Lab Manual and Workbook in Microbiology: Applications to Patient Care

337 Pages·2018·11.62 MB·English

by  Paul A. Granato

Twelfth Edition
Laboratory Manual
and Workbook in
MICROBIOLOGY
Applications to Patient Care
Paul A. Granato | Verna Morton | Josephine A. Morello
TWELFTH EDITION
Laboratory Manual and Workbook in
M
ICROBIOLOGY
A P P L I C A T I O N S T O P A T I E N T C A R E
Paul A. Granato, Ph.D., D(ABMM), F(AAM)
Professor Emeritus, Department of Pathology
SUNY Upstate Medical University
Director of Microbiology
Laboratory Alliance of Central New York
Syracuse, New York
Verna Morton, M.S., M.T. (ASCP)
Formerly Microbiology Section Head
Cox Health, Springfield, Missouri
Josephine A. Morello, Ph.D., D(ABMM), F(AAM)
Professor Emerita, Department of Pathology
Formerly Director of Hospital Laboratories and
Director of Clinical Microbiology Laboratories
The University of Chicago, Chicago, Illinois
LABORATORY MANUAL AND WORKBOOK IN MICROBIOLOGY:
APPLICATIONS TO PATIENT CARE, TWELFTH EDITION
Published by McGraw-Hill Education, 2 Penn Plaza, New York, NY 10121. Copyright © 2019 by McGraw-Hill
Education. All rights reserved. Printed in the United States of America. Previous editions © 2014, 2011, and
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Library of Congress Cataloging-in-Publication Data
Names: Granato, Paul A. | Morton, Verna. | Morello, Josephine A.
Title: Laboratory manual and workbook in microbiology : applications to patient care / Paul A. Granato, Ph.D.,
D(ABMM), F(AAM), professor emeritus, Department of Pathology. SUNY Upstate Medical University,
director of Microbiology, Laboratory Alliance of Central New York, Syracuse, New York, Verna Morton, M.S.,
M.T. (ASCP), formerly Microbiology section head, Cox Health, Springfield, Missouri, Josephine A. Morello,
Ph.D., D(ABMM), F(AAM), professor emerita, Department of Pathology, formerly director of hospital
laboratories and director of clinical microbiology laboratories, The University of Chicago, Chicago, Illinois.
Description: Twelfth edition. | New York, NY : McGraw-Hill Education, [2019] | Includes bibliographical
references and index.
Identifiers: LCCN 2017017678| ISBN 9781260002188 (alk. paper) | ISBN 1260002187 (alk. paper)
Subjects: LCSH: Medical microbiology—Laboratory manuals.
Classification: LCC QR46 .M75 2019 | DDC 616.9/041—dc23 LC record available at
https://lccn.loc.gov/2017017678
mheducation.com/highered
Contents
Preface vii 10. The Autoclave 63
Orientation to the Microbiology Laboratory xi
11. Disinfectants 71
Warning xi
Case Study: A Lesson Learned—Follow Proper
Safety Procedures and Precautions xi Procedures for the Disinfection of Equipment 76
General Laboratory Directions xii
12. Antimicrobial Agent Susceptibility Testing and
Resistance 81
PART ONE
Basic Techniques of Microbiology 1 Experiment 12.1 Disk Agar Diffusion Method 82
Experiment 12.2 Broth Dilution Method:
Exercises Determining Minimum Inhibitory
Concentration (MIC) 85
1. The Microscope 2
Experiment 12.3 Bacterial Resistance to
2. Handling and Examining Cultures 11 Antimicrobial Agents 87
3. Simple Stains and Wet-Mount
Preparations 21
PART THREE
Experiment 3.1 Preparing a Simple Stain 23 Diagnostic Microbiology in Action 93
Experiment 3.2 Preparing a Wet Mount 25
General Considerations 93
4. Gram Stain 29 Microbiology at the Bedside 93
Collection and Transport of Clinical Specimens
5. Acid-Fast Stain 35 for Culture 93
Precautions for Handling Specimens and
6. Culture Media 41 Cultures 95
Normal Flora of the Body 95
7. Streaking Technique to Obtain Pure
Principles of Diagnostic Microbiology 96
Cultures 45
Exercises
8. Subculture Techniques 51
13. Primary Media for Isolation of
Experiment 8.1 Subculture Technique
Microorganisms 97
(Picking Isolated Colonies for Pure Cultures) 51
14. Some Metabolic Activities of Bacteria 103
PART TWO
Experiment 14.1 Simple Carbohydrate
Destruction of Microorganisms 55
Fermentations 103
Physical Antimicrobial Agents 55 Experiment 14.2 Starch Hydrolysis 104
Chemical Antimicrobial Agents 56
Experiment 14.3 The Indole Test 106
Experiment 14.4 Use of Kligler Iron Agar 107
Exercises
9. Moist and Dry Heat 57 15. Activities of Bacterial Enzymes 111
Experiment 9.1 Moist Heat 57 Experiment 15.1 The Activity of Urease 111
Experiment 9.2 Dry Heat 58 Experiment 15.2 The Activity of Catalase 112
Experiment 9.3 Incineration 59 Experiment 15.3 The Activity of Oxidase 113
iii
Experiment 15.4 The Activity of Deoxyribonuclease Experiment 22.2 Laboratory Diagnosis of Bacterial
(DNase) 114 Pneumonia 183
Experiment 15.5 The Activity of a Deaminase 115 Experiment 22.3 Antimicrobial Susceptibility Test
of an Isolate from a Clinical Specimen 185
16. Antigen Immunoassays for Detection and
Identification of Microorganisms 119
23. The Enterobacteriaceae (Enteric Bacilli)
17. Principles of Nucleic Acid Assays and Multiplex and Other Clinically Important
Syndrome Panel Testing for the Diagnosis of Gram-Negative Bacilli 189
Infectious Disease 129 Experiment 23.1 Identification of Pure Cultures of
Enterobacteriaceae from the Normal Intestinal
18. Principles and Applications of MALDI-TOF
Flora 190
Mass Spectrometry for the Rapid Identification
of Bacteria and Fungi 139 Experiment 23.2 Isolation Techniques for Enteric
Pathogens 192
19. Staphylococci 145 Case Study: A Case of Diarrhea 195
Experiment 19.1 Isolation and Identification
Experiment 23.3 Identification Techniques for
of Staphylococci 146
Enteric Pathogens 196
Experiment 19.2 Staphylococci in the Normal
Experiment 23.4 Serological Identification of
Flora 149
Enteric Organisms 196
Case Study: Community-Acquired Methicillin-
Experiment 23.5 Techniques to Distinguish
Resistant Staphylococcus 150
Nonfermentative Gram-Negative Bacilli from
20. Streptococci, Pneumococci, and Enterobacteriaceae 197
Enterococci 153 Experiment 23.6 Rapid Methods for Bacterial
Experiment 20.1 Identification of Group A Identification 199
Streptococci (Streptococcus pyogenes) 157
Case Study: A Case of Pharyngitis 158 24. Clinical Specimens from the Intestinal
Tract 205
Experiment 20.2 The CAMP Test for Group B
Experiment 24.1 Culturing a Fecal Sample 205
Streptococci (Streptococcus agalactiae) 158
Experiment 24.2 Identification of an Unknown
Experiment 20.3 Identification of Pneumococci
Enteric Organism 206
(Streptococcus pneumoniae) 159
Case Study: A Case of Pneumonia with Experiment 24.3 Antimicrobial Susceptibility Test
Meningitis 162 of an Enteric Organism 208
Experiment 20.4 Identification of Enterococci 163
25. Urine Culture Techniques 211
Experiment 20.5 Streptococci in the Normal
Flora 164 Experiment 25.1 Examination and Qualitative
Culture of Voided Urine 214
21. Haemophilus, Corynebacteria,
Experiment 25.2 Quantitative Urine Culture 215
and Bordetella 169
Case Study: A Case of Cystitis 217
Experiment 21.1 Haemophilus 169
Experiment 21.2 Corynebacteria 172 26. Neisseria and Spirochetes 221
Experiment 21.3 Bordetella 175 Experiment 26.1 Neisseria 221
Case Study: A School-Associated Pertussis Experiment 26.2 Spirochetes 225
Outbreak 176 Case Study: A Case of a Patient with a Bull’s-Eye
Skin Lesion 227
22. Clinical Specimens from the Respiratory
Tract 181 27. Clinical Specimens from Blood 233
Experiment 22.1 Laboratory Diagnosis Case Study: Three Patients with Positive Blood
of a Sore Throat 181 Cultures 237
iv Contents
PART FOUR 30. Mycoplasmas, Rickettsiae, Chlamydiae, Viruses,
Microbial Pathogens Requiring and Prions 261
Special Laboratory Techniques;
31. Fungi: Yeasts and Molds 275
Serodiagnosis of Infectious Disease;
Principles and Practices of Infection
32. Protozoa and Animal Parasites 289
Prevention 241
33. Serodiagnosis of Infectious Disease 299
Exercises
34. Principles and Practices of Infection
28. Anaerobic Bacteria 242 Prevention 305
Case Study: A Case of Diarrhea in a Hospitalized Experiment 34.1 Nasal Surveillance Cultures for
Patient 250 MRSA 309
Selected Literature and Sources 313
29. Mycobacteria 253
Index 315
Experiment 29.1 Microscopic Morphology of
Mycobacterium tuberculosis 255
Experiment 29.2 Culturing a Sputum Specimen for
Mycobacteria 256
Case Study: A Case of Pneumonia in a Patient with
Chronic Alcoholism 257
Contents v

PREFACE
To the Student: Before each laboratory session, your responsibility is
to read through the introductory material of the exercise
You are about to begin the study of a fascinating group of
assigned and the procedures that you will follow, keeping
organisms that are of immense importance in human
in mind the Learning Objectives that precede each exer-
health and disease. Although infectious diseases were
cise. In this way, you will be able to understand the con-
once predicted to disappear with the widespread use of
cepts introduced and complete the work in the time
antimicrobial agents, the microbes have retaliated by
allotted. In some instances, you may work alone, or de-
means of their remarkable genetic versatility. Not only
pending on the instructor and class size, you may work in
have some species acquired resistance to almost all exist-
pairs or groups. In either case, you should be familiar
ing antimicrobial agents but, in part due to social and po-
with all procedures and the results of each experiment. At
litical unrest, some old diseases have returned. New
the end of each exercise, you will be asked a series of
diseases, even some forms of cancer, are now known to
questions. The question pages are set up so that they can
be caused by microbes. Thus, whether your career leads
be removed from the manual and handed in to the in-
you to direct patient contact as a nurse, or you pursue
structor without destroying the exercises. Be aware that
another patient care responsibility in a field of allied
not all answers to the questions are found in the exercise
health, knowledge of microbiology is essential to per-
material but will require supplementary reading in your
form your duties optimally and to protect both patients
textbook and other literature or a search on the internet.
and yourself from acquisition and transfer of sometimes
With the rapid pace of medical knowledge, the internet
deadly pathogens.
has become a valuable resource to your continuing edu-
In this context, this laboratory manual leads you
cation, but only if you learn to recognize valid sites, such
through a series of exercises that allow you to learn not
as those of the Centers for Disease Control and Prevention
only basic techniques for working with microbes similar to
(www.cdc.gov); the National Library of Medicine
those you will encounter in the clinical setting, but they
(www.nlm.nih.gov, which includes PubMed Central);
will teach you how to practice safety precautions in the Web MD (www.webmd.com); and ClinLab Navigator
laboratory and hospital environment. Your journey
(www.clinlabnavigator.com).
through these experiments will be guided by your in-
During our careers, we authors have been fasci-
structor, who will determine the time and materials avail-
nated by the widespread prominence of microbes and
able to perform each exercise. Most likely not every
their impact on health-related fields. We hope that your
exercise will be performed in your course, but enough of
introduction to microbiology will increase your appre-
them are available to provide you with a realistic idea of
ciation for their importance and need for respectful
your responsibilities. You will learn what happens to a
treatment.
patient specimen after it is collected for microbiology
laboratory analysis and how preliminary and completed
To the Instructor:
laboratory reports are conveyed to the physician.
Several of the procedures you will perform have This manual, now in its twelfth edition, maintains its
now been replaced by automated, immunological, or mo- original emphasis on the basic principles of diagnostic
lecular methods, as will be described. However, the exer- microbiology for students entering the allied health
cises in this manual will provide a basic understanding professions. The authors have emphasized the purposes
of microbes—their morphology, biochemical activity, and function of the clinical microbiology laboratory in
immunological characteristics, and susceptibility to anti- the diagnosis of infectious diseases. The exercises il-
microbial agents. lustrate as simply as possible the nature of laboratory
vii
procedures used for isolation and identification of in- Concern has been expressed that not all answers to
fectious agents, as well as the principles of asepsis, dis- the questions at the end of each exercise are found in the
infection, and sterilization. With the advent of introductory or procedural material. This omission is
automated, immunological, and molecular methods, purposeful to allow the students to think critically about
formerly standard techniques for culture identification the lessons and to explore alternative resources, as they
and antimicrobial susceptibility testing are being re- will need to do throughout their careers. They should be
placed by procedures that allow for more rapid test re- directed to their textbooks, and in this computer age, to
sults. These methods are described in each exercise the internet, with caution about possible unreliable
when appropriate, but we believe that the conventional sources of information. Government sources such as the
methods practiced herein will help improve students’ Centers for Disease Control and Prevention and the
basic understanding of the morphology, biochemical National Library of Medicine (see web addresses in To
activity, and immunological characteristics of impor- the Student) are usually accurate, but many other
tant microbial pathogens. sources are available. The question section of each exer-
Attention is also given to the role of the health pro- cise is set up so that it can be removed from the manual
fessional in regard to appropriate collection of clinical and handed in to the instructor without destroying the
specimens and the applications of aseptic and disinfec- exercises.
tant techniques as they relate to patient care. In this way, A complete Instructor’s Manual, available online
the student receives the foundation needed to interpret from McGraw-Hill Education, should greatly aid in-
patient-related information for the diagnosis and treat- structors’ preparation for this course. Included are notes
ment of infectious diseases. to instructors to help plan each exercise; formulae for
In this edition of the manual, each exercise has been preparation of reagents; preparation, storage, and sources
carefully reviewed for accuracy and currency and revised of media; and suggested answers to questions in the
when necessary to conform to changing practices in clini- manual. The URL and password for this site are avail-
cal laboratories. Two exercises from the eleventh edition, able from your McGraw-Hill Education representative.
Preparing a Hanging Drop and Pour-Plate Technique,
have been deleted because they play little role in current
What’s New in the 12th Edition
clinical laboratory practice. A description of the new mul-
tiplex syndrome panel testing technique using PCR has ∙ All figures and colorplates have been carefully
been added to the description of nucleic acid assays in a reviewed and changes made when necessary.
new exercise (Exercise 17). The MALDI-TOF instru- ∙ In Exercise 3, the hanging drop preparation has
ment, which is gaining widespread use, is described in a been deleted because it is seldom used in clinical
new Exercise 18. For details, see What’s New in the 12th practice.
Edition, which follows. Thought has been given to the ∙ Because former Exercise 6 (Special Stains) has
time and resources available to instructors and students. been deleted from this edition, a brief discussion of
Instructors may select among the exercises or parts of ex- capsules, flagella, and endospores has been included
ercises they wish to perform, according to the focus of in Exercise 5 to orient the student to these bacterial
their courses. structures.
A few exercises are primarily descriptive in nature ∙ In Exercise 6, rather than having students prepare
because the equipment, supplies, or reagents needed to culture media, the media are prepared beforehand
perform the relevant tests are beyond the resources by the instructor. However, the students learn how
available to most teaching laboratories. We considered to dispense them aseptically into Petri dishes or in
that the students can benefit from knowing that such tubes for sterilization.
techniques are in regular use, even though they may not ∙ In Exercise 8, the pour-plate technique has been
have the “hands-on” experience. If possible, it would deleted because it is seldom used in clinical
be worthwhile for the instructor to arrange a field practice.
trip to a nearby clinical microbiology laboratory so ∙ In Exercise 10, conditions of pressure, temperature,
that the students can view these diagnostic procedures and time currently used for steam pressure sterilization
firsthand. in most hospitals have been updated.
viii Preface
∙ In Exercise 12, a brief description of the problem Personalize Your Lab
of multidrug-resistant members of the Entero-
bacteriaceae has been added. The information in Craft your teaching resources to match the way you
teach! With McGraw-Hill CreateTM, www.mcgrawhillcre-
table 12.1, Zone Diameter Interpretive Table, has
ate.com, you can easily rearrange chapters, combine
been verified as conforming to the latest Perfor-
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mance Standards of the Clinical and Laboratory
content you have written like your course syllabus or teach-
Standards Institute.
ing notes. Find the content you need in Create by searching
∙ Exercise 16 (formerly Exercise 17) is now limited
through thousands of leading McGraw-Hill textbooks and
to a discussion of the principles of antigen immuno-
lab manuals. Arrange your book to fit your teaching style.
assays. Although no experiments are performed in
Create even allows you to personalize your book’s appear-
this exercise, the students are directed to experi-
ance by selecting the cover and adding your name, school,
ments they will be performing in later exercises.
and course information. Order a Create book and you’ll
∙ The information on nucleic acid assays is now
receive a complimentary print review copy in 3–5 business
found in Exercise 17, along with a discussion of
days or a complimentary electronic review copy (eComp)
Multiplex Syndrome Panel Testing Using PCR As-
via e-mail in minutes. Go to www.mcgrawhillcreate.com
says. This separation from the discussion of antigen
today and register to experience how McGraw-Hill Create
immunoassays allows emphasis to be placed on the
empowers you to teach your students your way.
differences between the technologies and the use of
each in the clinical microbiology laboratory.
Acknowledgements
∙ Exercise 18 is a new exercise discussing MALDI-
TOF Mass Spectrometry for the Rapid Identification We acknowledge the role of McGraw-Hill Education,
of Bacteria and Fungi. This instrument has come Lumina Datamatics, Inc., and Aptara, Inc. in the publication
into widespread use in the clinical laboratory. of this work. Their many courtesies—extended through
The exercise is accompanied by figures describing Marija Magner, Senior Portfolio Manager, Darlene
the instrument and workflow. Schueller, Product Developer, Mary Jane Lampe, Content
∙ In Exercise 20, the use of PCR for the rapid identifi- Product Manager, Shawntel Schmitt, Content Licensing
cation of Streptococcus pyogenes in throat specimens Specialist, Cara Douglass-Graff, Senior Digital Content
is described. The availability of effective vaccines Developer; and Sarita Yadav, Senior Project Manager,
for pneumococcal pneumonia has been added. Digital Publishing—have encouraged and guided this
Figure 20.1 showing latex agglutination has been new edition. They have been primarily responsible for its
replaced. production. For their skilled efforts and expert assistance,
∙ In Exercise 23, the advantage of molecular gene we thank Mathangi Anantharaman, Photo Researcher and
amplification methods in the detection of enteric Rajkishore Singh, Cover Designer.
pathogens is emphasized.
P. A. G.
∙ In Exercise 26, the importance of the meningococcal
vaccine in reducing the incidence of disease has been V. M.
added.
J. A. M.
∙ In Exercise 27, the importance of microarray assays
for rapid identification of bacteria in blood cultures
and the detection of antibiotic-resistance genes is
described.
∙ Exercise 30 includes a discussion of the availability
of nucleic acid amplification tests for organisms in
this exercise. A section on the recent Ebola virus
and Zika virus outbreaks has been added.
∙ Exercise 31 includes the use of nucleic acid
and MALDI-TOF MS technologies for yeast
identification.
Preface ix