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 Harley−Prescott: Front Matter Preface © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

PREFACE
Take interest, I implore you, in those sacred dwellings which one designates
by the expressive term: laboratories. Demand that they be multiplied, that
they be adorned. These are the temples of the future—temples of well-being
and of happiness. There it is that humanity grows greater, stronger, better.
Louis Pasteur
(French chemist, founder of microbiology, 1822–1895)

There are many excellent microbiology laboratory in a manner that will complement the textbook and
manuals on the market and many others that are make the study of microbiology both exciting and
called “in-house” productions because they are writ- challenging. According to an old Chinese proverb:
ten for a microbiology course at a particular school.
Tell me and I will forget.
Why another microbiology manual? The answer is
Show me and I might remember.
straightforward. Many instructors want a manual
Involve me and I will understand.
that is directly correlated with a specific textbook.
As a result, this laboratory manual was designed These words convey our basic philosophy that it is ex-
and written to be used in conjunction with the text- periences in the microbiology laboratory and the sci-
book Microbiology, fifth edition, by Lansing M. entific method that help develop students’ critical
Prescott, John P. Harley, and Donald A. Klein; how- thinking and creativity and that increase their appreci-
ever, it can be used with other textbooks with slight ation of the mechanisms by which microbiologists an-
adaptation. alyze information. The laboratory accomplishes this
Since this manual correlates many of the micro- by having students become intensely and personally
biological concepts in the textbook with the various involved in the knowledge they acquire.
exercises, comprehensive introductory material is The array of exercises was chosen to illustrate the
not given at the beginning of each exercise. Instead, basic concepts of general microbiology as a whole
just enough specific explanation is given to com- and of the individual applied fields. The protocols
plement, augment, reinforce, and enhance what is vary in content and complexity, providing the instruc-
in the textbook. We feel that time allocation is an tor with flexibility to mold the laboratory syllabus to
important aspect of any microbiology course. Stu- the particular needs of the students, available time and
dents should not be required to reread in the labora- equipment, and confines and scope of the course. Fur-
tory manual an in-depth presentation of material thermore, it provides a wide spectrum of individual
that has already been covered satisfactorily in exercises suitable for students in elementary and ad-
the textbook. vanced general microbiology as well as those in vari-
Each exercise has been designed to be modular ous allied health programs.
and short. This will allow the instructor to pick and In 1997, the American Society for Microbiology,
choose only those exercises or parts of exercises through its Office of Education and Training, adopted
that are applicable to a specific course. Several ex- a Laboratory Core Curriculum representing themes
ercises usually can be completed in a two- or three- and topics considered essential to teach in every intro-
hour laboratory period. The exercises have also ductory microbiology laboratory, regardless of its em-
been designed to use commonly available equip- phasis. An instructor might add items appropriate to
ment, with the least expense involved, and to be allied health, applied, environmental, or majors mi-
completed in the shortest possible time period. crobiology courses.
Considering the above parameters, the purpose of The Laboratory Core is not meant to be a syllabus
this laboratory manual is to guide students through a or outline. The core themes and topics are meant to
process of development of microbiological technique, frame objectives to be met somewhere within the in-
experimentation, interpretation of data, and discovery troductory microbiology laboratory. Depending on the

v
 Harley−Prescott: Front Matter Preface © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

specific emphasis of the course, a single lab session d. extrapolating plate counts to obtain correct
could meet multiple core objectives, focus on one ob- CFU or PFU in the starting sample
jective, or emphasize a topic that is not in the lab core
6. Use standard microbiology laboratory
but is important to that particular course.
equipment correctly, including

Laboratory Skills a. using the standard metric system for

weights, lengths, diameters, and volumes
A student successfully completing basic microbiol- b. lighting and adjusting a laboratory burner
ogy will demonstrate the ability to c. using an incubator
1. Use a bright-field light microscope to view and
interpret slides, including Laboratory Thinking Skills
a. correctly setting up and focusing the A student successfully completing basic microbiol-
microscope ogy will demonstrate an increased skill level in
b. proper handling, cleaning and storage of the
1. Cognitive processes, including
microscope
c. correct use of all lenses a. formulating a clear, answerable question
d. recording microscopic observations b. developing a testable hypothesis
c. predicting expected results
2. Properly prepare slides for microbiological
d. following an experimental protocol
examination, including
2. Analysis skills, including
a. cleaning and disposal of slides
b. preparing smears from solid and liquid a. collecting and organizing data in a
cultures systematic fashion
c. performing wet-mount and/or hanging drop b. presenting data in an appropriate form
preparations (graphs, tables, figures, or descriptive
d. performing Gram stains paragraphs)
c. assessing the validity of the data (including
3. Properly use aseptic techniques for the transfer
integrity and significance)
and handling of microorganisms and instruments,
d. drawing appropriate conclusions based on
including
the results
a. sterilizing and maintaining sterility of
3. Communications skills, including
transfer instruments
b. performing aseptic transfer a. discussing and presenting laboratory results
c. obtaining microbial samples or findings in the laboratory
4. Use appropriate microbiological media and 4. Interpersonal and citizenry skills, including
test systems, including a. working effectively in groups or teams so
a. isolating colonies and/or plaques that the task, results, and analysis are shared
b. maintaining pure cultures b. effectively managing time and tasks to be
c. using biochemical test media done simultaneously, by individuals and
d. accurately recording macroscopic within a group
observations c. integrating knowledge and making informed
judgments about microbiology in everyday
5. Estimate the number of microorganisms in a
life
sample using serial dilution techniques, including
Laboratories typically supplement and integrate
a. correctly choosing and using pipettes and closely with the lecture content in ways that are unique to
pipetting devices each instructor. Consequently, the laboratory content that
b. correctly spreading diluted samples for is considered essential for laboratory work by one instruc-
counting tor may be covered in lecture portion of the course by an-
c. estimating appropriate dilutions other instructor, making it difficult to define specific top-

vi Preface
 Harley−Prescott: Front Matter Preface © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

ics that should be integral in all microbiology laborato- Bergey’s Manual of Systematic Bacteriology in
ries. As a result, the ASM Laboratory Core Curriculum the identification of unknown bacteria.
Committee developed themes, which are broadly based PART SEVEN, Environmental Factors Affecting
and will enable instructors to have the flexibility to use a Growth of Microorganisms, acquaints students
wide variety of laboratories to meet the suggested core. with some of the various physical and chemical
A student successfully completing basic microbi- agents that affect microbial growth.
ology will demonstrate mastery of the basic principles PART EIGHT, Environmental and Food
of the following themes and complete laboratory activ- Microbiology, is concerned with the
ities that focus on one or more of the topics under each environmental aspects of water, milk, and food.
theme. PART NINE, Medical Microbiology, presents an
overview of some pathogenic microorganisms,
Theme 1. Integrating themes—impact of
and acquaints students with basic procedures used
microorganisms on the biosphere and humans;
in isolation and identification of pathogens from
microbial diversity
infected hosts, including those from the student’s
Theme 2. Microbial cell biology, including cell
own body.
structure and function, growth and division, and
PART TEN, Survey of Selected Eucaryotic
metabolism
Microorganisms, presents an overview that is
Theme 3. Microbial genetics, including mutations
intended to help students appreciate the
Theme 4. Interactions of microorganisms with
morphology, taxonomy, and biology of the fungi.
hosts (humans, other animals, plants), including
PART ELEVEN, Microbial Genetics and
pathogenicity mechanisms and antimicrobial
Genomics, presents six experiments designed to
agents
illustrate the general principles of bacterial
In order to meet the above themes, topics, and genetics and genomics.
skills (The American Society for Microbiology Labo-
The format of each exercise in this manual is in-
ratory Core Curriculum), this manual consists of 66
tended to promote learning and mastery in the shortest
exercises arranged into 11 parts covering the following
possible time. To this end, each experiment is de-
basic topics:
signed as follows:
PART ONE, Microscopic Techniques, introduces
the students to the proper use and care of the
different types of microscopes used in the Safety Considerations
microbiology laboratory for the study of This laboratory manual endeavors to include many
microorganisms. of the safety precautionary measures established by
PART TWO, Bacterial Morphology and Staining, the Centers for Disease Control and Prevention
presents the basic procedures for visualization and (CDC), Atlanta, Georgia; the Occupational Safety
differentiation of microorganisms based on cell and Health Administration (OSHA); and the Envi-
form and various structures. ronmental Protection Agency (EPA). Efforts are
PART THREE, Basic Laboratory and Culture made to instruct the student on safety, and all exer-
Techniques, acquaints students with proper cises will contain precautionary procedures that
laboratory procedures in preparing these agencies are enforcing in hospitals, nursing
microbiological media and in culture techniques homes, commercial laboratories, and industry. A
that are used in isolating microorganisms. safety considerations box is included for each ex-
PART FOUR, Biochemical Activities of Bacteria, ercise to help both the instructor and student prepare
introduces some of the biochemical activities themselves for the possibility of accidents.
that may be used in characterizing and Both the instructor and student should keep in
identifying bacteria. mind at all times that most technical programs, such
PART FIVE, Rapid Multitest Systems, acquaints as a microbiology laboratory, carry some measure of
students with some of the multitest systems that associated risk. The microbiology laboratory is a
can be used to identify bacteria. place where infectious microorganisms are handled,
PART SIX, Unknown Identification, contains two examined, and studied with safety and effectiveness.
exercises that guide students through the use of However, any of the microorganisms we work with

Preface vii
 Harley−Prescott: Front Matter Preface © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

may be pathogenic in an immunocompromised per- Pronunciation Guide

son. Therefore, rather than modifying the objectives
This section contains the phonetic pronunciations for
in this laboratory manual to avoid any risk, the au-
all organisms used in the exercise. If students take the
thors propose that instructors and students imple-
time to sound out new and unfamiliar terms and say
ment the Centers for Disease Control and Preven-
them aloud several times, they will learn to use the
tion (CDC) principles of biosafety throughout. One
vocabulary of microbiologists.
way we propose is to simply modify the “Universal
Precautions” (see pp. xiii–xiv) so the wording is ap- Why Are the Above Bacteria, Slides, or Other
propriate for the classroom by simply changing Microorganisms Used in This Experiment?
“laboratory worker” to “student.” In addition, a
The authors have chosen specific viruses, bacteria,
written safety policy consistent with CDC guide-
fungi, protozoa, algae, and various prepared slides for
lines and adopted by your institution’s governing
each exercise. This microbial material has been se-
body will protect you, your institution, and the stu-
lected based on cost, ease of growth, availability, reli-
dents. As in any laboratory, safety should be a major
ability, and most importantly, the ability to produce
part of the curriculum. Students should be required
the desired experimental results. In order to communi-
to demonstrate their knowledge of safety before
cate these guidelines to the student, this section ex-
they begin each laboratory exercise.
plains why the authors have chosen the microbial ma-
Materials per Student or Group of Students terial being used and also gives additional
biochemical, morphological, and taxonomic informa-
To aid in the preparation of all exercises, each proce- tion about the microorganism(s) that the student
dure contains a list of the required cultures with Amer- should find helpful when performing the experiment.
ican Type Culture Collection catalog numbers (Ameri-
can Type Culture Collection, 12301 Parklawn Drive, Medical Application
Rockville, Maryland 29852–1776; www.ATCC.org; Many students using this laboratory manual are either
703-365-2700), media, reagents, and other equipment in one of the allied health disciplines, such as nursing,
necessary to complete the exercise in the allocated lab or in a preprofessional program such as premed, pre-
time either per student or group of students. Appen- dent, or prevet and need to know the clinical relevance
dixes H and I provide recipes for reagents, stains, and of each exercise performed. To satisfy this need, a Med-
culture media. Appendix J describes the maintenance ical Application section is included for some of the
of microorganisms and supply sources. medically oriented exercises. Medical applications are
described for most clinical procedures as a specific ap-
Learning Objectives plication of the purpose of the exercise. For example, a
Each exercise has a set of learning objectives that procedure can be used for the identification of a partic-
define the specific goals of the laboratory session. It ular microorganism or used in combination with other
is to the student’s advantage to read through this list exercises in a diagnosis. For these exercises, some im-
before coming to class. In like manner, these objec- portant pathogens with their diseases and their need for
tives should be given special attention during the the test being performed in the exercise are listed.
laboratory exercise. Upon conscientious completion
of the exercise, the student should be able to meet all Principles
of the objectives for that exercise. Before leaving the This section contains a brief discussion of the micro-
class, students should check the objectives once biological principles, concepts, and techniques that
again to see that they can master them. If problems underlie the experimental procedures being performed
arise, consult the instructor. in the exercise.

Suggested Reading in Textbook Procedure

These cross-references have been designed to save the Explicit instructions are augmented by diagrams to aid
student’s time. By referring the student to sections, students in executing the experiment as well as interpret-
paragraphs, tables, charts, figures, and boxes within ing the results. Where applicable, actual results are shown
the textbook, unnecessary duplication is avoided. so that the student can see what should be obtained.

viii Preface
 Harley−Prescott: Front Matter Preface © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

Hints and Precautions the different types of dilution. This includes a variety of
practice problems. Answers are provided.
Additional information on what to watch out for, what
can go wrong, and helpful tidbits to make the experiment Instructor’s Guide
work properly are presented in accompanying boxes.
An instructor’s guide has been prepared for the labora-
Laboratory Report tory manual and is available on our web site at
Various pedagogical techniques are used for recording www.mhhe.com/prescott5. This guide provides answers
the obtained results. This part of the exercise can be to the questions in this manual.
turned in to the instructor for checking or grading.
Finally, it is our hope that this manual will serve
Review Questions as a vehicle to (1) introduce the complexity and diver-
Review questions are located at the end of each labo- sity of microorganisms and their relationships to one
ratory report. These were written so that students can another; (2) provide a solid foundation for further
test their understanding of the concepts and tech- study for those electing a career in science; and
niques presented in each exercise. (3) convey something of the meaning, scope, and ex-
citement of microbiology as a significant perspective
Dilution Ratios Used in This Manual from which to view the world.
According to the American Society for Microbiology
Style Manual, dilution ratios may be reported with ei- We appreciate the many comments offered to us
ther colons (:) or shills (/), but note there is a difference over the years by both faculty and students. In our desire
between them. A shill indicates the ratio of a part to a to continue to improve this laboratory manual, we invite
whole; e.g., d means 1 of 2 parts, with a total of 2 parts. constructive comments from those using it. Please con-
A colon indicates the ratio of 1 part to 2 parts, with a tact us through the Cell and Molecular Biology Editor,
total of 3 parts. Thus, d equals 1:1, but 1:2 equals h. McGraw-Hill Publishers (www.mhhe.com/prescott5).
John P. Harley
Dilution Problems Lansing M. Prescott
Since dilution problems are such an integral part of any
microbiology course, Appendix A gives an overview of

Preface ix
 Harley−Prescott: Front Matter Acknowledgments © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

AC K N OW L E D G M E N T S

Our special thanks go to the following reviewers, Raymond B. Otero

whose comments proved very helpful to us: Eastern Kentucky University
Ghayasuddin Ahmad Norbert A. Pilewski
Seton Hall University Duquesne University School of Pharmacy
Alberta M. Albrecht Marcia Pierce
Manhattanville College Eastern Kentucky University
Mary A. Anderson Ralph J. Rascati
Gustavus Adolphus College Kennesaw State College
Susan T. Bagley Jackie Reynolds
Michigan Tech University Richland College
Paul Blum Nancy Ricker
University of Nebraska–Lincoln Capilano College
Geoffrey W. Gearner Ivan Roth
Morehead State University University of Georgia
Robert J. Kearns Julie J. Shaffer
University of Dayton University of Nebraska at Kearney
Dana Kolibachuk Thomas Terry
Rhode Island College University of Connecticut
David Mardon Robert Twarog
Eastern Kentucky University University of North Carolina
Glendon Miller
Wichita State University A special thanks also goes to Kay Baitz, KEY Scien-
Rita Moyes tific Products, 1402 Chisholm Trail, Suite D, Round
Texas A&M University Rock, Texas 78681, for all of her help with the KEY
products.

x
 Harley−Prescott: Front Matter Orientation to the © The McGraw−Hill
Laboratory Exercises in Laboratory: Rules of Companies, 2002
Microbiology, Fifth Edition Conduct and General
Safety

O R I E N TAT I O N TO T H E
L A B O R AT O RY:
RU L E S O F C O N D U C T
AND GENERAL SAFETY

Many of the microorganisms used in this course may h. identification and proper disposal of
be pathogenic for humans and animals. As a result, different types of waste
certain rules are necessary to avoid the possibility of i. never applying cosmetics, including contact
infecting yourself or other people. Anyone who lenses, or placing objects (fingers, pencils)
chooses to disregard these rules or exhibits careless- in the mouth or touching the face
ness that endangers others may be subject to immedi- j. reading and signing a laboratory safety
ate dismissal from the laboratory. If doubt arises as to agreement indicating that the student has
the procedure involved in handling infectious mate- read and understands the safety rules of the
rial, consult your instructor. laboratory
In 1997, the American Society for Microbiology, k. good lab practice, including returning
through its Office of Education and Training, adopted materials to proper locations, proper care
the following on laboratory safety. Each point is con- and handling of equipment, and keeping the
sidered essential for every introductory microbiology bench top clear of extraneous materials
laboratory, regardless of its emphasis.
2. Protective procedures, including
A student successfully completing basic micro-
biology will demonstrate the ability to explain and a. tying long hair back, wearing personal
practice safe protective equipment (eye protection, coats,
closed shoes; glasses may be preferred to
1. Microbiological procedures, including
contact lenses), and using such equipment in
a. reporting all spills and broken glassware to appropriate situations
the instructor and receiving instructions for b. always using appropriate pipetting devices
cleanup and understanding that mouth pipetting is
b. methods for aseptic transfer forbidden
c. minimizing or containing the production of
3. Emergency procedures, including
aerosols and describing the hazards
associated with aerosols a. locating and properly using emergency
d. washing hands prior to and following equipment (eye-wash stations, first-aid kits,
laboratories and at any time contamination is fire extinguishers, chemical safety showers,
suspected telephones, and emergency numbers)
e. never eating or drinking in the laboratory b. reporting all injuries immediately to the
f. using universal precautions (see inside front instructor
and end covers of this laboratory manual) c. following proper steps in the event of an
g. disinfecting lab benches prior to and at the emergency
conclusion of each lab session

xi
 Harley−Prescott: Front Matter Orientation to the © The McGraw−Hill
Laboratory Exercises in Laboratory: Rules of Companies, 2002
Microbiology, Fifth Edition Conduct and General
Safety

In addition, institutions where microbiology lab- principle it is intended to convey. Also, read the appro-
oratories are taught will priate sections in your textbook that pertain to the ex-
periment being performed, this will save you much
1. train faculty and staff in proper waste stream
time and effort during the actual laboratory period.
management
All laboratory experiments will begin with a brief
2. provide and maintain necessary safety equipment discussion by your instructor of what is to be done,
and information resources the location of the materials, and other important in-
3. train faculty, staff, and students in the use of formation. Feel free to ask questions if you do not un-
safety equipment and procedures derstand the instructor or the principle involved.
4. train faculty and staff in the use of MSDS. The Much of the work in the laboratory is designed to
Workplace Hazardous Materials Information be carried out in groups or with a partner. This is to aid
System (WHMIS) requires that all hazardous in coverage of subject matter, to save time and ex-
substances, including microorganisms, be labeled pense, and to encourage discussion of data and results.
in a specific manner. In addition, there must be a Many of the ASM’s recommended precautions are
Material Safety Data Sheet (MSDS) available to represented by the specific safety guidelines given in-
accompany each hazardous substance. MSDS side the cover of this laboratory manual.
sheets are now supplied with every chemical sold
by supply houses. The person in charge of the
microbiology laboratory should ensure that
I have read the above rules and understand
adherence to this law is enforced.
their meaning
All laboratory work can be done more effectively
and efficiently if the subject matter is understood be- ___________________________
fore coming to the laboratory. To accomplish this, read Signature
the experiment several times before the laboratory be- ___________________________
gins. Know how each exercise is to be done and what Date

xii Orientation to the Laboratory: Rules of Conduct and General Safety

 Harley−Prescott: Front Matter Summary of Universal © The McGraw−Hill
Laboratory Exercises in Precautions and Companies, 2002
Microbiology, Fifth Edition Laboratory Safety
Procedures

S U M M A RY O F U N I V E R S A L
PRECAUTIONS AND
L A B O R AT O RY S A F E T Y
P RO C E D U R E S

Universal Precautions instruments after procedures. To prevent needlestick

injuries, needles should not be recapped, purposely
Since medical history and examination cannot reliably
bent or broken by hand, removed from disposable
identify all patients infected with HIV or other blood-
syringes, or otherwise manipulated by hand. After
borne pathogens, blood and body-fluid precautions
they are used, disposable syringes and needles,
should be consistently used for all patients.
scalpel blades, and other sharp items should be
1. All health-care workers should routinely use placed in puncture-resistant containers for disposal.
appropriate barrier precautions to prevent skin 4. Although saliva has not been implicated in HIV
and mucous-membrane exposure when contact transmission, to minimize the need for emergency
with blood or other body fluids of any patient is mouth-to-mouth resuscitation, mouthpieces,
anticipated. Gloves should be worn for touching resuscitation bags, or other ventilation devices
blood and body fluids, mucous membranes, or should be available for use in areas in which the
non-intact skin of all patients, for handling items need for resuscitation is predictable.
or surfaces soiled with blood or body fluids, and 5. Health-care workers who have exudative lesions
for performing venipuncture and other vascular or weeping dermatitis should refrain from all
access procedures. Gloves should be changed direct patient care and from handling patient-care
after contact with each patient. Masks and equipment.
protective eyewear or face shields should be worn 6. The following procedure should be used to clean up
during procedures that are likely to generate spills of blood or blood-containing fluids: (1) Put on
droplets of blood or other body fluids to prevent gloves and any other necessary barriers. (2) Wipe
exposure of mucous membranes of the mouth, up excess material with disposable towels and
nose, and eyes. Gowns or aprons should be worn place the towels in a container for sterilization.
during procedures that are likely to generate (3) Disinfect the area with either a commercial
splashes of blood or other body fluids. EPA-approved germicide or household bleach
2. Hands and other skin surfaces should be washed (sodium hypochlorite). The latter should be diluted
immediately and thoroughly if contaminated with from 1:100 (smooth surfaces) to 1:10 (porous or
blood or other body fluids. Hands should be dirty surfaces); the dilution should be no more than
washed immediately after gloves are removed. 24 hours old. When dealing with large spills or
3. All health-care workers should take precautions to those containing sharp objects such as broken glass,
prevent injuries caused by needles, scalpels, and first cover the spill with disposable toweling. Then
other sharp instruments or devices during saturate the toweling with commercial germicide or
procedures; when cleaning used instruments; during a 1:10 bleach solution and allow it to stand for at
disposal of used needles; and when handling sharp least 10 minutes. Finally clean as described above.

xiii
 Harley−Prescott: Front Matter Summary of Universal © The McGraw−Hill
Laboratory Exercises in Precautions and Companies, 2002
Microbiology, Fifth Edition Laboratory Safety
Procedures

Precautions for Laboratories 4. Mechanical pipetting devices should be used for

manipulating all liquids in the laboratory. Mouth
Blood and other body fluids from all patients should be
pipetting must not be done,
considered infective.
5. Use of needles and syringes should be limited to
1. All specimens of blood and body fluids should be situations in which there is no alternative, and the
put in a well-constructed container with a secure recommendations for preventing injuries with
lid to prevent leaking during transport. Care needles outlined under universal precautions should
should be taken when collecting each specimen to be followed.
avoid contaminating the outside of the container 6. Laboratory work surfaces should be
and of the laboratory form accompanying the decontaminated with an appropriate chemical
specimen. germicide after a spill of blood or other body fluids
2. All persons processing blood and body-fluid and when work activities are completed.
specimens should wear gloves. Masks and 7. Contaminated materials used in laboratory tests
protective eyewear should be worn if mucous- should be decontaminated before reprocessing or be
membrane contact with blood or body fluids is placed in bags and disposed of in accordance with
anticipated. Gloves should be changed and hands institutional policies for disposal of infective waste.
washed after completion of specimen processing. 8. Scientific equipment that has been contaminated
3. For routine procedures, such as histologic and with blood or other body fluids should be
pathologic studies or microbiologic culturing, a decontaminated and cleaned before being repaired
biological safety cabinet is not necessary. in the laboratory or transported to the manufacturer.
However, biological safety cabinets should be 9. All persons should wash their hands after
used whenever procedures are conducted that completing laboratory activities and should remove
have a high potential for generating droplets. protective clothing before leaving the laboratory.
These include activities such as blending, 10. There should be no eating, drinking, or smoking in
sonicating, and vigorous mixing. the work area.

xiv Summary of Universal Precautions and Laboratory Safety Procedures

 Harley−Prescott: I. Microscopic Techniques Introduction © The McGraw−Hill
Laboratory Exercises in Companies, 2002
Microbiology, Fifth Edition

PA RT O N E

Microscopic Techniques

The most important discoveries of the laws, staining characteristics, and motility of different microorgan-
methods and progress of nature have nearly isms. Therefore, proficiency in using the different micro-
always sprung from the examination of the scopes is essential to all aspects of microbiology and must be
smallest objects which she contains. mastered at the very beginning of a microbiology course.
Jean Baptiste Pierre Antoine Monet de Lamarck The next five exercises have been designed to accomplish
(French naturalist, 1744–1829) this major objective.
After completing at least exercise 1, you will, at
the minimum, be able to demonstrate the ability to

M icrobiologists employ a variety of light microscopes

in their work: bright-field, dark-field, phase-contrast,
and fluorescence are most commonly used. In fact, the same
use a bright-field light microscope. This will meet
the American Society for Microbiology Core Cur-
riculum skill number 1 (see pp. vi–viii): (a) correctly
microscope may be a combination of types: bright-field and setting up and focusing the microscope; (b) proper
phase-contrast, or phase-contrast and fluorescence. You will handling, cleaning, and storage of the microscope;
use these microscopes and the principles of microscopy ex- (c) correct use of all lenses; and (d) recording micro-
tensively in this course as you study the form, structure, scopic observations.

Leeuwenhoek was a manic observer, who tried to look at

everything with his microscopes.

Those little animals were everywhere! He told the Royal

Society of finding swarms of those subvisible things in
his mouth—of all places: “Although I am now fifty years
old,” he wrote, “I have uncommonly well-preserved teeth,
because it is my custom every morning to rub my teeth
very hard with salt, and after cleaning my teeth with a
quill, to rub them vigorously with a cloth. . . .”
From his teeth he scraped a bit of white stuff, mixed
it with pure rainwater, stuck it in a little tube onto the
needle of his microscope, closed the door of his study—
As he brought the tube into focus, there was an
unbelievable tiny creature, leaping about in the water of
the tube. . . . There was a second kind that swam
forward a little way, then whirled about suddenly, then
Antony van Leeuwenhoek (1632–1723) tumbled over itself in pretty somersaults. . . . There was
a menagerie in his mouth! There were creatures shaped
Leeuwenhoek was a master at grinding lenses for his micro- like flexible rods that went to and fro . . . there were
scopes. Working in Delft, Holland, in the mid-1600s, he is spirals that whirled through the water like violently
considered the greatest early microscopist. animated corkscrews. . . .
—Paul de Kruif
Microbe Hunters (1926)

1
 Harley−Prescott: I. Microscopic Techniques 1. Bright−Field Light © The McGraw−Hill
Laboratory Exercises in Microscope and Companies, 2002
Microbiology, Fifth Edition Microscopic Measurement
of Organisms

E X E RC I S E
1
Bright-Field Light Microscope
and Microscopic Measurement of Organisms
SAFETY CONSIDERATIONS
Slides and coverslips are glass. Be careful with them. Do Medical Application
not cut yourself when using them. The coverslips are
very thin and easily broken. Dispose of any broken glass In the clinical laboratory, natural cell size, arrangement and
in the appropriately labeled container. If your micro- motility are important characteristics in the identification of
scope has an automatic stop, do not use it as the stage a bacterial pathogen.
micrometer is too thick to allow it to function properly.
It may result in a shattered or broken slide or lens.

Materials per Student

Why Are Prepared Slides
compound microscope Used in This Exercise?
lens paper and lens cleaner
immersion oil Because this is a microbiology course and most of the mi-
prepared stained slides of several types of bacteria croorganisms studied are bacteria, this is an excellent place
(rods, cocci, spirilla), fungi, algae, and protozoa to introduce the student to the three basic bacterial shapes:
cocci, rods, and spirilla. By gaining expertise in using the
glass slides
bright-field light microscope, the student should be able to
coverslips
observe these three bacterial shapes by the end of the lab
dropper with bulb period. In addition, the student will gain an appreciation for
newspaper or cut-out letter e’s the small size and arrangement of procaryotic cell structure.
tweezers One major objective of this exercise is for the student
ocular micrometer to understand how microorganisms can be measured under
stage micrometer the light microscope and to actually perform some mea-
surements on different microorganisms. By making mea-
Learning Objectives surements on prepared slides of various bacteria, fungi,
Each student should be able to algae, and protozoa, the student will gain an appreciation
for the size of different microorganisms discussed through-
1. Identify all the parts of a compound microscope out both the lecture and laboratory portions of this course.
2. Know how to correctly use the microscope—
especially the oil immersion lens
3. Learn how to make and examine a wet-mount
preparation Principles
4. Understand how microorganisms can be measured The bright-field light microscope is an instrument
under the light microscope that magnifies images using two lens systems. Initial
5. Calibrate an ocular micrometer magnification occurs in the objective lens. Most mi-
6. Perform some measurements on different croscopes have at least three objective lenses on a ro-
microorganisms tating base, and each lens may be rotated into align-
ment with the eyepiece or ocular lens in which the
Suggested Reading in Textbook final magnification occurs. The objective lenses are
1. The Bright-Field Microscope, section 2.2; see identified as the low-power, high-dry, and oil immer-
also figures 2.3–2.6. sion objectives. Each objective is also designated by
2. See tables 2.1 and 34.1 other terms. These terms give either the linear magni-

2
 Harley−Prescott: I. Microscopic Techniques 1. Bright−Field Light © The McGraw−Hill
Laboratory Exercises in Microscope and Companies, 2002
Microbiology, Fifth Edition Microscopic Measurement
of Organisms

Figure 1.1 The Oil Immersion Objective. An oil immersion Figure 1.2 Preparation of a Wet-mount Slide. (a) Add a
objective lens operating in air and with immersion oil. Light rays drop of water to a slide. (b) Place the specimen (letter e) in the
that must pass through air are bent (refracted), and many do not water. (c) Place the edge of a coverslip on the slide so that it
enter the objective lens. The immersion oil prevents the loss of touches the edge of the water. (d) Slowly lower the coverslip to
light rays. prevent forming and trapping air bubbles.

Slide Air Oil Cover

glass

fication or the focal length. The latter is about equal (a) (b)

to or greater than the working distance between the

specimen when in focus and the tip of the objective
lens. For example, the low-power objective is also
called the 10×, or 16 millimeter (mm), objective; the
high-dry is called the 40×, or 4 mm, objective; and
the oil immersion is called the 90×, 100×, or 1.8 mm
objective. As the magnification increases, the size of
the lens at the tip of the objective becomes progres-
sively smaller and admits less light. This is one of the
(c) (d)
reasons that changes in position of the substage con-
denser and iris diaphragm are required when using
different objectives if the specimens viewed are to be 3. Cut a lowercase e from a newspaper or other
seen distinctly. The condenser focuses the light on a printed page. Prepare a wet-mount as illustrated in
small area above the stage, and the iris diaphragm con- figure 1.2. Place the glass slide on the stage of the
trols the amount of light that enters the condenser. microscope and secure it firmly using stage clips.
When the oil immersion lens is used, immersion oil If your microscope has a mechanical stage device,
fills the space between the objective and the specimen. place the slide securely in it. Move the slide until
Because immersion oil has the same refractive index the letter e is over the opening in the stage.
as glass, the loss of light is minimized (figure 1.1). The 4. With the low-power objective in position, lower
eyepiece, or ocular, at the top of the tube magnifies the tube until the tip of the objective is within
the image formed by the objective lens. As a result, the 5 mm of the slide. Be sure that you lower the tube
total magnification seen by the observer is obtained by while looking at the microscope from the side.
multiplying the magnification of the objective lens by 5. Look into the microscope and slowly raise the
the magnification of the ocular, or eyepiece. For exam- tube by turning the coarse adjustment knob
ple, when using the 10× ocular and the 43× objective, counterclockwise until the object comes into
total magnification is 10 × 43 = 430 times. view. Once the object is in view, use the fine
adjustment knob to focus the desired image.
6. Open and close the diaphragm, and lower and raise
Procedure for Basic Microscopy: Proper Use
the condenser, noting what effect these actions
of the Microscope
have on the appearance of the object being viewed.
1. Always carry the microscope with two hands. Place Usually the microscope is used with the substage
it on the desk with the open part away from you. condenser in its topmost position. The diaphragm
2. Clean all of the microscope’s lenses only with should be open and then closed down until just a
lens paper and lens cleaner if necessary. Do not slight increase in contrast is observed (table 1.1).
use paper towels or Kimwipes; they can scratch 7. Use the oil immersion lens to examine the stained
the lenses. Do not remove the oculars or any other bacteria that are provided (figure 1.3a–d). The
parts from the body of the microscope. directions for using this lens are as follows: First locate

Bright-Field Light Microscope and Microscopic Measurement of Organisms 3

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119

bodies the back

and very

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young existence was

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from though Atlantic

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East Dominion OR
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prehensile followed herds

in India

such was

by forest the

came this in

the banded 241

this wide the

the OF