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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

 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 1.3 Examples of Bacterial Shapes as Seen with the Bright-field Light Microscope. (a) Staphylococcus aureus cocci; singular,
coccus (×1,000). (b) Bacillus subtilis rods or bacilli; singular, bacillus (×1,000). (c) A single, large spirillum; plural, spiralla (Spirillum volutans;
×1,000). (d) Numerous, small spirilla (Rhodospirillum rubrum; ×1,000).

(a) (b)

(c) (d)

the stained area with the low-power objective and then

turn the oil immersion lens into the oil and focus with
the fine adjustment. An alternate procedure is to get
the focus very sharp under high power, then move the
revolving nosepiece until you are halfway between the
high-power and oil immersion objectives. Place a
small drop of immersion oil in the center of the
illuminated area on the slide. Continue revolving the
nosepiece until the oil immersion objective clicks into
place. The lens will now be immersed in oil. Sharpen
the focus with the fine adjustment knob. Draw a few
of the bacteria in the spaces provided.

4 Microscopic Techniques
 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

Table 1.1 Troubleshooting the Bright-Field Light Microscope

Common Problem Possible Correction
No light passing through the ocular Check to ensure that the microscope is completely plugged into a good receptacle
Check to ensure that the power switch to the microscope is turned on
Make sure the objective is locked or clicked in place
Make sure the iris diaphragm is open
Insufficient light passing through the ocular Raise the condenser as high as possible
Open the iris diaphragm completely
Make sure the objective is locked or clicked in place
Lint, dust, eyelashes interferring with view Clean ocular with lens paper and cleaner
Particles seem to move in hazy visual field Air bubbles in immersion oil; add more oil or make certain that oil immersion objective is in the oil
Make sure that the high-dry objective is not being used with oil
Make sure a temporary coverslip is not being used with oil. Oil causes the coverslip to float since the coverslip
sticks to the oil and not the slide, making viewing very hazy or impossible

8. After you are finished with the microscope, place the stage micrometer would appear as illustrated
the low-power objective in line with the ocular, in figure 1.4b.
lower the tube to its lowest position, clean the oil 2. When in place, the two micrometers appear as
from the oil immersion lens with lens paper and shown in figure 1.4c. Turn the ocular in the body
lens cleaner, cover, and return the microscope to tube until the lines of the ocular micrometer are
its proper storage place. parallel with those of the stage micrometer (figure
1.4d ). Match the lines at the left edges of the two
Principles of Microscopic Measurement micrometers by moving the stage micrometer.
It frequently is necessary to accurately measure the size 3. Calculate the actual distance in millimeters
of the microorganism one is viewing. For example, size between the lines of the ocular micrometer by
determinations are often indispensable in the identifica- observing how many spaces of the stage
tion of a bacterial unknown. The size of microorganisms micrometer are included within a given number of
is generally expressed in metric units and is determined spaces on the ocular micrometer. You will get the
by the use of a microscope equipped with an ocular mi- greatest accuracy in calibration if you use more
crometer. An ocular micrometer is a small glass disk ocular micrometer spaces to match with stage
on which uniformly spaced lines of unknown distance, micrometer lines.
ranging from 0 to 100, are etched. The ocular microme- Because the smallest space on the stage
ter is inserted into the ocular of the microscope and then micrometer equals 0.01 millimeter or 10 Ȗm
calibrated against a stage micrometer, which has uni- (figure 1.4b), you can calibrate the ocular
formly spaced lines of known distance etched on it. The micrometer using the following:
stage micrometer is usually divided into 0.01 millimeter 10 spaces on the ocular micrometer = Y spaces
and 0.1 millimeter graduations. The ocular micrometer on the stage micrometer.
is calibrated using the stage micrometer by aligning the Since the smallest space on a stage micrometer =
images at the left edge of the scales. 0.01 mm, then
The dimensions of microorganisms in dried,
fixed, or stained smears tend to be reduced as much as 10 spaces on the ocular micrometer = Y spaces on
10 to 20% from the dimensions of the living microor- the stage micrometer × 0.01 mm, and 1 space on
ganisms. Consequently, if the actual dimensions of a the ocular micrometer = Y spaces on the stage
microorganism are required, measurements should be × 0.01 mm
micrometer 10 .
made in a wet-mount.
For example, if 10 spaces on the ocular
Procedure micrometer = 6 spaces on the stage micrometer,
Calibrating an Ocular Micrometer then
6 × 0.01 mm
1. If you were to observe the ocular micrometer 1 ocular space = 10 ,
without the stage micrometer in place, it would
appear as shown in figure 1.4a. In like manner, 1 ocular space = 0.006 mm or 6.0 Ȗm.

Bright-Field Light Microscope and Microscopic Measurement of Organisms 5

 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

This numerical value holds only for the Calibrate for each of the objectives on your
specific objective-ocular lens combination used microscope and record below. Show all
and may vary with different microscopes. calculations in the space following the table; also
show your calculations to your instructor.

Low power (10× objective) 1 ocular space = ______ mm

Figure 1.4 Calibrating an Ocular Micrometer.
High-dry power (40× objective) 1 ocular space = ______ mm

Oil immersion (90× objective) 1 ocular space = ______ mm

HINTS AND PRECAUTIONS

(1) Forcing the fine or coarse adjustment knobs on the mi-
(a) (b) croscope beyond their gentle stopping points can render
the microscope useless. (2) A general rule for you to note
is that the lower the magnification, the less light should be
directed upon the object. (3) The fine adjustment knob on
0 20 40 60 80 100
the microscope should be centered prior to use to allow
for maximum adjustment in either direction. (4) If a slide
is inadvertently placed upside down on the microscope
Space = 0.1 mm
0.01 mm
stage, you will have no difficulty focusing the object
under low and high power. However, when progressing to
Image of ocular micrometer Image of stage micrometer oil immersion, you will find it impossible to bring the ob-
with uniformly spaced lines with uniform lines at standard ject into focus. (5) Slides should always be placed on and
known intervals
removed from the stage when the low-power (4× or 10×)
objective is in place. Removing a slide when the higher
objectives are in position may scratch the lenses. (6) A
80
Ocular 60
note about wearing eyeglasses. A microscope can be fo-
micrometer 40 cused; therefore, it is capable of correcting for near- or
20
0 farsightedness. Individuals who wear eyeglasses that cor-
Stage
(c) rect for near- or farsightedness do not have to wear their
micrometer
glasses. The microscope cannot correct for astigmatism;
thus, these individuals must wear their glasses. If eye-
glasses are worn, they should not touch the oculars for
proper viewing. If you touch the oculars with your
glasses, they may scratch either the glasses or the oculars.
(7) Because lens cleaner can be harmful to objectives, be
sure not to use too much cleaner or leave it on too long.
The distance between the lines of an ocular microme-
ter is an arbitrary measurement that has meaning only if
0 20 40 60 80 100
the ocular micrometer is calibrated for the specific objec-
tive being used. If it is necessary to insert an ocular mi-
crometer in your eyepiece (ocular), ask your instructor
whether it is to be inserted below the bottom lens or
(d)
placed between the two lenses. Make sure that the etched
graduations are on the upper surface of the glass disk that
you are inserting. With stained preparations such as
Gram-stained bacteria, the bacteria may measure smaller
than they normally are if only the stained portion of the
cell is the cytoplasm (gram-negative bacteria), whereas
Superposition of scales allows those whose walls are stained (gram-positive bacteria)
calibration of ocular scales will measure closer to their actual size.
(10 ocular units = 0.07 mm)

6 Microscopic Techniques
 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

Laboratory Report 1 Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————

Bright-Field Light Microscope

(Basic Microscopy)

Parts of a Compound Microscope

1. Your microscope may have all or most of the features described below and illustrated in figure 2.3 in your
textbook. By studying this figure and reading your textbook, label the compound microscope in figure LR1.1
on the next page. Locate the indicated parts of your microscope and answer the following questions.
a. What is the magnification stamped on the housing of the oculars on your microscope? _______________
b. What are the magnifications of each of the objectives on your microscope? ________________________
_____________________________________________________________________________________
c. Calculate the total magnification for each ocular/objective combination on your microscope.

Ocular × Objective = Total Magnification

___________________ _______________ __________________________________

___________________ _______________ __________________________________

___________________ _______________ __________________________________

___________________ _______________ __________________________________

d. List the magnification and numerical aperture for each objective on your microscope.

Magnification of Objective Numerical Aperture (NA)

____________________________________ ____________________________________

____________________________________ ____________________________________

____________________________________ ____________________________________

____________________________________ ____________________________________

e. With some compound microscopes, loosening a lock screw allows you to rotate the body tube 180°.
What is the advantage of being able to rotate the body tube? ____________________________________
_____________________________________________________________________________________
f. Note the horizontal and vertical scales on the mechanical stage. What is the function of these scales?
_____________________________________________________________________________________
g. Where is the diaphragm on your microscope located? _________________________________________
_____________________________________________________________________________________

7
 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 LR1.1 Modern Bright-Field Compound Microscope.

8 Microscopic Techniques
 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

How can you regulate the diaphragm? ______________________________________________________

_____________________________________________________________________________________
h. Locate the substage condenser on your microscope. What is its function, and how can it be regulated?
_____________________________________________________________________________________
_____________________________________________________________________________________
i. Can the light intensity of your microscope be regulated? Explain. ________________________________
_____________________________________________________________________________________

Microscopic Measurement of Microorganisms

2. After your ocular micrometer has been calibrated, determine the dimensions of the prepared slides of the
following microorganisms.

Microorganism Length Width Magnification

Bacterium
name ________________________ ________________________ ____________ __________________

Fungus name ___________________ ________________________ ____________ __________________

Alga name _____________________ ________________________ ____________ __________________
Protozoan name_________________ ________________________ ____________ __________________

3. Draw and label, as completely as possible, the microorganisms that you measured.

Genus and species: ________________________ Genus and species: ___________________________

Magnification: ×
___________________________ Magnification: ×
_______________________________

Genus and species: ________________________ Genus and species: ___________________________

×
Magnification: ___________________________ ×
Magnification: _______________________________

Bright-Field Light Microscope (Basic Microscopy) 9

 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

Review Questions

1. Differentiate between the resolving power and magnifying power of a lens. What is meant by the term
“parfocal”?

2. Why is the low-power objective placed in position when the microscope is stored or carried?

3. Why is oil necessary when using the 90× to 100× objective?

4. What is the function of the iris diaphragm? The substage condenser?

5. What is meant by the limit of resolution?

10 Microscopic Techniques
 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

6. How can you increase the bulb life of your microscope if its voltage is regulated by a rheostat?

7. In general, at what position should you keep your microscope’s substage condenser lens?

8. What are three bacterial shapes you observed?

9. How can you increase the resolution on your microscope?

10. In microbiology, what is the most commonly used objective? Explain your answer.

11. In microbiology, what is the most commonly used ocular? Explain your answer.

12. If 5× instead of 10× oculars were used in your microscope with the same objectives, what magnifications
would be achieved?

Bright-Field Light Microscope (Basic Microscopy) 11

 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

13. Why is it necessary to calibrate the ocular micrometer with each objective?

14. In the prepared slides, which organism was the largest?

15. When identifying microorganisms, why should a wet-mount be used when making measurements?

16. What is a stage micrometer?

17. Complete the following for the 10 × objective:

a. _____ ocular micrometer divisions = _____ stage micrometer divisions

b. _____ ocular micrometer divisions = 1 stage micrometer division = _____ mm

c. One ocular micrometer division = _____ stage micrometer divisions = _____ mm

18. Complete the following on units of measurement:

Unit Abbreviation Value
a. 1 centimeter ____________ 10–2 meter
b. 1 millimeter mm ____________
c. ____________ Ȗm 10–6 meter
d. 1 nanometer ____________ 10–9 meter
e. 1 angstrom ____________ 10–10 meter

12 Microscopic Techniques
 Harley−Prescott: I. Microscopic Techniques 2. The Hanging Drop Slide © The McGraw−Hill
Laboratory Exercises in and Bacterial Motility Companies, 2002
Microbiology, Fifth Edition

E X E RC I S E
2
The Hanging Drop Slide and Bacterial Motility

Pronunciation Guide
SAFETY PRECAUTIONS
Be careful with the Bunsen burner flame. Slides and Bacillus cereus (bah-SIL-lus SEE-ree-us)
coverslips are glass. Do not cut yourself when using Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jin-
them. Dispose of any broken glass in the appropriately OH-sah)
labeled container. Discard contaminated depression Spirillum volutans (spy-RIL-lum VOL-u-tans)
slides in a container with disinfectant.

Materials per Student Why Are the Above Bacteria Used

24- to 48-hour tryptic soy broth cultures of in This Exercise?
Pseudomonas aeruginosa (ATCC 10145,
The major objectives of this exercise are to allow students
small, motile bacillus), Bacillus cereus (ATCC to gain expertise in making hanging drop slides and observ-
21768, large, motile bacillus), and Spirillum ing the motility of living bacteria. To accomplish these ob-
volutans (ATCC 19554, spiral, motile jectives, the authors have chosen three bacteria that are
bacterium) easy to culture and vary in size, shape, arrangement of fla-
microscope or phase-contrast microscope gella, and types of motion. Specifically, Pseudomonas
lens paper and lens cleaner aeruginosa (L. aeruginosa, full of copper rust, hence
immersion oil green) is a straight or slightly curved rod (1.5 to 3.0 Ȗm in
clean depression slides and coverslips length) that exhibits high motility by way of a polar flagel-
petroleum jelly (Vaseline) lum; Bacillus cereus (L. cereus, waxen, wax colored) is a
inoculating loop large (3.0 to 5.0 Ȗm in length) rod-shaped and straight
toothpicks bacillus that moves by peritrichous flagella; and Spirillum
volutans (L. voluto, tumble about) is a rigid helical cell (14
Bunsen burner
to 60 Ȗm in length) that is highly motile since it contains
large bipolar tufts of flagella having a long wavelength and
Learning Objectives about one helical turn. P. aeruginosa is widely distributed
in nature and may be a saprophytic or opportunistic animal
Each student should be able to
pathogen. B. cereus is found in a wide range of habitats and
1. Make a hanging drop slide in order to observe is a significant cause of food poisoning. S. volutans occurs
living bacteria in stagnant freshwater environments.
2. Differentiate between the three bacterial species
used in this exercise on the basis of size, shape,
arrangement, and motility Principles
Many bacteria show no motion and are termed non-
Suggested Reading in Textbook motile. However, in an aqueous environment, these
1. Flagella and Motility, section 3.6; see also same bacteria appear to be moving erratically. This er-
figures 3.31–3.36. ratic movement is due to Brownian movement.

13
 Harley−Prescott: I. Microscopic Techniques 2. The Hanging Drop Slide © The McGraw−Hill
Laboratory Exercises in and Bacterial Motility Companies, 2002
Microbiology, Fifth Edition

Brownian movement results from the random motion shape, size, arrangement, and motility. Be careful
of the water molecules bombarding the bacteria and to distinguish between motility and Brownian
causing them to move. movement.
True motility (self-propulsion) has been recog- 6. Discard your coverslips and any contaminated
nized in other bacteria and involves several different slides in a container with disinfectant solution.
mechanisms. Bacteria that possess flagella exhibit fla- 7. Complete the report for exercise 2.
gellar motion. Helical-shaped spirochetes have axial
fibrils (modified flagella that wrap around the bac-
terium) that form axial filaments. These spirochetes
move in a corkscrew- and bending-type motion. Figure 2.1 Preparation of a Hanging Drop Slide.
Other bacteria simply slide over moist surfaces in a
form of gliding motion.
The above types of motility or nonmotility can be Toothpick
observed over a long period in a hanging drop slide.
Hanging drop slides are also useful in observing the Vaseline ring
general shape of living bacteria and the arrangement Slide concavity
(a)
of bacterial cells when they associate together (see
figure 1.3). A ring of Vaseline around the edge of the
coverslip keeps the slide from drying out. Inoculating loop

Drop of bacterial culture

Procedure (b) Coverslip

1. With a toothpick, spread a small ring of Vaseline

around the concavity of a depression slide (figure
Move slide to coverslip
2.1a). Do not use too much Vaseline. (c)
Drop of bacterial culture
2. After thoroughly mixing one of the cultures, use Vaseline

the inoculating loop to aseptically place a small

drop of one of the bacterial suspensions in the (d) Turn slide over
center of a coverslip (figure 2.1b).
3. Lower the depression slide, with the concavity
facing down, onto the coverslip so that the drop
protrudes into the center of the concavity of the
slide (figure 2.1c). Press gently to form a seal. HINTS AND PRECAUTIONS
4. Turn the hanging drop slide over (figure 2.1d) and (1) Always make sure the specimen is on the top side of
place on the stage of the microscope so that the the slide. (2) Particular care must be taken to avoid
drop is over the light hole. breaking the coverslip since it is more vulnerable when
5. Examine the drop by first locating its edge under supported only around its edges. (3) With depression
low power and focusing on the drop. Switch to slides, the added thickness of the slide and coverslip may
the high-dry objective and then, using immersion preclude the use of the oil immersion objective with
oil, to the 90 to 100× objective. In order to see the some microscopes. (4) If your microscope is equipped
with an automatic stop, it may be necessary to bring the
bacteria clearly, close the diaphragm as much as
image into focus by using the coarse adjustment knob.
possible for increased contrast. Note bacterial

14 Microscopic Techniques
 Harley−Prescott: I. Microscopic Techniques 2. The Hanging Drop Slide © The McGraw−Hill
Laboratory Exercises in and Bacterial Motility Companies, 2002
Microbiology, Fifth Edition

Laboratory Report 2 Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————

The Hanging Drop Slide and Bacterial Motility

1. Examine the hanging drop slide and complete the following table with respect to the size, shape, and motility
of the different bacteria.

Bacterium Size Shape Type of Motility Cell Arrangement

B. cereus ____________ __________________ _____________________ ________________________

P. aeruginosa ____________ __________________ _____________________ ________________________
S. volutans ____________ __________________ _____________________ ________________________

2. Draw a representative field for each bacterium.

B. cereus P. aeruginosa S. volutans

Magnification: ×
___________ Magnification: ×
___________ Magnification: ×
___________

15
 Harley−Prescott: I. Microscopic Techniques 2. The Hanging Drop Slide © The McGraw−Hill
Laboratory Exercises in and Bacterial Motility Companies, 2002
Microbiology, Fifth Edition

Review Questions

1. Why are unstained bacteria more difficult to observe than stained bacteria?

2. What are some reasons for making a hanging drop slide?

3. Describe the following types of bacterial movement:

a. Brownian movement
b. flagellar motion
c. gliding motion

4. Why do you have to reduce the amount of light with the diaphragm in order to see bacteria in a hanging drop
slide?

5. Can the hanging drop slide be used to examine other microorganisms? Explain which ones.

6. Which of the bacteria exhibited true motility on the slides?

7. How does true motility differ from Brownian movement?

16 Microscopic Techniques
 Harley−Prescott: I. Microscopic Techniques 3. Dark−Field Light © The McGraw−Hill
Laboratory Exercises in Microscope Companies, 2002
Microbiology, Fifth Edition

E X E RC I S E
3
Dark-Field Light Microscope

SAFETY CONSIDERATIONS
Gently scrape the gum line or gingival sulcus with a flat Why Is the Following Bacterium
toothpick so that you obtain a small amount of surface
Used in This Exercise?
scrapings and not lacerated gum tissue or impacted
food. Slides and coverslips are glass. Do not cut your- Treponema denticola (M.L. n, denticola, tooth dweller)
self when using them. Dispose of any broken glass in often is a part of the normal microbiota of the oral mucosa;
the appropriately labeled container. Do not throw used thus, this spirochete is readily available and does not have
toothpicks in the wastebasket. Place them in the appro- to be cultured. Most species stain poorly if at all with
priate container for disposal. Gram’s or Giemsa’s methods and are best observed with
dark-field or phase-contrast microscopy. Thus, T. denticola
is an excellent specimen to observe when practicing the use
Materials per Group of Students of a dark-field microscope, and also allows the student to
continue practicing the wet-mount preparation. T. denticola
dark-field light microscope
is a slender, helical cell, 6 to 16 Ȗm in length. In a wet-
flat toothpicks
mount, the bacteria show both rotational and translational
lens paper and lens cleaner movements due to two or three periplasmic flagella inserted
immersion oil at each end of the protoplasmic cylinder. Young cells rotate
slides and coverslips rapidly on their axis. Thus, by using T. denticola, the stu-
prepared slides of spirochetes (e.g., Treponema dent is also able to observe bacterial motility.
denticola), radiolarians, protozoa
tweezers
Principles
Learning Objectives
The compound microscope may be fitted with a dark-
Each student should be able to field condenser that has a numerical aperture (resolv-
1. Understand the principles behind dark-field ing power) greater than the objective. The condenser
microscopy also contains a dark-field stop. The compound micro-
2. Correctly use the dark-field microscope scope now becomes a dark-field microscope. Light
3. Make a wet-mount and examine it for spirochetes passing through the specimen is diffracted and enters
with the dark-field microscope the objective lens, whereas undiffracted light does
not, resulting in a bright image against a dark back-
ground (figures 3.1–3.2). Since light objects against a
Suggested Reading in Textbook
dark background are seen more clearly by the eye
1. The Dark-Field Microscope, section 2.2; see also than the reverse, dark-field microscopy is useful in
figures 2.7 and 2.8. observing unstained living microorganisms, microor-
ganisms that are difficult to stain, and spirochetes
Pronunciation Guide (figure 3.2), which are poorly defined by bright-field
microscopy.
Treponema denticola (trep-o-NE-mah dent-A-cola)

17
 Harley−Prescott: I. Microscopic Techniques 3. Dark−Field Light © The McGraw−Hill
Laboratory Exercises in Microscope Companies, 2002
Microbiology, Fifth Edition

Figure 3.1 Dark-field Microscopy. Dark-field microscopy Figure 3.2 Photomicrograph of Treponema pallidum, as
can best visualize transparent, unstained specimens, which display Seen with Dark-field Microscopy (×500).
only low contrast in bright-field. In this dark-field
photomicrograph (×100), a mixture of radiolarian shells is shown.
Notice their many unique and beautiful shapes.

Procedure 6. Nonpathogenic spirochetes (T. denticola) may be

part of the normal microbiota of the oral mucosa.
1. Place a drop of immersion oil directly on the To make a wet-mount of these, gently scrape your
dark-field condenser lens. gum line with a flat toothpick. Stir the scrapings
2. Position one of the prepared slides so that the into a drop of water on a slide. Gently lower a
specimen is directly over the light opening. coverslip (see figure 1.2) to prevent trapping air
3. Raise the dark-field condenser with the height bubbles. Examine with the dark-field microscope
control until the oil on the condenser lens just and draw several spirochetes in the space
touches the slide. provided in the report for exercise 3.
4. Lock the 10× objective into position. Focus with
the coarse and fine adjustment knobs until the
spirochetes come into sharp focus. Do the same
HINTS AND PRECAUTIONS
with the 40× objective.
(1) It is good practice to always clean the condenser
5. Use the oil immersion objective lens to observe
lens before placing a drop of oil on it. (2) Make sure the
the spirochetes. Draw several in the space prepared slide is placed right side up (coverslip up) on
provided in the report for exercise 3. the stage. (3) If you have trouble focusing with the oil
immersion lens, don’t flounder—ask for help from your
instructor. (4) Always make sure that the substage con-
denser diaphragm is wide open for adequate illumina-
tion of the specimen.

18 Microscopic Techniques
 Harley−Prescott: I. Microscopic Techniques 3. Dark−Field Light © The McGraw−Hill
Laboratory Exercises in Microscope Companies, 2002
Microbiology, Fifth Edition

Laboratory Report 3 Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————

Dark-Field Light Microscope

1. Drawing of spirochetes from a prepared slide. Drawing of spirochetes from a wet-mount.

Magnification: × __________________________ Magnification: × _____________________________

Genus and species: ________________________ Genus and species: ____________________________
Shape: __________________________________ Shape: _____________________________________
2. Label the following parts of a dark-field microscope. Use the following terms: dark-field stop, specimen,
Abbé condenser, and objective.

19
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