknowledgment is made to Robert Ingersoll Howes, Jr., for hours of effort in
developing a recovery process for the Los Alamos Medical Center, summer of 1959.
Preserving Gross Specimens in a Pliable Condition a4

58°C. This was followed by 6-8 hours washing and then processing
overnight in the Technicon for embedding the next morning. The re-
sults were creditable, good enough for tissue identification and some
pathological reading. The tissues in this run averaged 10—25 cm. in size
and were fixed in either formalin or a mercuric chloride fixative. A
longer stay in detergent might be required for larger pieces. No pre-
tense is being made that results are exceptional: considerable shrinkage
remains in the cells, and staining is not brilliant, but it is better than
after trisodium phosphate. If, however, it is essential to check back on a
tissue concerning its identity, a malignancy, or some other “‘matter of
life or death,” the author suggests trying this method of recovery of
otherwise irretrievable tissue. It might save the day.

Storing Gross Specimens

Sealing tissues for storage has been a serious problem. Bottled storage
has risks; any seal can spring a leak, and resulting evaporation culmi-
nates in desiccated tissue. Storing in plastic bags can be a more reliable
system than the use of bottles. A heavy quality of polyethylene plastic
is recommended. Lighter grades can split or unseal, whereas the heavy
grade, once well sealed, almost supports itself without collapsing. ‘The
plastic can be cut to any size and sealed on all four edges, if necessary,
with the “Pack Rite Poly Jaw Sealer’ of Pack-Rite Machines, 407
E. Michigan St., Milwaukee, Wisconsin. A small amount of formalin
included in the bag will keep the tissue moist so long as the bag remains
sealed. A quantity of small containers can be sealed together in a large
bag, affording additional protection against drying. Plastic tags with
data can be enclosed or attached to the outside. Storing in bags also
saves storage space, preservative and containers.”
See also: Lieb (1959); Gordon (1953); and Broadway and Koelle (1960).

Preserving Gross Specimens in a Pliable Condition

Palkowski (1960) Method
Sometimes it is economical to preserve gross specimens, pathological
ones in particular, for future teaching or demonstration purposes. Fix
as soon as possible in modified Kaiserling Solution: 3-4 hours.
* Polyethylene plastic bags may be purchased from a number of companies, in addition
to the one listed here: Falcon Plastics Company, 6020 W. Washington Blvd., Culver City,
California. Available are 9 sizes of bags, from 4” x 6” to 10” X 15” in 2 thicknesses—0.002
in. and 0.004 in.; units of 100 in a package, 500 to 200 in a case, depending on size of bag.
428 General Laboratory Aids (sEc. 2)

chlorate yhyanate:) ).% .. nhl Ree ees 5 300.0 gm

potassiumpsmlfates |: .... 51: Alen eee! 2s 6.0 gm.
potassiumiyinitmake 4:0... 2). Sa «3 114.0 gm
sodium Sulfategeeyes: <::.).+:ih 2 ee: Ze 54.0 gm.
sodium) chiegidemns.7:: .:. >. cake ek 3 66.0 gm
sodium bicarbonate +...) :.!s..1 He See ees 60.0 gm.
formaliny Fyre tees i: Sey hie BO ee 300.0 ml.
distilledsweatensires 2)... 22 . sri, 2) eee meee te a8 10,000.0 ml.
Because of contained air, lungs will float on the fixative. Submerge
them under cotton soaked with fixative.
Drain off excess Kaiserling and place specimens in four times their
volume of 80% alcohol: 18—24 hours.
Deep freeze at —29°C=, sealed in air-tight polyethylene bags not
much larger than the specimen.

The specimens will keep indefinitely this way, retain their color, and
become pliable after thawing. After use, they can be returned to deep
freeze with little deterioration.

Removing Laboratory Stains from Hands

and Glassware
STAIN TREATMENT

basic fuchsin—difficult to remove; try strong acetic acid in 95% alcohol,

or dilute HCl.
carmine—strong ammonia water or weak HC]; if stain resists, use them
alternately.
chromic acid—dilute sulfurous acid in water, or concentrated sodium
thiosulfate and a few drops of sulfuric acid added.
fast green and other similar acid stains—ammonia water.
hematoxylin—weak acid or lemon juice.
hemoglobin—fresh stains: lukewarm to cool water (never hot); older
stains: soften with borax solution, dilute ammonia or tincture of
green soap, finally treat with 2% aqueous oxalic acid.
zodine—sodium thiosulfate. .
iron alum stains on glassware—(1) strong NaOH (sticks) in water, fol-
lowed by (2) strong HCl.
methylene blue—acid alcohol or tincture of green soap.
most dyes—tincture of green soap.
osmic acid on glassware—3% H2Oz (Carr and Bacsich, 1958).
Removing Laboratory Stains from Hands and Glassware 429

picric acid—lithium iodide or carbonate, aqueous.

potassium permanganate—dilute sulfurous acid, HCI, oxalic acid or hy:
posulfite.
safranin and gentian violet—difficult to remove; try acid alcohol.
silver—Lugol’s or tincture of iodine, followed by sodium thiosulfate.
Verhoeff's—4% aqueous citric acid (Hull and Wegner, 1952).

Teaching Films
Motion pictures can be of considerable value to instructors of labora-
tory methods. See especially: “Pathology Film Reviews” in Laboratory
Investigation (Vol. 9, No. 5, Part II, September-October, 1960). This
lists motion pictures for education, research and recruitment, reviewing
the content of the films, type of audience, where distributed or loaned.
Samples are: “Autopsy Technique,” “Compound Microscope,” ““Cyto-
technologist’s Role,” and “Career: Medical Technologist.”
References

ACKERMAN, G. ApoLpH. “A combined al- aze of tissue.” Stain Technology, 34

kaline phosphatase-PAS staining method.” (1959), 9-13.
Stain Technology, 33 (1958), 269-271. ARENSBURGEFR, KONSTANTIN E. and MARKELL,
ADAMSTONE, F. B. and Taytor, A. B. “The Epwarp k. “A simple combination direct
rapid preparation of frozen tissue sec- smear and fecal concentrate for perma-
tions.” Stain Technology, 23 (1948), 109. nent stained preparations.” American
AbEY, W. R., RUDOLPH, ALICE F., HINE, I. F. Journal of Clinical Pathology, 34 (1960),
and Harritt, NAncy J. “Glees staining 50-51.
of monkey hypothalamus: a critical ap- ARMSTRONG, J. A. “Histochemical differ-
praisal of normal and experimental mate- entiation of nucleic acids by means of
rial.” Journal of Anatomy, 92 (1958), 219- induced fluorescence.” Experimental Cell
235. Research, 11 (1956), 640-643.
AGRELL, IvArR P. S. “Whole mounts of ARNOLD, JAMES S. and JEE, WEBSTER S. S.
small embryos attached directly to glass “Preparing bone sections for radioauto.-
slides.” Stain Technology, 33 (1958), 265- raphy.” Stain Technology, 29 (1954 A),
267. 4S -On.
ALBRECHT, Mitprep. “Mounting frozen sec- and “Embedding and_ sec-
tions with gelatin.” Stain Technology, tioning undecalcified bone and its appli-
29 (1954), 89-90. cation to radioautography.” Stain Tech-
—-—. “A simple mechanical aid in stain- nology, 29 (1954 B), 225-239.
ing frozen sections in quantity.” Slain ARNOLD, ZACH M. “A rapid method for
Technology, 31 (1956), 231-233. concentrating small organisms for sec-
ALLEN, ANTON M. ‘““Two methods for color- tioning.” Stain Technology, 27 (1952),
ing mast cells of mammalian tissues.” 199-200.
American Journal of Clinical Pathology, ArzAC, J. P. “A simple histochemical re-
33 (1960), 461-469. action for aldehydes.” Stain Technology,
AMERICAN PuBLIc HEALTH ASSOCIATION. 29, (1950), 187-194:
Diagnostic Procedures for Virus and Rick- ATKINSON, WILLIAM B. “Studies on the
ettsial Diseases. Publication Office, 1790 preparation and recoloration of fuchsin
Broadway, N.Y. (1956). sulfurous acid.” Stain Technology, 27
AMMERMAN, FRANK. “A chrome-alum prep- (1952), 153-160.
aration for delicate and difficult fixa- AusTIn, A. P. “Iron alum aceto-carmine
tions.” Stain Technology, 25 (1950), 197- staining for chromosomes and other ana-
199. tomical features of Rhodophyceae.” Stain
ANDERSON, F. D. “Dichromate-chlorate per- Technology, 34 (1959), 69-75.
fusion prior to staining degeneration in
brain and spinal cord.” Stain Technol- Baker, J. R. “A new method for mito-
ogy, 34 (1959), 65-67. chondria.” Nature, 30 (1932), 134.
ANTHONY, ADAM and CLATER, MERLIN. ‘‘Al- . “The structure and chemical com-
cohol-xylene versus Dioxane in the shrink- position of the Golgi clement.” Quarterly

431
432 References

Journal of Microscopical Science, 85 after electron microscopy.” Stain Tech-

(1944), 1-72. nology, 34 (1959), 348-349.
Cytological Technique. Methuen’s . “Carbon coated grids for electron
Monographs on Biological Subjects. Lon- microscopy.” Stain Technology, 35 (1960),
don, Methuen and Company, Ltd. (1945). 287-289.
. Principles of Biological Technique. BAYLEY, JOHN. “ ‘Cracking’ and ‘chatter’
London, Methuen and Company, Ltd., or, in paraffin sections.” Journal Pathology
New York, John Wiley and Co. (1958). and Bacteriology, 70 (1955), 548-549.
BANGLE, RAYMOND. “Gomori’s paraldehyde BEAMER, PARKER R. and FIRMINGER, HAR-
fuchsin stain.” Journal of Histochemistry LAN I. “Improved methods for demon-
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 Index

Abbe condenser, 90 Alizarine red S method: for calcium, 237;

Abbreviations, 407 for embryos, 172
Abopon mounting medium, 122 Alkaline alcohol, formula, 410
Accelerators, 109 Alkaline phosphatase, 341
Accentuators, 109 Altmann’s fixative, 16
Acetate-acetic acid buffer, Walpole’s, 415 Altmann’s method for mitochondria, 263;
Acetate buffer, Gomori’s, 414 Altmann-Benda method, 264
Acetic acid: in fixatives, 5; N solution, 408; Alum cochineal, 105
rinse, 147 Aminopetidase, 345
Acetocarmine or aceto-orcein methods: for Ammerman’s fixative, 16
chromosomes, 367; permanent mounts, Ammoniacal silver solutions, 176
369; restoration of deteriorated slides, Ammonium hydroxide N solutions, 408
370; solutions, 367; temporary mounts, Amoebae, fixing and staining, 384; parasitic:
368 section technic, 390; smears, 386
Acetone: in fixing solutions, 6; for enzymes, Amphibian larvae, fixing, 379
339 Amyloid: definition, 146; metachromatic
Achromatic objective, 89 methods, 278; staining of, 274
Acid alcohol, formula, 410 Anesthetizing invertebrates, 371
Acid alizarine blue: in azan stain, 151; in Aniline alcohol, 149
quad stain, 161 Aniline blue: counterstain, 130, 231; Ko-
Acid-fast staining of bacteria: discussion, neff’s, 160; Mallory’s, 147, 149; Masson’s,
304; methods, 312 153
Acid fuchsin: counterstain, 129, 231; in Aniline dyes, description, 109
Mallory’s triple connective tissue stain, Aniline-fuchsin, Altmann’s, 263
147; in Masson stain, 152, 154; in Penta- Aniline-xylol, see Carbol-xylol
chrome stain, 163 Anisotropic, 99
Acid mucopolysaccharides: 269; metachro- Annelides: anesthetizing, 372; fixation, 375
matic methods, 280; staining of, 269 Annular ring diaphragms, 97
Acid N solutions, 408 Antigen-antibodies, Coon’s fluorescent tech-
Acidophilic, definition, 110 nic, 330
Acid phosphatase, 343 Apathy’s gum syrup, 119
Acid reagents for decalcification, 25 Apochromatic objective, 89
Acridine orange: for fungi, 324; for exfoli- Aqueous mounting media, 118; mounting
ative cytology, 359; for mucin, 274; for technics, 122
neurological tissue, 202 Arachnids: killing and fixing, 376; whole
Acriflavine dihydrochloride, 296 mount preparation, 379
Adhesives: albumen, 412; blood serum, 414; Araldite (epoxy resin), electron microscopy,
gelatine, 412 401
Agar for freezing method, 63 Areolar tissue, 145
Albumen fixative (Mayer’s): formula, 412; Argentaffin reaction: discussion, 247; for
use of, 58 enterochromaffin cells, 248; for uric acid,
Alcian blue method: for acid mucopoly- 247
saccharides, 269; in pentachrome stain, Arginine test, 353
162 Arthropods: killing and fixing, 372, 377;
Alcohol dilution for dehydration and hydra- whole mount preparation, 379
tion, 31 Artifacts, 4
Alcohol for anesthetizing invertebrates, 372 Ascaris, uteri fixation, 379
Aldehyde fuchsin stain, 168; with PAS for Asphyxiation, anesthetizing invertebrates,
pituitary, 287 372
Aldehyde reactions, 266 Astrocyte impregnation, 193
458
Index 459

Auramin staining of tubercle bacilli, 316 Blood smears: thick, 221; thin, 219; recovery
Aurantia counterstain, 264 of old or precipitated smears, 221 :
Automatic tissue processors, 42 Bodian method for nerve fibers and end-
Autoradiography, 395 ings, 203
Auxochrome, 111 Body fluids, 29, 38, 356
Axis cylinders, staining, 201, 214 Bone: formation, 172; decalcified, 170; hand
Azan stain, 149 ground, undecalcified, 171; matrix stain-
Azocarmine, 149 ing, 148
Azo-coupling methods: acid phosphatase, Bone marrow: description, 218; staining,
344; alkaline phosphatase, 342; amino- 227
peptidase, 345; enterochromaffin cells, Borax carmine staining, 382
252; esterase, 347; succinic dehydrogenase, Bordeaux red counterstain, 130
350 Boric acid-Borax buffer, Holmes’, 416
Azure A-Schiff reagent, 302 Bouin-Duboscq fixative, 13
Azure B, with phloxine and methylene blue, Bouin’s fixative, 13
229 Boutons, see Pericellular end-bulbs
Azure II, in Maximow’s stain, 227 Brazilin, source, 106
Azures in polychroming, 110 Brightfield microscopy, 88
Brown and Brenn’s stain for bacteria, 307
Bryozoa: anesthetizing, 372; fixation, 375;
Bacteria: description, 304; acid fast stains, staining, 377
312; capsule staining, 317; fluorescent Buffer ingredients, molecular weights, 414
stain, 316; Gram stains, 317 Buffer solutions, 414
Barbitol buffer, Gomori’s, 416 Butanol, see Butyl alcohol
Barnstead still, 52 Butyl alcohol, use as dehydrant, 31
Basement membranes: definition, 145; stain- Butyl methacrylate for electron microscopy,
ing, 159, 162 402
Basic fuchsin: capsule staining, 317; Mal-
lory’s stain, 147; Negri body staining, 328;
resorcin-fuchsin, 162; Schifl’s reagent, 293, Cajal silver methods, see Ramon y Cajal
294 Calcium: removal, 25; staining, 236
Basophilic, definition, 110 Canada balsam, 116
Basophils (leukocytes), staining, 220 Capsule staining, 317
Bauer Feulgen method for glycogen, 301 Carbohydrates, see Saccharides
Benda’s staining solution, 265 Carbol-crystal violet, 309
Benzene clearing for paraffin method, 33 Carbol-fuchsin, formula and use, 312, 314
Benzidine method for hemoglobin, 238 Carbol-new fuchsin, 313, 315
Berberine, source, 106 Carbol-xylol, formula, 410
Berlese mounting medium, 120 Carbon, identification, 245
Berlin blue method for iron, 233 Carbon dioxide, use of, 62
Best’s carmine for glycogen, 267 Carbowax, see Water soluble waxes
Biebrich scarlet: counterstain, 130; for sex Carmine: for amoebae, 391; borax carmine,
chromatin, 366 382; carmalum, 383; source, 105
Bielschowsky-Foot silver method for reticu- Carmine (Best’s) for glycogen, 267
lum, 179 Carnoy’s fixative, 16
Bielschowsky methods of silver impregna- Carnoy-Lebrun, 17
tion, 176, 201 Cartilage staining, 152, 276, 280
Bile pigment staining, 240, 245 Castaneda’s method for rickettsia, 325
Bilirubin, 232 C.C., Commission Certified, 113
Biliverdin, 232 Cedarwood oil for clearing, 34
Biological Stain Commission, 112 Celestin blue, 230, 284; in Mallory’s stain,
Bioloid mounting resin, 118 149
Biopsies, surgical, freezing method, 63 Cell blocks, 29, 38, 228
Birefringent plates, 98 Celloidin technic, see Nitrocellulose method
Bismarck brown staining of mast cells, 277 Cellosolve, 34
Blocking: freezing method, 63; nitrocellu- Cellulose tape mounting, 394
lose method, 73; paraffin method, 51 Certification label, 113
Blood parasites, staining, 219, 221 Cestodes: fixation, 374; staining, 382, 384
Blood serum adhesive, 414 Champy fixative, 17
460 Index

Champy-Kull modification of Altmann- Coon’s fluorescent technic for antigen-anti-

Benda for mitochondria, 264 bodies, 331
Chelating agents for decalcification, 27 Corals: anesthetizing, 371; fixation, 373
Chick embryos, fixing and staining, 378 Corrosive sublimate, see Mercuric chloride
Chitin staining, 148 Counterstains: definition, 114; listing of, 129
Chloral hydrate for anesthetizing, 372 Cover glasses, microscopic: cleaning, 57; de-
Chloral hydrate silver method: for nerve scription, 56; mounting, 115, 122; ringing,
fibers and endings, 205; for pericellular 123
end-bulbs, 206 Creosote-xylol, see Carbol-xylol
Chloretone for anesthetizing, 372 Cresyl violet for Nissl substance, 195
Chlorinity, definition, 409 Critical illumination, Kohler method, 91
Chloroform: for anesthetizing, 372; for Cryostat cold chamber, 337
clearing, 34; for double embedding, 84 Crystallization of paraffin, 37
Chromaffin material, 250 Crystal violet staining, 307-310; for amyloid,
Chromatization, after fixation, 23 278; capsule staining, 317; trichrome
Chromic acid, see Chromium trioxide method, 158
Chromium hematoxylin phloxine method Cyanol reaction for hemoglobin, 239
for pancreas, 290
Chromium trioxide: in fixatives, 6, 400; with Dafano fixative, 17
Schiff’s reagent, 301, 321 Dafano method for Golgi, 260
Chromogen, 111 Dark field microscopy, 96
Chromophores, 111 Darrow red nuclear stain, 139
Chromosomes: permanent mounts, 369; res- Decalcification: acid reagents, 25; chelating
toration of slides, 370; squash and smear agents, 27; combined with other processes,
technics, 367; temporary mounts, 368 28; electrolysis method, 26; ion-exchange
Chromotrope 2R, in trichrome method, 157, method, 26
158, 159, 389 Decalcification-dehydration, 29
Chrysoidin staining of mast cells, 277 Deformalization, after fixation, 24
Churg and Prado trichrome method, 158 Degenerating axons: Nauta and Gygax
Ciliates, anesthetizing, 372 method, 208; Marchi method, 210
C.1I. number, 113 Dehydrants, 31
Citric acid-citrate decalcification, 25 Dehydration and clearing combined for
Cleaning slides, 423 paraffin method: Dioxane method, 39;
Cleaning solution for glassware, 412 Tetrahydrofuran method, 40
Clearing for paraffin method, 33 Dehydration combined with fixation, 29
Clinical microtome, 43, 64 Dehydration: for embedding, nitrocellulose
CMC-10 mounting medium, 121 method, 71; paraffin method, 30; during
Cocaine for anesthetizing, 372 staining, 114, 131
Cochineal, source, 105 Delafield’s hematoxylin: formula, 124; pro-
Coelenterates: anesthetizing, 372; fixation, gressive method, 130; regressive method,
373 133
Cold chamber (cryostat), 337 Del Rio Hortega methods of silver impreg-
Collagen combined with reticulum and nation, 176; for neurological elements,
elastin staining, 186 194; for reticulum, 180
Collagenic fibers (collagen): definition, 145; Desoxyribonucleic acid, polysaccharides
staining, 148, 150, 152, 153, 155, 159, 160, and protein, triple stain for, 302
162, 164, 165, 167, 187, 231 Dialyzed iron, 271
Collodion, see Nitrocellulose Diamond knives for electron microscopy, 403
Colloidal iron for acid mucopolysaccharides, Diaphane mounting medium, 122
270 Diaphragm: iris, 90; annular ring, 97
Colophonium, 225 Diazo-safranin method for enterochromaffin
Color index number, 113 cells, 250
Colorless tissue, special treatment for em- Dieterle’s method for spirochetes, 317
bedding, 32 Difficulties in sectioning: freezing method,
Commission Certified, 113 64; nitrocellulose method, 76; paraffin
Congo red: counterstain, 130; amyloid stain, method, 54
274 Diluting alcohol for dehydration and hydra-
Connective tissue methods, 145 tion, 31
Controlled illumination, 90 Di-nitrosoresorcinol method for iron, 236
.
Index 461

Dioxane: as dehydrant, 31; double embed- Farrant’s mounting medium, 119

ding, 85; paraffin method, 39; toxicity, 39 Fast green: counterstain, 130; in Feulgen
DNA, desoxyribose nucleic acid, methods stain, 294; Masson stain, 153; for mito-
for, 294, 360 chondria, 262; in Quad stain, 161; for sec
Dopa oxidase reaction, 351 chromatin, 366
Double embedding methods, 83 Fat staining: discussion, 253; methods, 254
Dryers, hot air slide, 60 Ferric-ferricyanide: for enterochromaffin,
Dry ice holder, 62 251; for melanin, 242
Drying resin mounts, 118 Ferrous iron, Turnbull blue method, 235
Dyes, see Stains Feulgen technics: discussion, 292; methods,
293, 301
Earthworms: anesthetizing, 372; fixation, Fibrin: definition, 146; staining, 156, 159,
375 164
Echinodermata, fixing, 377 Field’s method and stain for blood smears,
Egg albumen: formula, 412; use, 58 221
Ehrlich’s hematoxylin, 124 Film mounts: of sections, 392; smears and
Elastin combined with reticulum and colla- tissue cultures, 393
gen staining, 186 Films, teaching, 429
Elastic fibers (elastin): definition, 145; stain- Fite formaldehyde method for bacteria, 314
ing of, 152, 160, 162, 164, 165, 166, 167, Fixation, 3; with decalcification, 28; for
168, 169, 187 enzymes, 339; perfusion, 22; post-fixation,
Electrolysis method for decalcification, 26 23; precautions and preparations, 11;
Electrolytic dissociation of dyes, 111 washing after, 12
Electron microscopy: description, 103, 397; Fixatives (adhesives), 412
fixatives for, 399; knives for, 403; section- Fixing solutions: chemicals used, 5; for elec-
ing, 403 tron microscopy, 399; for enzymes, 339;
Elementary and inclusion bodies, 306 routine solutions, 13; special solutions, 16
Embedding: double methods, 83; electron Flemming’s fixatives, 17
microscopy, 401; for enzymes, 339; gela- Flukes: anesthetizing, 372; fixation, 373;
tine, 66; nitrocellulose, 72; paraffin, 36; staining, 382
polyester resin, 85; water soluble waxes, Fluorescent methods: for amyloid, 275; for
80 antigen-antibodies, 331; apparatus, 102;
Embryos: alizarine red S method, 172; fixing for DNA, 295; for exfoliative cytology,
and staining, 382 359; for fat, 257; for fungi, 323; mounting
Endogenous pigments, 232 media, 122; for mucin, 273; for neurologi-
Enterochromafhin cells, 250; azo-coupling cal tissue, 202; for tubercle bacilli, 316
method, 252; dtazo-safranin method, 250; Fluorochromes, 101; uses for, 103
ferric-ferricyanide method, 251; silver Foreign stains, sources, 113
method, 248 Formaldehyde: pigment, 7, 244; use in fixa-
Enzymes, acetone fixation and embedding tives, 7, 14
of, 339 Formalin, see Formaldehyde
Eosin-azure stain (Maximow’s), 227 Formic acid decalcification, 25; N solutions,
Eosin; counterstaining, 129; eosin-orange, 408
129; Putt’s, 129; with hematoxylin, 131 Formol-alcohol fixative, 18
Eosinophils (leukocytes), 220, 229 Formulas, fixatives, 13
Epoxy embedding, electron microscopy, 401 Freeze-drying for embedding, 336
Erythrocytes, 218, 220, 228 Freeze-substitution, see Ice-solvent method
Erythrosin B counterstain, 130 Freezing method, 62; surgical biopsies, 63;
Esterase and Lipase, 347 rapid staining, 67
Ethanol, see Ethyl alcohol Freon, use of, 62
Ether for anesthetizing invertebrates, 372 Fungi staining, 300, 320
Ethyl alcohol: as dehydrant, 31; fixatives, 6
Euparol, 116 Gallocyanin: formula, 128; for Nissl sub-
Exfoliative cytology, 356; — fluorescent stance, 197; for sex chromatin, 365; stain-
method, 359; Papanicolaou method, 357 ing procedure, 138
Exogenous pigments, 232 Garnet GBC salt: to purify, 346; use, 252,
‘ 345
Faded slides, restaining, 424 Gastropods, anesthetizing, 372
Fanz knife sharpener (A. H. Thomas), 46 Gelatine embedding, freezing method, 66
462 Index

Gelatine fixative (adhesive): formula, 412; Helly’s fixative, 14

use, 65 Hematoidin, 232
Gendre’s fixative, 18, 268 Hemalum, see Hematein
Giemsa method: frozen sections, 69; for Hematein (hemalum): formula, 129; source,
rickettsia, inclusion bodies, and trachoma, 105; staining procedure, 139; for whole
327 mounts, 384
Giemsa stain: Jenner-Giemsa, 225, 325; Wol- Hematologic elements: description, 218;
bach’s modification, 224; Wright-Giemsa, methods, 219
223 Hematoxylin: destaining, 108; discussion,
Gilson’s fixative, 18 105; progressive staining, 130; regressive
Glass knives for electron microscopy, 403 staining, 133; substitutes, 128; testing
Glass marking pencils, 57 solutions, 128
Glass plate for sharpening microtome Hematoxylin solutions: Delafield’s, 124;
knives, 45 Ehrlich’s, 124; Hansen’s Iron, 127;
Globus’ hydrobromic acid, 194 Harris’, 125; Heidenhain’s, 127; Jans-
Glycerol jelly, Kaiser’s; formula, 119; for sen’s, 126; Mallory’s Iron Chloride, 128;
whole mounts, 381 Mayer’s, 125; Papamiltiades’, 125; Phos-
Glycogen: staining, 267, 301; removal, 300 photungstic acid, 125; Weigert’s Iron, 126
Glynn’s method for bacteria, 309 Hemoglobin, methods for staining, 238
Gold chloride stock solution formula, 410 Hemosiderin: description, 232; identifica-
Golgi apparatus, 259 tion, 245
Golgi method of silver impregnation, 198 Hermann’s fixative, 19
Gomori’s buffers: acetate, 414; barbitol, Hiss’ method for capsule staining, 317
416; phosphate, 417; tris maleate, 420 Histochemistry, definition and discussion,
Gomori’s: chromium hematoxylin phloxine, 335
291; iron reaction, 233; methenamine sil- Hollande Bouin’s fixative, 15
ver nitrate for fungi, 322; trichrome Holmes’ boric acid-borax buffer, 416
method, 156 Hones, care and use, 46
Goodpasture’s fuchsin, 311 Hookworms, fixation, 374
Gram staining of bacteria: discussion, 304; Hortega silver methods, see Del Rio Hor-
methods, 306 tega
Gram Weigert method, 307 Hot air slide dryers, 60
Gray and Wess’ mounting medium, 120 Hotchkiss-McManus method for fungi, 320
Grenacher’s borax carmine staining, 382 Hot nitrocellulose method, 75
Gridley’s: method for fungi, 321; silver Hyalin(e): definition, 146; staining of, 148,
method for reticulum, 181 156, 159, 275, 280
Groat’s variation of Weigert’s hematoxylin, Hydra: anesthetizing, 372; fixation, 373;
126 staining, 382
Gross specimens: reclaiming and_ storing, Hydration during staining, 114, 130
426; preserving in pliable form, 427 Hydrobromic acid, 194
Ground substance: definition, 145; staining Hydrochloric N solutions, 408
of, 152, 164 Hyrax mounting medium, 118
Gum damar, 116 Hypertonic solution, definition, 409
Gum mastic, 317 Hypotonic solution, definition, 409
Gum sandarac, 116
Gum syrup, Apathy’s, 119 Ice-solvent method, 337
Illumination, for microscope: controlled,
Hale’s tris buffer, 419 90; critical (Kohler), 91
Halmi’s mixture, 289 Impregnation with silver: discussion, 175;
Hansen’s iron trioxyhaematin, formula, 127 loose sections, 60; neurological methods,
Harris’ hematoxylin: formula, 125; pro- 189; reticulum methods, 179
gressive method, 130; regressive method, Inclusion body staining, 225, 326
133 Indifferent salts, 10
Haupt’s gelatine fixative, 413 Indigo, source, 106
Heidelberger’s Victoria blue, 191 Infiltration: electron microscopy, 402; paraf-
Heidenhain’s (Mallory) azan stain, 149 fin, 35; polyester resin, 86; nitrocellulose,
Heidenhain’s hematoxylin: destaining 71; water soluble waxes, 81
methods, 135; formula, 127; staining pro- Insects: killing and fixing, 372, 376; whole
cedure, 134 mount preparation, 379
Index 463

Interference and phase microscopy, 97 Light green counterstain, 130, 286, 321, 323,
Intestinal protozoa: sections, 390; smears, 389
387 Lillie’s: quick method for bacteria, 310;
Invertebrates: anesthetizing and narcotizing silver method for reticulum, 183; vari-
agents for, 371; special handling, 372; ation of Orth’s, 15; variation of Weigert’s
staining, 377; whole mounts, 579 hematoxylin, 126
Ion-exchange-resin method for decalcifica- Lipase and esterase, 347
tion, 26 Lipid (lipoid) staining, sce Fat staining
Iris diaphragm, 90 Lipofuscins: description, 232; methods for,
Iron: masked (occult), definition, 232; meth- 241
ods for, 233 Lison’s ‘““Gendre fluid,” 268
Iron chloride hematoxylin, Mallory’s; for- Litmus, source, 106
mula, 128; staining procedure, 138 Locke’s solution, 411
Iron hematoxylin: Groat’s, 126, 135; Han- Loeffler’s methylene blue solution, 317
sen’s, 127; Heidenhain’s, 127, 134; Jans- Loose sections, 60
sen’s, 126; Mallory’s, 128, 138; Weigert’s, Low viscosity nitrocellulose, 70
126, 137 Lugol’s solution: formulas, 410; use, 131
Isopentane: use for freeze drying, 336; use Lustron 2020, 118, 123, 233
for ice-solvent method, 337 Luxol fast blue: with Holmes silver nitrate,
Isopropyl! alcohol, use as dehydrant, 31 214; with P.A.S., 212
Isotonic solution, definition, 409 Lymphocytes, staining, 220
Isotropic, 99

Janssen’s hematoxylin, 126 MacCallum-Goodpasture method for bac-

Jellyfish, fixation, 373
teria, 311
Maceration solutions, 10
Jenkin’s fluid, 29
Jenner-Giemsa stain, 225, 324 Mahon’s method for myelin, 217
Johnson’s fixative, 19 Malachite green counterstain, 314
Malaria staining, 219, 221, 225
Malarial pigment removal, 244
Kaiser’s glycerol jelly: formula, 119; use, 381 Mallory Heidenhain’s Azan stain, 149
Karo mounting medium, 121 Mallory’s iron chloride hematoxylin: for-
Keratin: definition, 146; staining, 153 mula, 128; staining procedure, 138
Kinyoun’s carbol fuchsin method for bac- Mallory’s triple connective tissue stain, 146
teria, 314 Mammalian embryos, fixing, 379
Kleermount, 118 Marchi method for degenerating axons, 210
Knife sharpeners, mechanical, 46, 49 Masked iron, 232
Knives, microtome, 44; cleaning and sharp- Massignani’s method for Negri bodies, 329
ening, 45; use of, 52 Masson's gelatine fixative, 412
Kohler method of illumination, 91 Masson trichrome stain, 152
Kolmer’s fixative, 19 Mast cells: description, 146; metachromatic
Koneff’s aniline blue stain, 160 methods, 281; staining, 276
Kopsch fixative, see Regaud Mayer’s albumen fixative: formula, 412;
Kornhauser’s quad stain, 16] use, 58
Kristensen’s decalcifying fluid, 26 Mayer’s carmalum staining, 383
Mayer’s hematoxylin: formula, 125; stain-
Labelling slides, 423 ing procedure, 133
Laboratory stains, removal, 428 Mayer’s mucicarmine for mucin, 273
Lactophenol mounting medium, 121 May-Grunwald, see Jenner-Giemsa
Laidlow’s silver method for reticulum, 182 Maximow’s eosin-azure method, 227
Lake, 107 Mcllvaine’s standard buffer, 418
Laminated thermoplastic mounts, 394 Measuring devices for microscope, 94
Lavdowsky fixative, 19 Medusae: fixation, 373; staining, 377
Lawless rapid method for intestinal pro- Melanin: definition, 232; methods for, 241,
tozoa, 389 250; removal of, 243
Leukocytes: fluorescent method, 360; Membranes, preparation, 29
staining, 220 Menthol for anesthetizing, 372
Levaditi method for spirochetes, 320 Mercuric chloride: use in fixatives, 8; pre-
Liesegang silver impregnation methods, 176 cipitate elimination, 9, 12
464 Index

Metachromasia: acid mucopolysaccharides, Mounting media formulas: Apathy’s gum

280; amyloid, 278; discussion, 278; mast syrup, 119; Berlese, 120; Farrant’s, 119;
cells, 281; mucin, 279 fluorescent, 122; Gray and Wess’, 120;
Metachroming, 110 histochemical, 355; Kaiser’s glycerol jelly,
Metanil yellow counterstain, 322 119; Karo medium, 121; lactophenol, 121;
Methacrylate embedding, electron micros- polyvinyl alcohol (PVA), 120; Yetwin’s
copy, 402 for nematodes and ova, 121
Methenamine silver nitrate for: entero- Mounting sections: freezing method, 65;
chromaffin cells, 248; fungi, 322; uric acid, nitrocellulose method, 77; paraffin
248 method,51
Methyl benzoate, use in double embedding, Movat’s pentachrome stain, 162
83 Mucicarmine (Mayer’s) for mucin, 273
Methylene blue: with acid fast stains, 312; Mucin: metachromatic methods, 279; re-
Loeffler’s, 317; for Negri bodies, 328; with moval of, 300; staining, 273
phloxine, 229; for Rickettsia, 326 Mucopolysaccharide (acid): staining, 269;
Methyl green: counterstain, 263; for nu- metachromatic methods, 280
cleic acids, 297 Mucus staining, 148, 152, 153, 155, 156, 360
Methyl salicylate for clearing, 34 Muscle staining, 148, 150, 153, 155, 156, 159,
Microfilaria, fixation and staining, 375 160, 162, 280
Microglia: silver impregnation, 194; stain- Mussels, preparation for fixing, 376
ing, 191 Myelin staining, 212, 241; Luxol fast blue-
Micrometers, ocular and stage, 97 Holmes silver, 214; Luxol fast blue-PAS,
Microorganisms, discussion of, 304 212; Mahon method, 217; Ora’s method,
Microscope: compound, 88; dark field, 96; 215
electron, 103; fluorescent, 101; measuring
devices, 94; operation, 91; phase and in- Naphthalene yellow for fat, gross specimens,
terference, 97; polarizing, 98; ultraviolet, 257
100; X-ray, 99 Naphthol yellow S, 302
Microscope lamps, 90 Narcotizing invertebrates, 371
Microscopic cover glasses: cleaning, 57; de- Nassar and Shanklin’s silver impregnation
scription, 56; double cover glass mount- for neuroglia, 192
ing, 122; single glass mounting, 115 Natural dyes, 105
Microscopic slides: cleaning, 57; descrip- Nature of staining action, 111
tion, 56; labelling, 423; see also Mounting Nauta and Gygax method: degenerating
Microscopy: brightfield, 88; dark field, 96; axons, 208; pericellular end bulbs, 207
electron, 103, 403: fluorescent, 101; inter- Navashin’s fixative, 20
ference, 97; phase, 97; polarizing, 98; Negri body staining, 328
ultraviolet, 100; X-ray, 99 Nemathelminthes, fixation, 374
Microtome knives, 44 Nematodes: fixation, 374; mounting me-
Microtomes: kinds, 43, 62; use of in freezing dium for, 375
method, 62; nitrocellulose method, 74; Nemertinea, fixation, 374
paraffin method, 52. Nerve cells, fibers and endings: Bielschow-
Millon reaction for protein, 246 sky method, 201; Bodian method, 203;
Mitochondria: discussion, 259; methods chloral hydrate silver method, 205;
for, 262 Golgi’s rapid method, 198; Ramon _ y
Molds for paraffin embedding, 36 Cajal’s pyridine silver method, 200
Molecular solutions, definition, 407 Neurofibrils, Ramon y Cajal’s method, 197
Molecular weights of buffer ingredients, 414 Neurological elements, staining: astrocytes,
Molluscs: anesthetizing, 372; fixing, 376
193; degenerating axons, 208; fluorescent
method, 202; motor end plates, 211;
Monocytes, staining, 220
myelin, 212; nerve cells and fibers, 198;
Mordants and mordanting: discussion, 107;
fibers and endings, 203; neurofibrils, 197;
destaining with, 108, use, 107
neuroglia, 192; Nissl substance, 195;
Motor end plates, Cole method, 211
nomenclature, 189; oligodendroglia and
Mountants, See Mounting media microglia, 194; preparation for staining,
Mounting a cover glass, 115; aqueous 190; silver methods, 192; victoria blue
mounts, 122; difficulties, 116; two cover staining, 191
glass method, 123 Neutral red staining of mast cells, 276
Mounting media, 116; quick drying of, 118 Neutral stains, 110
Index 465

Neutrophils (leukocytes), staining, 220 Paraffin: discussion, 35; crystallization, 37;

New fuchsin: with orcinol, elastin stain, infiltration and embedding, 35
167; carbol-new fuchsin, 313, 315 Paraffin method: clearing, 34; dehydration,
Nile blue A: for fats and fatty acids, 258; 30; Dioxane with, 39; double embedding,
for melanin and lipofuscin, 241 83; infiltrating and embedding, 35;
Ninhydrin reaction for protein, 247 mounting slides, 57; sectioning, 52; Tet-
Niss] substance methods: cresyl violet, 195; rahydrofuran with, 40; timing schedule,
gallocyanin, 197; thionin, 196 41; tissue processors, 42; trimming and
Nitric acid: decalcification, 25; N solutions, blocking, 51
408 Paraffin sections: detaching, 60; difficulties
Nitrocellulose, for loose paraffin sections, 60 in sectioning, 54, 58; mounting, 51; static
Nitrocellulose method, 70; difficulties in elimination, 54; storing, 54
sectioning, 76; double embedding, 83; Paramecium, fixing and staining, 384
hot celloidin method, 75; serial sections, Parasites: description, 386; intestinal pro-
78; staining and mounting, 76 tozoa, smear technics, 386; section technic,
Nitrogen, liquid, use for freeze-drying, 336 390
Normal solutions: definition, 407; dilutions Parenchyma, definition, 145
for acids, 408 Parfocal, 92
Nuclear staining, red, 139 Parloidin, see Nitrocellulose
Nucleic acids, 293 PAS, see Periodic acid Schiff
Nudibranchs, anesthetizing, 371 Pencils, glass marking, 57
Penfield’s modification of del Rio Hortega’s
Obelia, staining, 377 silver impregnation, 194
Objectives of microscope, 89 Pentachrome stain, Movat’s, 162
Occult iron, see Masked iron Peracetic acid, 243
Ocular micrometer, 95 Percentage solutions, definition, 408
Ocular, pointer for, 94 Perenyl’s fixative, 20
Oil immersion objective, use and care, 93 Performic acid, 243
Oil of Wintergreen, see Methyl salicylate Perfusion, fixation by, 22
Oil red O for fat, 254 Pericellular end-bulbs (boutons): chloral
Oils for clearing, 34 hydrate-silver method, 206; Nauta and
Oligodendroglia silver impregnation, 194 Gygax method, 207
Omentum, see Subcutaneous tissue Periodic acid Schiff: discussion, 292; for
Orange G: counterstain, 130, 230, 285, 287; fungi, 300; for parasites, 386; methods,
in Mallory’s stain, 147; in Quad stain, 161 298
Ora’s method for myelin, 215 Periodic acid solutions, 296, 299, 300, 302
Orcein: stain for elastin, 161, 169; source Perl’s reaction, see Berlin blue
of, 106 Permount, 116
Orcinol-New fuchsin elastin stain, 167 Phase and interference microscopy, 97
Ordway-Machiavello method for rickettsia Phenol new fuchsin, formula and use, 313,
and inclusion bodies, 326 315
Orthochromatic action, 111 Phloxine B counterstain, 130, 291
Orth’s fixative, 15; Lillie’s variation, 15 Phloxine-Methylene blue stain, 229
Osmic acid staining, see Osmium tetroxide Phosphate buffer: Gomori’s, 417; Sorenson’s,
Osmium tetroxide in fixatives, 8, 399 417
Osmium tetroxide staining: of fat, 256; of Phosphine 3 R for fat staining, 257
Golgi apparatus, 259; of mitochondria, Phospholipids, Sudan black method, 258
266 Phosphomolybdic acid: in Mallory type
Ova, mounting medium for, 375 stain, 147; in Masson stain, 152, 154; in
Oxidation of hematoxylin, 105 Quad stain, 161
Phosphotungstic acid: in Mallory type stain,
Pancreas staining, 290 147, 150; in Masson stain, 152; in Penta-
Papamiltiades hematoxylin, formula, 125 chrome stain, 163; in Quad stain, 161
Papanicolaou method for exfoliative cy- Phosphotungstic acid hematoxylin: for-
tology, 357 mula, 125; staining procedure, 136
Paper mold for paraffin embedding, 37 Photoxylin, see Nitrocellulose
Pappenheim stain for rickettsia, 324 Physiological solutions: definitions, 409;
Paraffin blocks: hardening, 36; sectioning, formulas, 410
52; storing, 38; trimming-mounting, 51 Piccolyte, 116
466 Index

Picric acid: in fixatives, 9; destaining with, Protozoa, intestinal: smear technics, 386;
134; staining with, 165, 166, 230 section technic, 390
Picro-Gomori trichrome method, 159 Prussian blue, see Berlin blue
Picro-ponceau staining (Van Gieson), 165, Putt’s eosin, 129
166 PVA (polyvinyl alcohol) mounting medium,
Pigments: description, 232; removal of, 243 120, 355
Pike oil, 44 Pyridine silver impregnation, 200
Pinacyanole staining: freezing method, 67; Pyronin methyl green for nucleic acids, 297
sex chromatin, 362 Pyroxylin, see Nitrocellulose
Pituitary: discussion, 283; methods for, 284
Planaria, fixation, 373 Quad stain, Kornhauser’s, 161
Plasma cells, definition, 146 Quartz microscope accessories, 100
Plastic embedding, see Polyester resin Quenching, 338
method
Plastic mounts, 394 Radioautography, 395
Platelets, staining, 220 Ramon y Cajal’s: gold chloride for astro-
Platyhelminths: fixation, 373; staining, 377, cytes, 193; for neurofibrils, 197; pyridine
382, 384 silver, 200; silver impregnation methods,
Pointer for eyepiece, 94 176
Polarizing microscope, 98 Rapid method, Lawless, for intestinal pro-
Pollak’s rapid trichrome stain, 155 tozoa, 389
Polychroming, 110 Rapid staining method, biopsies, 67
Polyester resin embedding method, 85 Rays, ordinary and extraordinary, 99
Polyethylene glycol, see Water soluble Razor blade holder, Spencer (American
waxes Optical Company), 49
Poirrier’s blue, formula and use, 326 Reclaiming stored gross specimens, 426
Polymorphs, see Leukocytes Red blood cells, see Erythrocytes
Polysaccharides, DNA and proteins triple Refractive index, 88
stain, 302 Regaud’s fixative, 20
Polyvinyl alcohol (PVA) mounting medium, Regressive staining, 108
120 Removal of pigments, 243
Ponceau de xylidine, in Masson stain, 152, Removing laboratory stains, 428
154 Resin mounts: drying, 118; mounting cover
Ponceau 2R, in trichrome stain, 155, 286 glass, 115, 122
Ponceau S, staining, 165, 166 Resins, synthetic, 116
Porifera, fixation, 372 Resorcin-fuchsin: Weigert-Hart’s, 162; in
Post fixation treatments: chromatization, pentachrome stain, 162
23; decalcification, 24; deformalization, Restoration of slides: chromosome prepara-
24; mercuric chloride removal, 9, 12, 24; tions, 370; faded tissue slides, 424; blood
washing, 12 smears, 221
Potassium dichromate: chromatization, 23; Reticular fibers (reticulum): definition,
in fixatives, 9 145; silver impregnation, 179; staining,
Potassium: ferricyanide, 235; ferrocyanide, 150, 162, 165
233, Zoe Reticulum, combined with collagen and
Potassium permanganate for electron mi- elastin staining, 186
croscopy, 400 Rhodacyan method for pituitary, 289
Precipitate, mercuric chloride elimination,
Rickettsia staining, 324
Ringer’s solution, 411
oy U4
Ringing cements, 123
Progressive staining, 108
Ringing cover glasses, 123
Propane-isopentane, in ice-solvent method,
Rio Hortega silver methods, see Del Rio
337 Hortega
Propylene glycol, use in ice-solvent method, RNA, ribose nucleic acid: methods for, 297,
337 360; removal, 300 ;
Protargol, 203 Romanowsky stains, 110
Protein staining, 246; with DNA and poly- Rosin (Colophonium) solution, 225
saccharides, 302 Rossman’s fixative, 20
Protozoa: fixation, 384; cover glass mount- Rotary microtome, 43, 52
ing, 385; staining, 385 Rotifers, anesthetizing, 372
Index 467

Saccharides, description, 266 of, 111; orthochroming, 111; polychrom-

Saffron: in pentachrome stain, 163; source, ing, 110; progressive, 108, 130; regres-
106 sive, 108, 133; triple, 110
Safranin counterstain, 233 Staining procedures: freezing method, 67;
Salinity, definition, 409 general principles, 114; nitrocellulose, 76;
Sanfelice fixative, 21 paraffin method, 130
Scarba red nuclear stain, 140 Stain removal: from hands and glassware,
Schaudinn’s fixative, 21; PVA modification, 428; from slides, 424
389 Stains: C.J. number, 113; discussion, 104;
Schedule for timing paraffin method, 41 natural, 105; neutral, 110; nomenclature,
Schleifstein method, 328 111; Romanowsky, 110; solubilities, 420;
Schiff’s reagent: formulas, 293-295; Azure standardization, 112; synthetic, 109
A reagent, 302; fluorescent, 296 Standard buffer, MclIlvaine’s, 418
Schmorl technic, 251 Standardization of stains, 112
Scott’s solution: formula, 412; use, 131 Static elimination, paraffin sectioning, 54
Sea anemones: anesthetizing, 371; fixation, Sterling’s gentian violet, formu‘a and _ use,
ayia) 307, 311
Sea water, artificial, 411 Stieve’s fixative, 15
Section mounting: freezing method, 65; Stock solutions, 409
nitrocellulose method, 77; — paraffin Storing gross specimens, 426
method, 51 Stroma, definition, 145
Sectioning: electron microscopy, 403; freez- Strops, care and use, 47
ing method, 64; nitrocellulose, 74; paraf- Subcutaneous tissue, preparation and stain-
fin, 52 ing, 169
Sectioning difficulties: freezing method, 64; Succinic dehydrogenase, 350
nitrocellulose method, OF paraffin Sudan staining: for fat, 255; for phospholip-
method, 54 ids, 258
Sections detaching, 60 Sulfuric acid N solutions, 408
Serial sections: nitrocellulose, 78; paraffin, Surgical biopsies freezing method, 63
60 Susa’s fixative, 15
Serum, blood, adhesive, 414 Synthetic dyes, 109
Sex chromatin, methods for, 362 Synthetic resins, 116; quick drying of, 118
Sharpening microtome knives, 45
Silver impregnation: discussion, 175; loose Tapeworms: anesthetizing, 372; fixation,
sections, 178; neurological methods, 192; 373; staining, 377, 382, 384
reticulum methods, 179 Teaching films, 429
Sinha’s fixative, 21 Tellyesniczky fixative, see Formol-alcohol
Sledge microtome, 44 Testing hematoxylin solutions, 128
Slide dryers, hot air, 60 Tetrahydrofuran method for paraffin em-
Slide mounting: freezing method, 65; nitro- bedding, 40
cellulose method, 77; paraffin method, 51 ‘Thermoplastic mounts, 394
Slides, microscopic: cleaning, 57; descrip- Thick blood smears, preparation and stain-
tion, 56; labelling, 423; see also Mount- ing, 221
ing Thin blood smears, preparation and _ stain-
Slide warmer, 58 ing, 219
Sliding microtome, 43, 74 Thioflavine T, for amyloid, 275
Smith’s fixative, 21 Thionin: acid mucopolysaccharides, 280;
Snails, killing and fixing, 372, 376 bone, 170; freezing method, 68; mast
Sodium thiosulfate, 412 cells, 281; metachroming, 110; mucin,
Solubilities of stains, 420 280; Nissl substance, 197; sex chromatin,
Solution preparation, 407, 409 364
Sources of foreign stains, 113 Thymonucleic acid, see Nucleic acids
Spirochete staining, 317 Timing schedule, paraffin method, 41
Sponges, fixation, 372 ‘Tissue processors, automatic, 42
Stage micremeter, 95 Toluene clearing: for paraffin method, 33;
Stain commission, 112 after staining, 115
Staining action: acidophilic, 110; baso- Toluidine blue staining, 238; freezing
philic, 110; double, 109; general consider- method, 68; mast cells, 282; mucin, 279
ations, 104; metachroming, 110; nature Touch preparations, 29
468 Index

Trachoma staining, 327 Walpole’s acetate-acetic acid buffer, 415

Trematodes, fixation, 373 Warthin-Starry silver method for spiro-
Trichloracetic acid, use in fixatives, 10 chetes, 318
Trichrome PAS method for pituitary, 284 Washing after fixation, 12
Trichrome staining: Churg and_ Prado Water bath, use of, 58
method, 158; Gomori method, 156; Mas- Water soluble waxes, embedding and _ sec-
son method, 152; Picro-Gomori method, tioning, 80
159; Pollak rapid method, 155 Weaver’s gelatine fixative, 413
Trioxyhaematin, Hansen’s, formula, 127 Weigert-Hart resorcin-fuchsin stain, 162; in
Triple connective tissue stain, Mallory’s, pentachrome stain, 162
147 Weigert’s iron hematoxylin: formula, 126;
Tris buffer (Hale), 419 Groat’s variation, 126; Lillie’s variation,
Tris maleate buffer, Gomori’s, 420 126; staining procedure, 137
Tubercle bacilli, fluorescent method, 316 Whole mounts: unstained, 379; stained,
Tunicates: anesthetizing, 371, 372; stain- 382
ing, 377 Wilcox blood stain, method, 223
Turbellaria, anesthetizing, 372 Wilder’s silver method for reticulum, 184
Turnbull blue for ferrous iron, 235 Woodstain scarlet, in pentachrome stain,
‘Two cover glass mounting, 125 163
Two different stain methods on one slide, Worms: anesthetizing, 372; fixation, 373-
424 375
Wright-Giemsa stain, 223
Wright’s buffer, 220
Ultrathin sectioning, 403
Wright’s method and stain for blood
Ultraviolet microscopy, 100
smears, 219
Ungewitter modification of Bodian, 204
Uranium nitrate, Wilder’s silver method,
X-ray diffraction, 99
184
Xylene clearing: for paraffin method, 33;
Uric acid staining, 247
after staining, 115

Vacuum oven for paraffin infiltration, 35 Yetwin’s mounting medium for nematodes
Van Gieson staining, modified, 315; see also and ova, 121
Picro-ponceau Yolk staining, 148, 260
Verhoefl’s elastin stain, 166
Vernier reading, 94 Zenker’s fixative, 16
Victoria blue for neurological staining, 191 Zenker formol, see Helly’s fixative
Von Kossa method for calcium, 236 Ziehl-Neelsen method for bacteria, 312
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