BAILEY & SCOT T 'S
`
`Diagnostic
`Microbiology
`
`Te11t/J Lditio11
`
`Betty A. Forbes, PhD
`Professor of Pathology and Medicine
`Director, Clinical Microbiology Laboratories
`SUNY Health Science Center
`Syracuse, New York
`
`Daniel F. Sahm, PhD
`Chief Scientific Officer
`MRL Pharmaceutical Services
`Reston, Virginia
`
`Alice S. Weissfeld, PhD
`President, Microbiology Specialists Incorporated
`Adjunct Assistant Professor
`Department of Microbiology and Immunology
`Baylor College of Medicine
`Houston, Texas
`
`Photography by Ernest A. Trevino, MT (ASCP)
`Director of Operations
`Microbiology Specialists Incorporated
`Houston, Texas
`
`with 548 illustrations
`
`~T~ Mosby
`
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`
`L_
`
`ALL 2034
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`Page 1
`
`

`

`~ ... '1 Mosby
`
`Dedicated to Publishing Excellence
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`lf'"'9 A Times Mirror
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`TENTH EDITION
`
`Copyright© 1998 by Mosby, Inc.
`
`Previous editions copyrighted 1962, 1966, 1970, 1974, 1978, 1982, 1986, 1990, 1994
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`All rights reserved. No part of this publication may be re produced, stored in a retrieval system,
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`Printed in the United States of America
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`Library of Congress Cataloging-in-Publication Data
`Weissfeld , Alice S.
`Bailey & Scott's diagnostic microbi ology I Alice S. Weissfeld,
`Daniel F. Sahm , Betty A. Forbes; photography by Ernest Trevino.-
`10th ed.
`p. cm.
`Rev. ed. of: Bailey & Scott's diagnostic microbi ology I Ellen Jo
`Baron, Lance R. Peterson, Sydney M. Finegold. 9th ed. ©1994.
`Includes bibliographica l references and index.
`ISBN 0-8 151-2535-6
`1. Di agnostic microbiology.
`Betty A.
`Ill. Baron, Ellen Jo.
`IV. Title.
`Microbiologica l Techniques.
`1998
`
`II. Forbes,
`I. Sa hm, Daniel F.
`Ba iley & Scott's diagnostic
`
`QW 25 W433b 1998]
`
`97-46902
`CIP
`
`Page 2
`
`

`

`2 LABORATORY SAFETY
`
`M icrobiology laboratory safety practices were first
`
`published in 1913 in a textbook by Eyre. 2 They
`included admonitions such as the necessity to (1) wear
`gloves, (2) wash hands after working with infectious
`materials, (3) disinfect all instruments immediately after
`use, (4) use water to moisten specimen labels rather
`than the tongue, (5) disinfect all contaminated waste
`before discarding, and ( 6) report to appropriate person(cid:173)
`nel all accidents or exposures to infectious agents.
`into
`These guidelines are still
`incorporated
`safety programs in the late twentieth-century labo(cid:173)
`ratory.
`In addition, safety programs have been
`expanded to include not only the proper handling of
`biologic hazards encountered in processing patient
`specimens or handling infectious microorganisms but
`also fire safety; electrical safety; the safe handling,
`storage and disposal of chemicals and radioactive
`substances; and techniques for the safe lifting or
`moving of heavy objects. In areas of the country
`prone to natural disasters (e.g., earthquakes, hurri(cid:173)
`canes, snowstorms), safety programs also involve
`disaster preparedness plans that outline steps to take
`in an emergency. Although all microbiologists are
`responsible for their own health and safety, theidnsti(cid:173)
`tution and immediate supervisors are required to
`provide safety training to help familiarize microbiolo(cid:173)
`gists with known hazards and to avoid accidental
`exposure. Laboratory safety is considered such an
`integral part of overall laboratory services that federal
`law in the United States mandates preemployment
`safety training followed by at least quarterly safety in(cid:173)
`services. Microbiologists should find very little reason
`to be afraid while performing duties if the safety regu(cid:173)
`lations are internalized and followed without devia.(cid:173)
`tion. Investigation of the causes of accidents usu(cid:173)
`ally shows that accidents happen when individuals
`become sloppy in performing their duties or when
`they do not believe that they will be affected by
`departures from safety standards.
`
`STERILIZATION AND DISINFECTION
`
`Sterilization is a process whereby all forms of micro(cid:173)
`bial life, including bacterial spores, are killed. Steril-
`
`ization may be accomplished by physical or chemical
`means. Disinfection is a process whereby pathogenic
`organisms, but not necessarily all microorganisms or
`spores, are destroyed. As with sterilization, disinfec(cid:173)
`tion may be accomplished by physical or chemical
`methods.
`
`METHODS OF STERILIZATION
`
`The physical methods of sterilization include the
`following:
`
`• Incineration
`• Moist heat
`• Dry heat
`• Filtration
`• Ionizing (gamma) radiation
`
`Incineration is the most common method of
`treating infectious waste. Hazardous material is liter(cid:173)
`ally burned to ashes at temperatures of 870° to 980°
`C. Toxic air emissions and the presence of heavy
`metals in ash have limited the use of incineration in
`most large U.S. cities, however.
`Moist heat (steam under pressure) is used to
`sterilize biohazardous trash and heat-stable objects;
`an autoclave is used for this purpose. An autoclave is
`essentially a large pressure cooker. Moist heat in the
`form of saturated steam under 1 atmosphere (15 psi
`[pounds per square inch]) of pressure causes the
`irreversible denaturation of enzymes and structural
`proteins. The most common type of steam sterilizer in
`the microbiology laboratory is the gravity displace(cid:173)
`ment type shown in Figure 2-1. Steam enters at the
`top of the sterilizing chamber and, because steam is
`lighter than air, it displaces the air in the chamber and
`forces it out the bottom through the drain vent. The
`two common sterilization temperatures are 121° C
`(250° F) and 132° C (270° F). Items such as media,
`liquids, and instruments are usually autoclaved for 15
`minutes at 121° C. Infectious medical waste, on the
`other hand, is often sterilized at 132° C for 30 to 60
`minutes to allow penetration of the steam throughout
`the waste and the displacement of air trapped inside
`
`Page 3
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`

`

`CHAPTER 2 LABORATORY SAFETY
`
`21
`
`A
`
`B
`
`Water/Steam
`Elector
`
`FIGURE 2-1 Gravity displacement type autoclave. A, Typical Eagle Century Series sterilizer for laboratory applica(cid:173)
`t ions. B, Typical Eagle 3000 sterilizer piping diagram. The arrows show the entry of steam into the chamber and the
`displacement of air. (Courtesy AMSCO International Inc, a wholly owned subsid iary of STERIS Corp., Mentor, Ohio.)
`
`the autoclave bag. Moist heat is the fastest and
`simplest physical method of sterilization.
`Dry heat requires longer exposure times (1.5 to
`3 hours) and higher temperatures than moist heat
`(160° to 180° C). Dry-heat ovens are used to sterilize
`items such as glassware, oil, petrolatum, or powders.
`Filtration is the method of choice for antibiotic solu(cid:173)
`tions, toxic chemicals, radioisotopes, vaccines, and
`carbohydrates, which are all heat-sensitive. Filtration
`of liquids is accomplished by pulling the solution
`through a cellulose acetate or cellulose nitrate mem(cid:173)
`brane with a vacuum. Filtration of air is accom(cid:173)
`plished using high-efficiency-particulate-air (HEPA)
`filters designed to remove organisms larger than 0.3
`µm from isolation rooms, operating rooms, and
`biological safety cabinets (BSCs) . Ionizing radiation
`used in microwaves and radiograph machines are
`short wavelength and high-energy gamma rays. Ioniz(cid:173)
`ing radiation is used for sterilizing disposables such as
`plastic syringes, catheters, or gloves before use.
`The most common chemical sterilant is ethylene
`oxide (EtO), which is used in gaseous form for steriliz(cid:173)
`ing heat-sensitive objects. Formaldehyde vapor and
`vapor-phase hydrogen peroxide (an oxidizing agent)
`have been used to sterilize HEPA filters in BSCs. Gluter-
`
`aldehyde, which is sporocidal (kills spores) in 3 to 10
`hours, is used for medical equipment such as broncho(cid:173)
`scopes, because it does not corrode lenses, metal, or
`rubber. Peracetic acid, effective in the presence of
`organic material, has also been used for the surface ster(cid:173)
`ilization of surgical instruments. The use of gluteralde(cid:173)
`hyde or peracetic acid is called cold sterilization.
`
`METHODS OF DISINFECTION
`
`PHYSICAL METHODS OF DISINFECTION
`The three physical methods of disinfection are:
`
`• Boiling at 100° C for 15 minutes, which kills veg(cid:173)
`etative bacteria
`• Pasteurizing at 63 ° C for 30 minutes or 72° C for
`15 seconds, which kills food pathogens
`• Using nonionizing radiation such as ultraviolet
`(UV) light
`
`UV rays are long wavelength and low energy.
`They do not penetrate well and organisms m ust
`have direct surface exposure, such as the working
`surface of a BSC, for this form of disinfection to
`work.
`
`Page 4
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`

`

`CHAPTER 12 LABORATORY CULTIVATION AND ISOLATION OF BACTERIA
`
`159
`
`A
`
`B
`
`c
`
`D
`
`FIGURE 12-7 Growth characteristics of various bacteria in thioglycollate broth . A, Facultatively anaerobic gram(cid:173)
`negative bacilli (i.e., those that grow in the presence or absence of oxygen) grow throughout broth. B, Gram-positive
`cocci grow as "puff balls." C, Strictly aerobic organisms (i.e., those that require oxygen for growth), such as
`Pseudomonas aeruginosa, grow toward the top of the broth. D, Strictly anaerobic organisms (i.e., those that do not
`grow in the presence of oxygen) grow in the bottom of the broth.
`
`that are not enteric
`many gram-negative bacilli
`pathogens and inhibits gram-positive organisms. A
`phenol red indicator in the medium detects increased
`acidity from carbohydrate (i.e., lactose, xylose, and
`sucrose) fermentation. Enteric pathogens, such as
`Shigella spp., do not ferment these carbohydrates so
`their colonies remain colorless (i.e., the same approxi(cid:173)
`mate pink to red color of the uninoculated medium).
`Colonies of Salmonella spp. are also colorless on
`XLD, because of the decarboxylation of lysine, which
`results in a pH increase that causes the pH indicator to
`turn red. These colonies often exhibit a black center
`that results from Salmonella spp. producing H 2S.
`Several of the nonpathogens ferment one or more of
`the sugars and produce yellow colonies (Figure 12-8).
`
`Preparation of artificial media
`Nearly all media are commercially available as ready(cid:173)
`to-use agar plates or tubes of broth. If media are not
`purchased, laboratory personnel can prepare agars
`and broths using dehydrated powders that are recon(cid:173)
`stituted in water (distilled or deionized) according to
`
`manufacturer's recommendations. Generally, media
`are reconstituted by dissolving a specified amount of
`media powder, which usually contains all necessary
`components, in water. Boiling is often required to
`dissolve
`the powder, but specific manufacturer's
`instructions printed in media package inserts should
`be followed exactly. Most media require sterilization
`so that only bacteria from patient specimens will
`grow and not those that are contaminants from water
`or the powdered media . Broth media are distributed
`to individual tu bes before sterilization. Agar media
`are usually sterilized in large flasks or bottles capped
`with either plastic screw caps or plugs before being
`placed in an autoclave.
`
`MEDIA STERILIZATION The timing of autoclave steriliza(cid:173)
`tion should start from the moment the temperature
`reaches 121° C and usually requires a minimum of 15
`minutes. Once the sterilization cycle is completed,
`molten agar is allowed to cool to approximately 50°
`C before being distributed to individual petri plates
`(usually 25 mL of molten agar per plate). If other
`
`Page 5
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`

`

`160
`
`PART 2 SCIENTIFIC AND LABORATORY BASIS FOR CLINICAL MICROBIOLOGY
`
`FIGURE 12-8 Differential capabi lities of XLD agar for lactose-fermenting, gram-negative bacilli (e.g., E. coli, arrow A).
`non lactose fermenters (e.g ., Shigella spp., arrow 8) and H2S producers (e.g., Salmonella spp., arrow C).
`
`ingredients are to be added (e.g., supplements such as
`sheep blood or specific vitamins, nutrients, or antibi(cid:173)
`otics) they should be incorporated when the molten
`agar has cooled, just before distribution to plates.
`Delicate media components that cannot with(cid:173)
`stand steam sterilization by autoclaving (e.g., serum,
`certain carbohydrate solutions, certain antibiotics, and
`other heat-labile substances) can be sterilized by
`membrane filtration. Passage of solutions through
`membrane filters with pores ranging in size from 0.2 to
`0.45 µm in diameter will not remove viruses but does
`effectively remove most bacterial and fungal contami(cid:173)
`nants. Finally, all media, whether purchased or
`prepared, must be subjected to stringent quality control
`before being used in the diagnostic setting (for more
`information regarding quality control see Chapter 6).
`
`CELL CULTURES Although most bacteria grow readily
`on artificial media, certain pathogens require factors
`provided only by living cells. These bacteria are obli(cid:173)
`gate intracellular parasites that require viable host
`cells for propagation. Although all viruses are oblig(cid:173)
`ate intracellular parasites, chlamydiae, rickettsiae, and
`rickettsiae-like organisms are bacterial pathogens that
`require living cells for cultivation.
`The cultures
`for growth of
`
`these bacteria
`
`comprise layers of living cells growing on the surface of
`a solid matrix such as the inside of a glass tube or the
`bottom of a plastic flask. The presence of bacterial
`is detected by
`pathogens within the cultured cells
`specific changes in the cells' morphology. Alternatively,
`specific stains, composed of antibody conjugates, may
`be used to detect bacterial antigens within the cells. Cell
`cultures may also detect certain bacterial toxins (e.g.,
`Clostridium diffi.cile cytotoxin). Cell cultures are most
`commonly used in diagnostic virology. Cell culture
`maintenance and inoculation is addressed in Part 7.
`
`ENVIRONMENTAL REQUIREMENTS
`Optimizing the environmental conditions to support
`the most robust growth of clinically relevant bacteria
`is as important as meeting the organism's nutritional
`needs for in vitro cultivation. The four most critical
`environmental factors to consider include oxygen and
`carbon dioxide (C0 2) availability, temperature, pH,
`and moisture content of medium and atmosphere.
`
`Oxygen and carbon dioxide availability
`Most clinically relevant bacteria are either aerobic,
`facultatively anaerobic, or strictly anaerobic. Aerobic
`bacteria use oxygen as a terminal electron acceptor
`and grow well in room air. Most clinically significant
`
`Page 6
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`