Laboratory exercises in microbiology

302 Pages·2009·9.652 MB·English

by  Robert A Pollack, et al

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LABORATORY
EXERCISES IN
MICROBIOLOGY,
THIRD EDITION
Robert A. Pollack
Nassau Community College
Lorraine Findlay, PHD
Nassau Community College
Walter Mondschein
Nassau Community College
R. Ronald Modesto, PHD
C.W. Post Campus of Long Island University
JOHN WILEY & SONS, INC.
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SENIOR EDITOR Kevin Witt
ASSOCIATE EDITOR Merillat Staat
SENIOR PRODUCTION EDITOR Elizabeth Swain
SENIOR MARKETING MANAGER Wayne Parkins
SENIOR PHOTO EDITOR Jennifer MacMillan
COVER DESIGNER Jim O’Shea
EDITORIAL ASSISTANT Alissa Rufino
This book was set in 10.5/13 Times Roman by Aptara and printed and bound by Bind-Rite.
The cover was printed by Bind-Rite.
This book is printed on acid free paper. ∞
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PREFACE
D NEW FEATURES OF THE THIRD
Developed for use in an undergraduate
EDITION
microbiology laboratory course, this third edition of
Laboratory Exercises in Microbiology meets the needs
• Revised Exercises. The exercise on serology
of students majoring in diverse programs such as allied
(Exercise 18) now includes a serological test for
health or biological sciences. The manual contains a
Clostridium difficile, a major nosocomial con-
variety of interactive activities and experiments that
cern. Student charts and tables have been moved
teach students the basic concepts of microbiology and
to the Laboratory Report section so the laboratory
support the content covered during lectures.
instructor can more easily evaluate student
progress.
APPROACH AND ORGANIZATION • Many new and improved photomicrographs.
New enlarged photomicrographs are included in the
photographic atlas. As with the second edition, all
We are firmly committed to the idea that a microbiol-
plates in the Atlas are now cross-referenced within
ogy laboratory—and the manual used for it—should ex-
the text for easy study.
tend learning experience for students, and not be a re-
peat or reproduction of lecture material. With this in
mind we made every effort to avoid duplicating text and
illustrations that will be found in their lecture text. We LABORATORY FEATURES
have minimized the amount of textual material for stu-
dents to read at the start of the laboratory period. Rather, The self-contained laboratory exercises in this manual
labs are introduced in a clear and concise manner and are all designed to maximize the learning opportunity
maintain a student-friendly tone. This leaves plenty of and time spent during each laboratory period. Each
time for students to engage in activities and experiments Exercise begins with a Pre-Test and clearly defined list
that promote a deeper understanding of microbiological of Objectives, followed by a Materials List needed to
concepts and principles, to answer questions, and to complete the lab. Procedures and Results sections within
write up lab reports. each lab are easily identified. Included within lab exer-
The 27 Exercises are divided into five parts: cises are a variety of activities like Art Labeling,
General Microscopy and Aseptic Technique; Microbial Coloring,Identification Exercises,and Critical Thinking
Morphology,Differential Stains; Microbial Control and Questions that help summarize the concepts covered in
Biochemistry; Medical Microbiology; and Food and the lab. An inventory list that allows students to double
Environmental Microbiology. A Photographic Atlas of check their work is also a feature of most exercises.
67 full-color plates depicting laboratory techniques and Immediately following each Exercise is a glossary
results, and numerous micrographs, is included. of Working Definitions and Terms that provides a
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quick review and reinforcement for students as they Fitzgerald, Senior Editor, Mary O'Sullivan, Project
complete the lab reports that follow. The Laboratory Editor, Sarah Wolfman-Robichaud, Production Editor,
Report can be completed and turned into an instructor. Anna Melhorn, Senior Illustration Editor, and Teresa
Included in the lab reports are a mix of Fill-in-the Romito, Photo Editor.
Blank, Matching, and Multiple-Choice Questions. For this edition we would like to thank Kevin Witt,
Student work tables and charts are now included in the Senior Editor, for his ideas and support, Merillat Staat,
Laboratory Report so instructors can review results of Associate Editor, who worked closely with us to effec-
each completed exercise. tively manage the development and review process, and
Alissa Rufino for her editorial assistance. Elizabeth Swain
managed the production process with skill and care.
SUPPORTING MATERIALS
Jennifer Macmillan coordinated the photo program.
Madelyn Lesure created the effective design for the man-
A companion web site for instructors complements the
ual, while Jim O’Shea designed the cover, and Wayne
use of this manual. It includes the following:
Parkins directed the marketing campaign. Our thanks go
• Laboratory Materials List and Suggestions to all for their expertise and collaboration.
Finally, we wish to thank the following reviewers
• Reagents and Stain Formulations
who gave us such helpful feedback during the develop-
• Media Formulations ment process of the third edition:
Christine Bezotte, Elmira College
• Suggested Sequencing of laboratories for health sci-
Lynn Burgess, Dickinson State University
ence students and for non-health science students.
Janelle Hare, Morehead State University
• An Answer Key to the questions from the labora-
Jean Lu, University of Tennessee at Martin
tory reports.
Kathleen Page, Southern Oregon University
The supporting Web site can be accessed at www.wi- Frances Sailer, University of North Dakota-School
ley.com/college/pollack of Medicine and Health Sciences
Fadi Zaher, Gateway Technical College
Reviewers of Previous Editions included:
ACKNOWLEDGEMENTS
William H. Coleman, University of Hartford
Michael A. Davis,Central Connecticut State University
We wish to acknowledge the assistance provided by
Lloyd J. Hays, University of Texas at San Antonio
Dr. Aleta Labiento,PhD in Infectious Disease Control,who
Janice Ito, Leeward Community College, Hawaii
is currently Assistant Director in Undergraduate Health
Lisa Spring, Central Piedmont Community College
Programs, Department of Health Professions and Family
Studies, Hofstra University, in developing the Laboratory We invite all readers and users of this manual to
Operation and Safety Instructions located at the beginning send any comments and suggestions to us so that we
of the manual,as well as Exercise 1. Dr. Labiento is cur- can include them in planning for future editions.
rently an Associate Professor at York College,New York. Robert A. Pollack
We would also like to recognize the original pub- Lorraine Findlay, PHD
lishing team at Wiley who supported and guided our Walter Mondschein
efforts to produce this manual. Ronde Bradley,our Wiley Nassau Community College
sales representative, introduced us—and promoted our R. Ronald Modesto, PHD
work—to the editorial team, which included Patrick C.W. Post Campus of Long Island University
iv Preface
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CONTENTS
Preface iii Capsule Stain 65
Introduction: Laboratory Operations Hanging Drop Technique 65
and Safety vii
7 Fungi 71
8 Viruses—Visualization and
PART I
Enumeration 79
GENERAL MICROSCOPY AND
ASEPTIC TECHNIQUE 1 9 Parasitology 85
1 Laboratory Safety: Introduction
to the Microscope 3
PART III
2 Transfer and Isolation MICROBIAL CONTROL AND
BIOCHEMISTRY 95
Techniques 17
10 Microbial Sensitivity Testing
Microbes in the Environment 25
Ultraviolet Light Sensitivity 98
PART II
Heat Sensitivity 100
MICROBIAL MORPHOLOGY,
DIFFERENTIAL STAINS 31 Chemical Sensitivity 101
3 Cultural and Cellular Morphology Antibiotic Sensitivity:
33
Kirby-Bauer Technique 104
4 Bacterial Growth 43
11 Bacterial Biochemistry 111
5 Gram Stain 53
12 Gas Requirements of
Acid-Fast Stain 55 Microorganisms 121
6 Endospore Stain 63 13 Specialized Media 127
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PART IV 24 Identification of Microbes in
MEDICAL MICROBIOLOGY 137 Beef and Poultry and the
Quantitation of Microbial
14 Genetics 139
Numbers 227
15 Epidemiology 145
25 Soil Microbiology 233
16 Speciman-Handling Protocols 155
26 Microbial Ecology 241
17 Specific Laboratory Tests 167
27 Biofilms 247
18 Serology 175
Photographic Atlas PA-1
19 Identification of Enteric
Bibliography 255
Pathogens: Traditional
Methods 185 Appendix 1 Flow Chart for
the Identification of
20 Identification of Enteric
Enterobacteriaceae 257
Pathogens: Rapid Identification
Methods 195 Appendix 2 Gram-Negative Flow
Chart 262
21 Identification of a Bacterial
Unknown: The Gram-Negative Appendix 3 Gram-Positive Flow
Unknown 205 Chart 263
22 Identification of a Bacterial Photo Credits 264
Unknown: The Gram-Positive
Index 265
Cocci 213
PART V
FOOD AND ENVIRONMENTAL
MICROBIOLOGY 219
23 Identification and Quantitation of
Microbial Numbers in a Water
Sample 221
vi Contents
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Introduction:
Laboratory Operations
and Safety
Safety is an important consideration in any laboratory structor’s direction in cleaning, setting up, and put-
environment. In microbiology, we have the additional ting away your microscope. Be aware of the proper
concern that comes from using dangerous or potentially containers needed to place used slides,tubes,stains,
dangerous organisms called pathogens. The following chemicals, paper, and other items used in the lab.
section lists safety rules appropriate for any laboratory. Leave the laboratory area in good order and return
The procedures and techniques you will learn here will all materials and equipment to their original location.
continue to be useful to you in other laboratory courses,
at home,and in the workplace for years to come. Please
review and familiarize yourself with these procedures LABORATORY SAFETY
so that your laboratory experience will be an enjoyable
and safeone. Additional safety procedures and require-
1. Never put anything up to your mouth in the labora-
ments specific to microbiology will be reintroduced and
tory. A major way microbes enter the body (called a
reinforced in Exercise 1 and,where appropriate,through-
portal of entry) is by the mouth. Therefore, no eat-
out the manual.
ing, drinking, gum chewing, application of cosmet-
ics, or smoking is allowed in the laboratory.
GENERAL LABORATORY 2. Wear a laboratory coat or apron in class. This item of
OPERATING PROCEDURES equipment will protect you from various stains,chem-
icals, and microbes (including those in aerosols).
1. Be prompt. Microbiology laboratories require that When leaving the lab, either leave the lab coat in an
you master various techniques needed to handle and assigned cabinet or place it in a plastic bag or con-
manipulate microbes safely and efficiently. Instruc- tainer. This item of apparel is not for street use, re-
tion and demonstrations of these procedures will be gardless of what you may see on TV medical shows.
done at the beginning of each session. 3. Wash your hands after completing the laboratory ses-
2. Be prepared. If instructed to do so, read the intro- sion. Do not bring anything up to your mouth or eyes
ductory material ahead of time so that you will know without washing your hands first.
what to expect and what is expected of you. Make 4. Be aware of the locations of the fire extinguisher,
sure you have your laboratory manual, lab coat, eyewash station, deluge shower, and exits.
marking pen, and whatever else is required of you
5. Inform your instructor about any accidents,spills,or
for each session.
potential hazards.
3. Be responsible. Take care of your work area and
6. When in doubt,ask your instructor about a procedure.
equipment assigned to you. Wipe down your work
7. Do not wear sandals or open-toed shoes.
area with disinfectant solution before and after each
8. Do not apply makeup.
laboratory session. Keep your microscope clean and
in good working order. Follow your laboratory in- 9. Tie back long hair.
INTRODUCTION: LABORATORY OPERATIONS AND SAFETY vii
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HAND WASHING 2. Lather the soap up using friction to loosen and remove
dirt, dead skin, or other contaminants. Pay particular
Hand washing is one of the most important procedures attention to the areas between the fingers and the fin-
used to prevent the spread of microbes from one area to gernails. Using the thumb of one hand, rub the cuti-
another. Even plain soap can effectively remove signif- cle and nail bed of each finger of the opposite hand.
icant numbers of microbes from a work surface or a per- If the area under the nails is dirty, use a nail brush.
son, thus lowering the chances of infection. You do not 3. Rinse the soap from your hands; hold the hands in a
have to perform the extremely thorough 5 minutes of downward position.
scrubbing associated with operating room procedures.
4. Repeat steps 1–3 if your hands are excessively dirty
If you followed such a rigorous procedure in your every-
or if you suspect contamination with blood or blood
day routine, you would not have too much in the way
products.
of skin by the end of the day.
5. Wipe your hands with a paper towel. While holding
Proper hand washing involves scrubbing the hands
the paper towel in your hand,turn off the water; oth-
with soap and water for at least 30 seconds. The soap
erwise, you will recontaminate your hand.
loosens and sometimes kills the microbes,while the fric-
tion due to scrubbing removes them. Specifically, you
Always wash your hands:
should practice the following routine after each labora-
tory session: • After each laboratory session.
• After each patient/client contact.
1. Remove any rings and bracelets, storing them in a
• If contaminated with any potential infectious mate-
safe location during the lab session. Then place
rial, such as blood, other body fluids, excretions,
some liquid soap in the palm of your hand. (Bar
secretions, or microbial cultures.
soap is virtually never used in the clinical area be-
cause it may act as a source of microbial contami- • After any procedure in which you have to wear pro-
nation.) tective gloves.
viii HAND WASHING