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47Principles of Diagnostic Medical Microbiology
C H A P T E R 
Diagnostic medical microbiology is concerned with the etio-
logic diagnosis of infection. Laboratory procedures used in 
the diagnosis of infectious disease in humans include the 
following:
1. Morphologic identification of the agent in stains of 
specimens or sections of tissues (light and electron 
microscopy).
2. Detection of the agent in patient specimens by antigen 
testing (latex agglutination, enzyme immunoassay, etc) 
or nucleic acid testing (nucleic acid hybridization, poly-
merase chain reaction [PCR], sequencing, etc).
3. Culture isolation and identification of the agent. Sus-
ceptibility testing of the agent by culture or nucleic acid 
methods, where appropriate.
4. Demonstration of meaningful antibody or cell-mediated 
immune responses to an infectious agent.
In the field of infectious diseases, laboratory test results 
depend largely on the quality of the specimen, the timing and 
the care with which it is collected and transported, and the 
technical proficiency and experience of laboratory person-
nel. Although physicians should be competent to perform a 
few simple, crucial microbiologic tests (perform direct wet 
mounts of certain specimens, make a Gram-stained smear 
and examine it microscopically, and streak a culture plate), 
the technical details of the more involved procedures are usu-
ally left to trained microbiologists. Physicians who deal with 
infectious processes must know when and how to take speci-
mens, what laboratory examinations to request, and how to 
interpret the results.
This chapter discusses diagnostic microbiology for bac-
terial, fungal, and viral diseases. The diagnosis of parasitic 
infections is discussed in Chapter 46.
COMMUNICATION BETWEEN 
PHYSICIAN AND LABORATORY
Diagnostic microbiology encompasses the detection and 
characterization of thousands of agents that cause or are 
associated with infectious diseases. The techniques used to 
characterize infectious agents vary greatly depending on the 
clinical syndrome and the type of agent being considered, 
be it virus, bacterium, fungus, or parasite. Because no single 
test will permit isolation or characterization of all potential 
pathogens, clinical information is much more important for 
diagnostic microbiology than it is for clinical chemistry or 
hematology. The clinician must make a tentative diagnosis 
rather than wait until laboratory results are available. When 
tests are requested, the physician should inform the laboratory 
staff of the tentative diagnosis (type of infection or infectious 
agent suspected). Proper labeling of specimens includes such 
clinical data as well as the patient’s identifying data (at least 
two methods of definitive identification) and the requesting 
physician’s name and pertinent contact information.
Many pathogenic microorganisms grow slowly, and days 
or even weeks may elapse before they are isolated and identi-
fied. Treatment cannot be deferred until this process is com-
plete. After obtaining the proper specimens and informing 
the laboratory of the tentative clinical diagnosis, the clinician 
should begin treatment with drugs aimed at the organism 
SECTION VII DIAGNOSTIC MEDICAL MICROBIOLOGY 
AND CLINICAL CORRELATION
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742 SECTION VII Diagnostic Medical Microbiology and Clinical Correlation
thought to be responsible for the patient’s illness. As the labo-
ratory staff begins to obtain results, they will inform health 
care providers, who can then reevaluate the diagnosis and 
clinical course of the patient and perhaps make changes in 
the therapeutic program. This “feedback” information from 
the laboratory consists of preliminary reports of the results 
of individual steps in the isolation and identification of the 
causative agent.
DIAGNOSIS OF BACTERIAL AND FUNGAL 
INFECTIONS
Specimens
Laboratory examination usually includes microscopic study 
of fresh unstained and stained materials and preparation of 
cultures with conditions suitable for growth of a wide vari-
ety of microorganisms, including the type of organism most 
likely to be causative based on clinical evidence. If a micro-
organism is isolated, complete identification may then be 
pursued. Isolated microorganisms may be tested for suscepti-
bility to antimicrobial drugs. When significant pathogens are 
isolated before treatment, follow-up laboratory examinations 
during and after treatment may be appropriate.
A properly collected specimen is the single most impor-
tant step in the diagnosis of an infection, because the results 
of diagnostic tests for infectious diseases depend on the selec-
tion, timing, and method of collection of specimens. Bacteria 
and fungi grow and die, are susceptible to many chemicals, 
and can be found at different anatomic sites and in different 
body fluids and tissues during the course of infectious dis-
eases. Because isolation of the agent is so important in the for-
mulation of a diagnosis, the specimen must be obtained from 
the site most likely to yield the agent at that particular stage 
of illness and must be handled in such a way as to favor the 
agent’s survival and growth. For each type of specimen, sug-
gestions for optimal handling are given in the following para-
graphs and in the later section on diagnosis by anatomic site.
Recovery of bacteria and fungi is most significant if the 
agent is isolated from a site normally devoid of microorgan-
isms (a normally sterile area). Any type of microorganism 
cultured from blood, cerebrospinal fluid (CSF), joint fluid, the 
pleural cavity, or peritoneal cavity is a significant diagnos-
tic finding. Conversely, many parts of the body have normal 
microbiota (Chapter 10) that may be altered by endogenous or 
exogenous influences. Recovery of potential pathogens from 
the respiratory, gastrointestinal, or genitourinary tracts; from 
wounds; or from the skin must be considered in the context of 
the normal microbiota of each particular site. Microbiologic 
data must be correlated with clinical information in order to 
arrive at a meaningful interpretation of the results.
A few general rules apply to all specimens:
1. The quantity of material must be adequate.
2. The sample should be representative of the infectious 
process (eg, sputum, not saliva; pus from the underlying 
lesion, not from its sinus tract; a swab from the depth of 
the wound, not from its surface).
3. Contamination of the specimen must be avoided by using 
only sterile equipment and aseptic technique.
4. The specimen must be taken to the laboratory and exam-
ined promptly. Special transport media may be needed.
5. Meaningful specimens to diagnose bacterial and fungal 
infections must be secured before antimicrobial drugs 
are administered. If antimicrobial drugs are given before 
specimens are taken for microbiologic study, drug therapy 
may have to be stopped and repeat specimens obtained 
several days later.
The type of specimen to be examined is determined by 
the presenting clinical picture. If symptoms or signs point 
to involvement of one organ system, specimens are obtained 
from that source. In the absence of localizing signs or symp-
toms, repeated blood samples for culturing are taken first, and 
specimens from other sites are then considered in sequence, 
depending in part on the likelihood of involvement of a given 
organ system in a given patient and in part on the ease of 
obtaining specimens.
Microscopy and Stains
Microscopic examination of stained or unstained specimens 
is a relatively simple and inexpensive, but much less sensitive 
method than culture for detection of small numbers of bacte-
ria. A specimen must contain at least 105 organisms per mil-
liliter before it is likely that organisms will be seen on a smear. 
Liquid medium containing 105 organisms per milliliter does 
not appear turbid to the eye. Specimens containing102–103 
organisms per milliliter produce growth on solid media, and 
those containing 10 or fewer bacteria per milliliter may pro-
duce growth in liquid media.
Gram staining is a very useful procedure in diagnos-
tic microbiology. Most specimens submitted when bacte-
rial infection is suspected should be smeared on glass slides, 
Gram-stained, and examined microscopically. The materials 
and method for Gram staining are outlined in Table 47-1. On 
microscopic examination, the Gram reaction (purple-blue 
indicates gram-positive organisms; red, gram-negative) 
and morphology (shape: cocci, rods, fusiform, or other; 
see Chapter 2) of bacteria should be noted. In addition, the 
presence or absence of inflammatory cells and the type of 
cell are important to note and quantify. Likewise, the pres-
ence of material that does not appear inflammatory, such as 
squamous epithelial cells obtained from a respiratory sample 
or wound, may be useful for determining the adequacy of 
the sample collection. The appearance of bacteria on Gram-
stained smears does not permit identification of species. 
Reports of gram-positive cocci in chains are suggestive of, 
but not definitive for, streptococcal species; gram-positive 
cocci in clusters suggest a staphylococcal species. Gram-neg-
ative rods can be large, small, or even coccobacillary. Some 
nonviable gram-positive bacteria can stain as gram negative. 
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CHAPTER 47 Principles of Diagnostic Medical Microbiology 743
Typically, bacterial morphology has been defined using 
organisms grown on agar. However, bacteria in body fluids or 
tissue can have highly variable morphology.
Specimens submitted for examination for mycobacteria 
should be stained for acid-fast organisms. The most sensi-
tive fluorescent stains for mycobacteria detection, such as 
auramine-rhodamine, should be used. Confirmation of a 
positive fluorescent stain is usually performed using one of the 
nonfluorescent acid-fast stains, either Ziehl-Neelsen stain or 
Kinyoun stain (Table 47-1). These stains can be used as alter-
natives to the fluorescent stains for mycobacteria in laboratories 
that lack fluorescence microscopy (see Chapter 23). Immuno-
fluorescent antibody (IF) staining is useful in the identification 
of many microorganisms. Such procedures are more specific 
than other staining techniques but also more cumbersome to 
perform. The fluorescein-labeled antibodies in common use 
are made from antisera produced by injecting animals with 
whole organisms or complex antigen mixtures. The resultant 
polyclonal antibodies may react with multiple antigens on the 
organism that was injected and may also cross-react with anti-
gens of other microorganisms or possibly with human cells in 
the specimen. Quality control is important to minimize nonspe-
cific IF staining. Use of monoclonal antibodies may circumvent 
the problem of nonspecific staining. IF staining is most useful 
in confirming the presence of specific organisms such as Borde-
tella pertussis or Legionella pneumophila in colonies isolated on 
culture media. The use of direct IF staining on specimens from 
patients is more difficult and less specific and is largely being 
replaced by nucleic acid amplification techniques (NAATs).
TABLE 47-1 Gram and Acid-Fast Staining Methods
Gram stain
 (1) Fix smear using heat or methanol.
 (2) Cover with crystal violet stain (10–30 seconds).
 (3) Rinse with water. Do not blot.
 (4) Counterstain with Gram’s iodine stain (10–30 seconds).
 (5) Rinse with water. Do not blot.
 (6) Decolorize with gentle agitation in 30% acetone-alcohol 
(10–30 seconds, until stain no longer flows off slide).
 (7) Rinse with water. Do not blot.
 (8) Cover with safranin stain (10–30 seconds).
 (9) Rinse with water and air or blot dry.
Ziehl-Neelsen acid-fast stain
 (1) Fix smear using heat.
 (2) Cover with carbolfuchsin, stain gently for 5 minutes over direct 
flame (or for 20 minutes over a water bath). Do not permit 
slides to boil or dry out.
 (3) Rinse with deionized water.
 (4) Decolorize in 3.0% acid-alcohol (95% ethanol and 
3.0% hydrochloric acid) until only a faint pink color remains.
 (5) Rinse with water.
 (6) Counterstain for 1 minute with Löffler’s methylene blue stain.
 (7) Rinse with deionized water and let dry.
Kinyoun carbolfuchsin acid-fast stain
 (1) Formula: 4 g basic fuchsin, 8 g phenol, 20 mL 95% alcohol, 
100 mL distilled water.
 (2) Stain fixed smear for 3 minutes (no heat necessary) and 
continue as with Ziehl-Neelsen stain.
Stains such as calcofluor white, methenamine silver, 
Giemsa, and occasionally periodic acid-Schiff (PAS) and oth-
ers are used for tissues and other specimens in which fungi 
or parasites are present. Such stains are not specific for given 
microorganisms, but they may define structures so that mor-
phologic criteria can be used for identification. Calcofluor 
white binds to cellulose and chitin in the cell walls of fungi 
and fluoresces under long-wavelength ultraviolet light. It may 
demonstrate morphology that is diagnostic of the species (eg, 
spherules with endospores in Coccidioides immitis infection). 
Pneumocystis jirovecii cysts are identified morphologically in 
silver-stained specimens. PAS is used to stain tissue sections 
when fungal infection is suspected. After primary isolation 
of fungi, stains such as lactophenol cotton blue are used to 
distinguish fungal growth and to identify organisms by their 
morphology.
Specimens to be examined for fungi can be examined 
unstained after treatment with a solution of 10% potassium 
hydroxide, which breaks down the tissue surrounding the 
fungal mycelia to allow a better view of the hyphal forms. 
Phase contrast microscopy is sometimes useful in unstained 
specimens. Dark-field microscopy is used to detect Treponema 
pallidum in material from primary or secondary syphilitic 
lesions or other spirochetes such as Leptospira.
Culture Systems
For diagnostic bacteriology, it is necessary to use several 
types of media for routine culture, particularly when the 
possible organisms include aerobic, facultatively anaerobic, 
and obligately anaerobic bacteria. The specimens and cul-
ture media used to diagnose the more common bacterial 
infections are listed in Table 47-2. The standard medium for 
specimens is blood agar, usually made with 5% sheep blood. 
Most aerobic and facultatively anaerobic organisms will grow 
on blood agar. Chocolate agar, a medium containing heated 
blood with or without supplements, is a second necessary 
medium; some organisms that do not grow on blood agar, 
including pathogenic Neisseria and Haemophilus, will grow 
on chocolate agar. A selective medium for enteric gram-
negative rods (either MacConkey agar or eosin–methylene 
blue [EMB] agar) is a third type of medium used routinely. 
These agars contain indicators that allow differentiation of 
lactose-fermenting organisms from non–lactose-fermenting 
organisms. Specimens to be cultured for obligate anaerobes 
must be plated on anaerobic media, such as brucella agar, a 
highly supplemented medium with hemin and vitamin K or 
a selective medium containing substances that inhibit the 
growth of enteric gram-negative rods and facultatively anaer-
obic or anaerobic gram-positive cocci.
Many other specialized media are used in diagnostic 
bacteriology; choices depend on the clinical diagnosis and 
the organism under consideration. The laboratory staff selects 
the specific media on the basis of the information in the cul-
ture request. Thus, freshly made Bordet-Gengou or charcoal-
containing medium is used to culture for B pertussis in the 
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TABLE 47-2 Common Localized Bacterial Infections
Disease Specimen Culture Media Common Causative Agents
Usual Microscopic 
Findings Comments
Cellulitis of skin Punch biopsy BA, CA Streptococcus pyogenes, 
Staphylococcus aureusOccasionally gram-
positive cocci
Biopsy at leading edge of erythema may yield the 
organism
Impetigo Pus, Swab BA, CA S pyogenes, S aureus As for cellulitis (above) 
and pharyngitis 
(below)
Often contains skin flora
Skin ulcers, deep Punch biopsy; deep 
tissue aspirate or 
biopsy
BA, CA, MAC/EMB, 
ANA
Mixed flora Mixed flora Often contain skin flora and gastrointestinal flora in 
below-the-waist ulcers
Meningitis CSF BA, CA Neisseria meningitidis Gram-negative 
intracellular 
diplococci
Adolescents, young adults
Haemophilus influenzae Small gram-negative 
coccobacilli
Adolescents, young adults
Streptococcus pneumoniae Gram-positive cocci 
in pairs
Adolescents, young adults
Group B streptococci Gram-positive cocci in 
pairs and chains
Infants
Escherichia coli, other 
Enterobacteriaceae species
Gram-negative rods Infants
Listeria monocytogenes Gram-positive rods Immunocompromised, pregnant women, infants; 
β-hemolytic
Brain abscess Pus BA, CA, MAC/EMB, 
ANA
Mixed infection; anaerobic 
gram-positive and gram-
negative cocci and rods, 
aerobic gram-positive cocci
Gram-positive cocci or 
mixed flora
Specimen must be obtained surgically and 
transported under anaerobic conditions
Perioral abscess Pus BA, CA, MAC/EMB, 
ANA
S aureus, S pyogenes, 
Actinomyces
Mixed flora Usually mixed bacterial infection; often contains oral 
flora
Pharyngitis Swab BA, CA, special 
media for 
Corynebacterium 
diphtheriae
S pyogenes Not recommended β-Hemolytic
C diphtheriae Not recommended Clinically suspected cases; diphtheroid toxicity 
testing required
Whooping cough 
(pertussis)
NP swab, nasal 
wash/aspirate, 
BAL
Regan-Lowe agar Bordetella pertussis Not recommended Fluorescent antibody test identifies organisms from 
culture and occasionally in direct smears; PCR 
more sensitive than culture
Epiglottitis Swab BA, CA H influenzae Usually not helpful H influenzae is part of normal microbiota in 
nasopharynx
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Pneumonia Sputum, BAL BA, CA, MAC/EMB S pneumoniae Many PMNs, gram-
positive cocci in 
pairs or chains. 
Capsule can be seen
S pneumoniae is part of normal microbiota in 
nasopharynx. Blood cultures positive in 10–20% 
of patients
S aureus Gram-positive cocci in 
clusters
Uncommon cause of pneumonia. Usually 
β-hemolytic, coagulase-positive
Enterobacteriaceae and other 
gram-negative rods
Gram-negative rods Hospital-associated pneumonia. Alcoholic 
pneumonia
Add ANA Mixed anaerobes and aerobes Mixed respiratory tract 
flora; sometimes 
many PMNs
Aspiration pneumonia, often associated with pleural 
effusion/abscess
Chest empyema Pleural fluid BA, CA. MAC/EMB, 
ANA
Same as pneumonia, or mixed 
flora infection
Mixed flora Usually pneumonia; mixed aerobic and anaerobic 
flora derived from oropharynx
Liver abscess Abscess fluid BA, CA, MAC/EMB, 
ANA
E coli; Bacteroides fragilis; mixed 
aerobic or anaerobic flora
Gram-negative rods 
and mixed flora
Commonly enteric gram-negative aerobes and 
anaerobes; consider Entamoeba histolytica 
infection
Cholecystitis Bile BA, CA, MAC/EMB, 
ANA
Gram-negative enteric aerobes, 
also B fragilis
Gram-negative rods Usually gram-negative rods from gastrointestinal 
tract
Abdominal or 
perirectal 
abscess
Abscess fluid BA, CA, MAC/EMB, 
ANA
Gastrointestinal flora Mixed flora Aerobic and anaerobic bowel flora; often more than 
five species grown
Enteric fever, 
typhoid
Blood, stool, urine BA, CA, MAC/EMB, 
Hektoen, enteric 
agar
Salmonella serovar Typhi Not recommended Multiple specimens should be cultured; lactose 
negative, H
2
S positive
Enteritis, 
enterocolitis, 
bacterial 
diarrheas
Stool MAC/EMB, Hektoen, 
enteric agar, 
Campylobacter 
agar
Salmonella species Gram stain or 
methylene blue 
stain may show 
PMNs
Non–lactose-fermenting colonies on TSI slants: 
Nontyphoid salmonellae produce acid and gas in 
butt, alkaline slant, and H
2
S
Shigella species Gram stain or 
methylene blue 
stain may show 
PMNs
Non–lactose-fermenting colonies on TSI slants: 
Shigellae produce alkaline slant, acid butt without 
gas or H
2
S
Campylobacter jejuni “Gull wing–shaped” 
gram-negative rods 
and often PMNs
Incubate at 42°C in Campylobacter gas; colonies 
oxidase-positive; smear shows “gull wing–shaped” 
rods
Add TCBS agar; Vibrio cholerae Not recommended Clinically suspected cases; yellow colonies on TCBS. 
V cholerae is oxidase-positive
Add TCBS agar Other Vibrio species Not recommended Differentiate from V cholerae by biochemical and 
culture tests
Add CIN agar Yersinia enterocolitica Not recommended Enrichment at 4°C helpful; incubate cultures at 25°C
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TABLE 47-2 Common Localized Bacterial Infections (Continued)
Disease Specimen Culture Media Common Causative Agents
Usual Microscopic 
Findings Comments
Hemorrhagic colitis 
and hemolytic 
uremic 
syndrome
Stool MAC/EMB, 
MacConkey 
sorbitol agar
E coli O157:H7 and other 
serotypes
Not recommended Look for sorbitol-negative colonies; type with 
antisera for O antigen 157 and flagellar antigen 
7; also EIAs or PCR for shiga-like toxins
Urinary tract 
infection
Urine (clean catch 
midstream, 
bladder 
catheterization 
or suprapubic 
aspiration)
BA, MAC/EMB E coli; Enterobacteriaceae 
species; other gram-negative 
rods
Gram-negative 
rods seen on 
stained smear of 
uncentrifuged urine 
indicate more than 
105 organisms/mL
Semiquantitative culture for urine bacterial load; 
E coli Gram-negative rods indole-positive; others 
require further biochemical tests; urinalysis shows 
leukocytes and/or nitrates present
Urethritis/cervicitis Swab BA, CA, Modified 
Thayer-Martin 
agar
Neisseria gonorrhoeae Intracellular gram-
negative diplococci 
in PMNs. Specific for 
N gonorrhoeae in 
men; less reliable in 
women
Positive stained smear diagnostic in men. Nucleic 
acid tests more sensitive than culture
Culture rarely 
performed
Chlamydia trachomatis PMNs with no 
associated gram-
negative diplococci
Nucleic acid tests more sensitive than culture
Genital ulcers Swab, lymph node 
aspirate
BA, CA, modified 
Thayer-Martin 
agar
Haemophilus ducreyi 
(chancroid)
Mixed flora Difficult to culture, diagnosis often made clinically
Treponema pallidum (syphilis) Dark-field or 
fluorescent 
antibody 
examination shows 
spirochetes but 
rarely available
Culture not performed, diagnosis made serologically 
(rapid plasma reagin [RPR], venereal disease 
research laboratory [VDRL] test, specific 
treponemal antibody tests)
C trachomatis, 
lymphogranuloma 
venereum (LGV) serovars
PMNs with no 
associated gram-
negative diplococci
Diagnosis made with nucleic acid test for 
C trachomatis, LGV serovars diagnosed serologically
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Pelvic inflammatory 
disease
Cervical swab, 
pelvic aspirate
BA, CA, modified 
Thayer-Martin 
agar
N gonorrhoeae PMNs with associated 
gram-negative 
diplococci; mixed 
flora may be 
present
Nucleic acid tests more sensitive than culture
C trachomatis PMNs without 
associated gram-
negative diplococci
Nucleic acid test more sensitive than culture
Add ANA Mixed flora Mixed flora Usually mixed anaerobic and aerobic bacteria
Arthritis Synovial fluid BA, CA S aureus Gram-positive cocci in 
clusters
Coagulase-positive; usually β-hemolytic
Add modified 
Thayer-Martin 
medium
N gonorrhoeae Gram-negative 
diplococci in PMNs
Add ANA Others Variable morphology Includes streptococci, gram-negative rods, and 
anaerobes
ANA, anaerobe agar (Brucella agar); BA, blood agar; BAL, bronchoalveolar lavage fluid; CA, chocolate agar; CIN, cefsulodin-irgasan-novobiocin medium; EIA, enzyme immunoassay; EMB, eosin-methylene blue agar; MAC, 
MacConkey agar; TCBS, thiosulfate-citrate-bile salts-sucrose agar; TSI, triple sugar iron agar.
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diagnosis of whooping cough, and other special media are 
used to culture for Vibrio cholerae, Corynebacterium diphthe-
riae, Neisseria gonorrhoeae, and Campylobacter species. For 
culture of mycobacteria, specialized solid and liquid media 
are commonly used. These media may contain inhibitors of 
other bacteria. Because many mycobacteria grow slowly, the 
cultures must be incubated and examined periodically for 
weeks (see Chapter 23).
Broth cultures in highly enriched media are important 
for back-up cultures of biopsy tissues and body fluids such 
as CSF. Broth cultures may give positive results when there 
is no growth on solid media because of the small number of 
bacteria present in the inoculum (see above).
Many yeasts will grow well on blood agar. Biphasic and 
mycelial phase fungi grow better on media designed specifi-
cally for fungi. Brain–heart infusion agar, with and without 
antibiotics, and inhibitory mold agar have largely replaced 
the traditional use of Sabouraud’s dextrose agar to grow 
fungi. Media made with plant and vegetable materials, the 
natural habitats for many fungi, also grow many fungi that 
cause infections. Cultures for fungi are commonly done in 
paired sets, one set incubated at 25–30°C and the other at 
35–37°C. Table 47-3 outlines specimens and other tests to be 
used for the diagnosis of fungal infections.
In addition to the above standard and selective media, 
agars that incorporate antibiotics and chromogenic enzyme 
substrates that impart color to specific organisms of inter-
est, such as methicillin-resistant Staphylococcus aureus and 
various Candida species, among many others, are available. 
These media, while more expensive, do enhance sensitivity by 
inhibiting background microbiota and allowing the pathogen 
of interest to be more easily recognized. Typically, these chro-
mogenic agars are used for specimens such as surveillance 
cultures and cultures of urine.
Antigen Detection
Immunologic systems designed to detect antigens of micro-
organisms can be used in the diagnosis of specific infections. 
Immunofluorescent tests (direct and indirect fluorescent 
antibody tests) are one form of antigen detection and are dis-
cussed in separate sections in this chapter and in the chapters 
on the specific microorganisms.
Enzyme immunoassays (EIAs), including enzyme-
linked immunosorbent assays (ELISA), and agglutination 
tests are used to detect antigens of infectious agents present in 
clinical specimens. The principles of these tests are reviewed 
briefly here.
There are many variations of EIAs to detect antigens. One 
commonly used format is to bind a capture antibody, specific 
for the antigen in question, to the wells of plastic microdilu-
tion trays. The specimen containing the antigen is incubated 
in the wells followed by washing of the wells. A second anti-
body for the antigen, labeled with enzyme, is used to detect 
the antigen. Addition of the substrate for the enzyme allows 
detection of the bound antigen by colorimetric reaction. 
A significant modification of EIAs is the development of 
immunochromatographic membrane formats for antigen 
detection. In this format, a nitrocellulose membrane is used 
to absorb the antigen from a specimen. A colored reaction 
appears directly on the membrane with sequential addition 
of conjugate followed by substrate. In some formats, the anti-
gen is captured by bound antibody directed against the anti-
gen. These assays have the advantage of being rapid and also 
frequently include a built-in internal control. EIAs are used 
to detect viral, bacterial, chlamydial, protozoan, and fungal 
antigens in a variety of specimen types such as stool, CSF, 
urine, and respiratory samples. Examples of these are dis-
cussed in the chapters on the specific etiologic agents.
In latex agglutination tests, an antigen-specific antibody 
(either polyclonal or monoclonal) is fixed to latex beads. 
When the clinical specimen is added to a suspension of the 
latex beads, the antibodies bind to the antigens on the micro-
organism forming a lattice structure, and agglutination of 
the beads occurs. Coagglutination is similar to latex agglu-
tination except that staphylococci rich in protein A (Cowan I 
strain) are used instead of latex particles; coagglutination is 
less useful for antigen detection compared with latex aggluti-
nation but is helpful when applied to identification of bacteria 
in cultures such as Streptococcus pneumoniae, Neisseria men-
ingitidis, N gonorrhoeae, and β-hemolytic streptococci.
Latex agglutination tests are primarily directed at the 
detection of carbohydrate antigens of encapsulated microor-
ganisms. Antigen detection is used most often in the diagnosis 
of group A streptococcal pharyngitis. Detection of cryptococ-
cal antigen is useful in the diagnosis of cryptococcal meningitis 
in patients with AIDS or other immunosuppressive diseases.
The sensitivity of latex agglutination tests in the diag-
nosis of bacterial meningitis is not better than that of Gram 
stain, which is approximately 100,000 bacteria per milliliter. 
For that reason, the latex agglutination test is not recom-
mended for direct CSF specimen testing.
Serological Testing
Detection of specific antibodies to infectious agents can be 
useful for diagnosis of acute or chronic infections, and for 
investigating the epidemiology of infectious disease. During 
the course of illness, IgM antibody is first produced, followed 
by appearance of IgG antibody. Caution must be used when 
interpreting positive IgM results, as these assays demonstrate 
cross-reactivity and can be falsely positive. Serology is most 
useful when acute and convalescent sera are tested to show 
increases in antibody titers over time.
There are a variety of serological assays available, includ-
ing direct immunofluorescence, agglutination, complement 
fixation (CF), EIA, and ELISA formats. There are also non-
specific immunoassays available, such as the heterophile test 
for EBV mononucleosis and rapid plasma reagin for syphilis. 
Several of these tests can measure antibody titer by perform-
ing dilutions of patient serum to determine the lowest titer at 
which reactivity is seen.
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TABLE 47-3 Common Fungal Infections and Nocardiosis: Agents, Specimens, and Diagnostic Tests
Specimen Serologic and Other Tests Comments
Invasive (deep-seated) mycoses
Aspergillosis: Aspergillus fumigatus, other Aspergillus species
 Pulmonary Sputum, BAL Culture, serum/BAL galactomannan 
assays
Must distinguish colonization from infection
 Disseminated Biopsy specimen, blood As above Aspergillus is difficult to grow from blood and body fluids of patients 
with disseminated infection.
Blastomycosis: Blastomyces dermatitidis
 Pulmonary Sputum, BAL Culture, serology Serology useful to determine exposure; definitive diagnosis requires 
culture; yeast show broad-based budding.
 Oral and cutaneous ulcers Biopsy or swab specimen Culture, serology
 Bone Bone biopsy Culture, serology
Coccidioidomycosis: Coccidioides immitis
 Pulmonary Sputum, BAL Culture, serology Serology often more sensitive than culture; positive immunodiffusion 
can be followed by complementation fixation titers; C immitis can 
grow on routine bacterial cultures and pose laboratory exposure 
risk.
 Disseminated Biopsy specimen, CSF As above
Histoplasmosis: Histoplasma capsulatum
 Pulmonary Sputum, BAL Culture, serology, urine antigen test Serology useful to determine exposure; definitive diagnosis requires 
culture.
 Disseminated Bone marrow, biopsy specimen As above Pathology shows small intracellular yeast forms distinguished from 
Leishmania by the absence of kinetoplast.
Nocardiosis: Nocardia asteroides complex and other Nocardia species
 Pulmonary Sputum, BAL Culture, modified acid-fast stain Nocardiae are bacteriathat clinically behave like fungi; weakly acid-
fast, branching, filamentous gram-positive rods.
 Subcutaneous Aspirate or biopsy of abscess
 Brain Material from brain abscess
Paracoccidioidomycosis (South American blastomycosis): Paracoccidioides brasiliensis
Biopsy specimen Culture, serology Serology useful to determine exposure; definitive diagnosis requires 
culture.
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TABLE 47-3 Common Fungal Infections and Nocardiosis: Agents, Specimens, and Diagnostic Tests (Continued)
Specimen Serologic and Other Tests Comments
Sporotrichosis: Sporothrix schenckii
 Skin and subcutaneous 
 nodules
Biopsy specimen Culture, serology Soil and gardening exposure
 Disseminated Biopsy specimen
Zygomycosis: Rhizopus species, Mucor species, others
 Rhinocerebral Nasal-orbital tissue Culture Nonseptate hyphae seen in microscopic sections
 Cutaneous; pulmonary and 
 disseminated
Sputum, BAL, biopsy specimens Culture
Yeast infections
Candidiasis: Candida albicans and other Candida speciesa
 Mucous membrane Oral swab, vaginal swab, biopsy 
specimen
Culture, yeast wet mount Vaginal candidiasis diagnosed clinically and by Gram stain (Nugent 
criteria)
 Skin Swab, biopsy specimen Culture
 Systemic Blood, biopsy specimen, urine Culture Candida and other yeast species grow well in routine bacterial cultures.
Cryptococcosis: Cryptococcus neoformans
 Pulmonary Sputum, BAL Culture, cryptococcal antigen Most common in immunocompromised patients
 Meningitis CSF Culture, cryptococcal antigen
 Disseminated Bone marrow, bone, blood, other Culture, cryptococcal antigen
Primary skin infections
Dermatophytosis: Microsporum species, Epidermophyton species, Trichophyton species
Hair, skin, nails from infected sites Culture Requires specialized dermatophyte agars
aCandida tropicalis, Candida parapsilosis, Candida glabrata, and other Candida species.
BAL, bronchoalveolar fluid; CF, complement fixation; CSF, cerebrospinal fluid; EIA, enzyme immunoassays.
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Western Blot Immunoassays
These assays are usually performed to detect antibodies against 
specific antigens of a particular organism. This method is 
based on the electrophoretic separation of major proteins of 
the organism in question in a two-dimensional agarose gel. 
Organisms are mechanically or chemically disrupted, and 
resultant solubilized antigen of the organism is placed in a 
polyacrylamide gel. An electric current is applied, and major 
proteins are separated out on the basis of size (smaller pro-
teins travel faster). The protein bands are transferred to strips 
of nitrocellulose paper. Following incubation of the strips with 
a patient’s specimen containing antibody (usually serum), the 
antibodies bind to the proteins on the strip and are detected 
enzymatically in a fashion similar to the EIA methods 
described earlier. Western blot tests are used as specific tests 
for antibodies in HIV infection and Lyme disease.
Molecular Diagnostics
A. Nucleic Acid Hybridization Probes
The principle behind hybridization probe molecular assays is 
the hybridization of a characterized nucleic acid probe to a 
specific nucleic acid sequence in a test specimen followed by 
detection of the paired hybrid. For example, single-stranded 
probe DNA (or RNA) is used to detect complementary RNA 
or denatured DNA in a test specimen. The nucleic acid probe 
typically is labeled with enzymes, antigenic substrates, che-
miluminescent molecules, or radioisotopes to facilitate detec-
tion of the hybridization product. By carefully selecting the 
probe or making a specific oligonucleotide and performing 
the hybridization under conditions of high stringency, detec-
tion of the nucleic acid in the test specimen can be extremely 
specific. Such assays are currently used primarily for rapid 
confirmation of a pathogen once growth is detected (eg, 
the identification of Mycobacterium tuberculosis in culture 
using a DNA probe). In situ hybridization involves the use of 
labeled DNA probes or labeled RNA probes to detect comple-
mentary nucleic acids in formalin-fixed paraffin-embedded 
tissues, frozen tissues, or cytologic preparations mounted on 
slides. Technically, this can be difficult and is usually per-
formed in histology laboratories and not clinical microbiol-
ogy laboratories. However, this technique has increased the 
knowledge of the biology of many infectious diseases, espe-
cially the hepatitides and oncogenic viruses, and is still use-
ful in infectious diseases diagnosis. A novel technique that is 
somewhat of a modification of in situ hybridization makes 
use of peptide nucleic acid probes. Peptide nucleic acid probes 
are synthesized pieces of DNA in which the sugar phosphate 
backbone of DNA (normally negatively charged) is replaced 
by a polyamide of repetitive units (neutral charge). Individual 
nucleotide bases can be attached to the now neutral back-
bone, which allows for faster and more specific hybridization 
to complementary nucleic acids. Because the probes are syn-
thetic, they are not subject to degradation by nucleases and 
other enzymes. These probes can be used for detection of 
S aureus, enterococci, certain Candida spp., and some gram-
negative bacilli from positive blood culture bottles. The probe 
hybridization is detected by fluorescence and is called peptide 
nucleic acid–fluorescence in situ hybridization (PNA-FISH).
B. Bacterial Identification Using 16S rRNA Probe 
Hybridization
The 16S rRNA of each species of bacteria has stable (con-
served) portions of the sequence. Many copies are present in 
each organism. Labeled probes specific for the 16S rRNA of 
a species are added, and the amount of label on the double-
stranded hybrid is measured. This technique is widely used 
for the rapid identification of many organisms. Examples 
include the most common and important Mycobacterium 
species, C immitis, Histoplasma capsulatum, and others.
Molecular diagnostic assays that use amplification of 
nucleic acid have become widely used and are evolving rap-
idly. They have been used on a variety of sample types includ-
ing direct patient specimens, positive cultures, and isolated 
organisms. These amplification systems fall into several basic 
categories as outlined below.
C. Target Amplification Systems
In these assays, the target DNA or RNA is amplified many 
times. The polymerase chain reaction (PCR) is used to 
amplify extremely small amounts of specific DNA present in 
a clinical specimen, making it possible to detect what were 
initially minute amounts of the DNA. PCR uses a thermo-
stable DNA polymerase to produce a twofold amplification 
of target DNA with each temperature cycle. Conventional 
PCR, also referred to as end detection PCR, utilizes three 
sequential reactions—denaturation, annealing, and primer 
extension—as follows. The DNA extracted from the clinical 
specimen along with sequence-specific oligonucleotide prim-
ers, nucleotides, thermostable DNA polymerase, and buffer 
are heated to 90–95°C to denature (separate) the two strands 
of the target DNA. The temperature in the reaction is low-
ered, usually to 45–60°C depending on the primers, to allow 
annealing of the primers to the target DNA. Each primer 
is then extended by the thermostable DNA polymerase by 
adding nucleotides complementary to the target DNA yield-
ing the twofold amplification. The cycle is then repeated 
30–40 times to yield amplification of the target DNA seg-
ment by more than 1010-fold. The amplified segment often 
can be seen in an electrophoretic gel or detected by Southern 
blot analysis using labeled DNA probes specific for the seg-
ment or by a variety of proprietary commercial techniques. 
More recently, real-time PCR protocols have replaced these 
end detection methods (see below).
PCRcan also be performed on RNA targets, which is 
called reverse transcriptase PCR. The enzyme reverse tran-
scriptase is used to transcribe the RNA into complementary 
DNA for subsequent PCR amplification.
PCR assays are available commercially for identifica-
tion of a broad range of bacterial and viral pathogens such as 
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Chlamydia trachomatis, N gonorrhoeae, M tuberculosis, cyto-
megalovirus (CMV), HIV-1, hepatitis C virus, and many oth-
ers. There are many other laboratory-developed PCR assays 
that have been implemented by individual laboratories to 
diagnose infections. Such assays are the tests of choice to diag-
nose many infections—especially when traditional culture 
and antigen detection techniques do not work well. Examples 
include testing of CSF for herpes simplex virus (HSV) to diag-
nose herpes encephalitis and testing of nasopharyngeal sam-
ples to diagnose B pertussis infection (whooping cough).
A major consideration for laboratories that perform PCR 
assays is to prevent contamination of reagents or specimens 
with target DNA from the environment, which can obscure 
the distinction between truly positive results and falsely posi-
tive ones because of the contamination.
E. Signal Amplification Techniques
These assays strengthen the signal by amplifying the label (eg, 
fluorochromes, enzymes) that is attached to the target nucleic 
acid. The branched DNA (bDNA) system has a series of pri-
mary probes and a branched secondary probe labeled with 
enzyme. Multiple oligonucleotide probes specific for the tar-
get RNA (or DNA) are fixed to a solid surface such as a micro-
dilution tray. These are the capture probes. The prepared 
specimen is added, and the RNA molecules are attached to 
the capture probes on the microdilution tray. Additional tar-
get probes bind to the target but not to the tray. The enzyme-
labeled bDNA amplifier probes are added and attach to the 
target probes. A chemiluminescent substrate is added, and 
light emitted is measured to quantitate the amount of tar-
get RNA present. Examples of the use of this type of assay 
include the quantitative measurement of HIV-1, hepatitis C 
virus, and hepatitis B virus.
F. Amplification Methods: Non–PCR-Based
The transcription-mediated amplification (TMA) and the 
nucleic acid sequence–based amplification (NASBA) sys-
tems amplify large quantities of RNA in isothermal assays 
that coordinately use the enzymes reverse transcriptase, 
RNase H, and RNA polymerase. An oligonucleotide primer 
containing the RNA polymerase promoter is allowed to bind 
to the RNA target. The reverse transcriptase makes a single-
stranded cDNA copy of the RNA. The RNase H destroys 
the RNA of the RNA–cDNA hybrid, and a second primer 
anneals to the segment of cDNA. The DNA-dependent DNA 
polymerase activity of reverse transcriptase extends the 
DNA from the second primer, producing a double-stranded 
DNA copy, with intact RNA polymerase. The RNA polymerase 
then produces many copies of the single-stranded RNA. Detec-
tion of C trachomatis, N gonorrhoeae, and M tuberculosis and 
quantitation of HIV-1 viral loads are examples of the use of 
these types of assays.
Strand displacement assays (SDA) are isothermal ampli-
fication assays that employ use of restriction endonuclease 
and DNA polymerase. The restriction endonuclease “nicks” 
the DNA at specific sites allowing DNA polymerase to initiate 
replication at the nicks on the target molecule and simultane-
ously displacing the nicked strand. Displaced single strands 
then serve as templates for additional amplification.
Loop-mediated isothermal amplification (LAMP) gets 
its name from the fact that the final amplification product 
consists of a structure that contains multiple loops (repeats) of 
the target sequence. The reaction is isothermal and consists of 
autocycling strand displacement DNA synthesis using Bst DNA 
polymerase and four to six primers. Amplification products 
can be detected in real time by precipitating DNA by adding 
magnesium pyrophosphate to the reaction creating turbidity 
that can be read visually or by using a spectrophotometer. This 
method is very sensitive, detecting as few as 10 target copies 
per reaction. A commercial assay using LAMP technology for 
the detection of Clostridium difficile in stool samples and other 
pathogens in a variety of specimen types is available.
G. Real-Time PCR
Technologic advances, which have lead to “real-time ampli-
fication,” have streamlined nucleic acid amplification plat-
forms, improved the sensitivity of amplification tests, and 
drastically reduced the potential for contamination. Dramatic 
improvements in the chemistry of nucleic acid amplification 
reactions have resulted in homogeneous reaction mixtures 
in which fluorogenic compounds are present in the same 
reaction tube in which the amplification occurs. A variety 
of fluorogenic molecules are used. These include nonspecific 
dyes such as SYBR green, which binds to the minor groove 
of double-stranded DNA, and amplicon-specific detection 
methods using fluorescently labeled oligonucleotide probes, 
which fall into three categories: TaqMan or hydrolysis probes; 
fluorescence energy transfer (FRET) probes; and molecular 
beacons. The signal from these probes is proportional to the 
amount of product DNA present in the reaction and is plot-
ted against the PCR cycle. Use of a threshold value allows 
determination of positive and negative reactions. The signal 
is measured through the closed reaction tube using fluores-
cent detectors; hence, the assay is performed in “real time.” 
Since the reaction tube does not need to be opened to analyze 
the PCR products on a gel, there is much less risk of amplicon 
carry-over to the next reaction. When used with a standard 
curve, real-time PCR assays can be quantitative, allowing for 
determination of organism concentration. These assays are 
commonly used for viral load quantification of HIV, hepatitis 
C virus, hepatitis B virus, and CMV. The reader is referred 
to the Persing et al reference for more detailed information 
about real-time PCR and other molecular methods.
H. PCR-Sequencing
The product of a PCR reaction can be sequenced and com-
pared to a database for identification of organisms or resis-
tance mutations. PCR primers are designed to hybridize 
to conserved genomic regions, with the sequence of inter-
est amplified between the primers. A variety of sequencing 
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CHAPTER 47 Principles of Diagnostic Medical Microbiology 753
methods can be used, the discussion of which is beyond the 
scope needed here.
For bacterial identification, sequencing of the 16S rRNA 
gene is commonly used. This gene has highly conserved 
regions interspersed with variable sequences, making it ideal 
for amplifying and differentiating many bacterial species. 
Other conserved gene targets are also used for bacterial iden-
tification, including rpoB, sodA, and hsp65. Similarly, fun-
gal identification can be performed using PCR-sequencing 
of 28S rRNA gene and ribosomal RNA gene internal tran-
scribed spacer elements.
PCR-sequencing is also used for strain typing and detec-
tion of specific resistance mutations in viruses (see Diagnosis 
of Viral Infections section below). Its use is expanding to gene 
characterization in other organisms, such as the detection of 
certain mutations causing rifampin or isoniazid resistance in 
M tuberculosis.
I. Microarrays
Nucleic acid microarrays involve the use of multiple oligonu-
cleotide probes to detect the complementary target sequence 
in amplified DNA or RNA. The arrays can have from tens 
to hundreds of thousands of probes (high-density microar-
rays), and yield substantial information about the genetic 
makeup of specific organisms. Patient samples or clinicaliso-
lates are subject to DNA amplification labeling followed by 
hybridization, washing, and detection of labeled DNA bound 
to specific probes. Microarrays can be used to detect micro-
organisms directly from patient samples or positive blood 
cultures through the use of conserved targets such as 16S 
ribosomal DNA probes. They can also provide genetic pro-
filing of isolated organisms, yielding information about the 
genotype, virulence factors, or resistance markers present in 
the organism.
J. High-Throughput Sequencing
High-throughput sequencing (also known as next-generation 
or “deep” sequencing) involves the simultaneous sequencing 
of a large number of DNA molecules (known as a library). 
The source of the library can be an organism isolate or direct 
patient sample. Several different instrument platforms are 
available, and can generate thousands to millions of sequence 
reads per sample. Bioinformatic algorithms are then used 
to classify, assemble, and compare the sequence to known 
organism databases. High-throughput sequencing can be 
used to assemble entire organism genomes, define the micro-
biome, detect infectious agents, or look for low-level sequence 
variants, known as quasispecies. Database comparison can be 
used to classify the organism subtype or determine the pres-
ence of markers of drug resistance or virulence.
Mass Spectrometry
Mass spectrometry (MS), a technology used to analyze pro-
teins or DNA, has revolutionized the approach to microbial 
identification in clinical laboratories. MS employs methods 
such as ionization radiation to disrupt material, forming 
charged particles that are identified in various ways on the 
basis of mass or mass-to-charge ratio. Applications in micro-
biology have been made possible by advances in technology, 
such as matrix-assisted laser desorption ionization time-of-
flight mass spectroscopy (MALDI-TOF MS). Several different 
methods are briefly described below.
MassTag PCR incorporates a tag of known mass 
(a library of 64 mass tags is commercially available) into the 
PCR product. Most frequently used in multiplex PCR reac-
tions, the tags are released by ultraviolet (UV) irradiation and 
analyzed by MS. The identity of the desired target or targets is 
determined by the size of the tag(s).
PCR electrospray ionization mass spectrometry (PCR-
ESI-MS) uses a unique principle. Briefly, for particular 
microbes, a set of PCR primers is designed that amplifies 
key regions of the microbial genome. Multiple PCR reac-
tions are conducted in a microtiter plate for analysis of each 
sample, and some of the wells contain more than one primer 
pair. Following PCR, the microtiter plate is placed on a fully 
automated instrument and ESI-MS analysis is performed. 
The mass spectrometer is an analytical tool that effectively 
weighs the amplicons, or mixture of amplicons, with suffi-
cient mass accuracy that the composition of A, G, C, and 
T can be deduced for each amplicon in the PCR reaction. The 
determined composition is then interrogated using a propri-
etary software database.
The above two platforms allow for direct detection of 
the nucleic acid of microbes directly from clinical samples 
without a culture step because they use a PCR amplification 
reaction. Another application is using MALDI-TOF to iden-
tify bacteria and yeast isolates recovered in clinical cultures. 
These platforms target the highly abundant ribosomal pro-
teins of bacteria and yeast. The assays involve making a thin 
smear from a colony or broth culture onto a metallic slide of 
the organism to be identified and applying an acid matrix to 
it. The slide is placed into the instrument where the organ-
ism mixture is hit by laser pulses. Charged protein fragments 
are produced and accelerated through an electrostatic field 
in a vacuum tube until they contact the mass spectrometer’s 
detector. Molecules of different masses and charges “fly” at 
different speeds (“time of flight”). A spectral signature, gen-
erally in the range of 1000–20,000 mass-to-charge ratio (m/z), 
is generated. This spectral signal is compared to others in the 
proprietary databases of each instrument for genus or species 
assignment of the organism. The reader is referred to the Patel 
reference for additional details.
THE IMPORTANCE OF NORMAL 
BACTERIAL AND FUNGAL MICROBIOTA
Organisms such as M tuberculosis, Salmonella serovar Typhi, 
and Brucella species are considered pathogens whenever they 
are found in patient specimens. However, many infections are 
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754 SECTION VII Diagnostic Medical Microbiology and Clinical Correlation
caused by organisms that are permanent or transient mem-
bers of the normal microbiota. For example, Escherichia coli 
is part of the normal gastrointestinal microbiota, but is also 
the most common cause of urinary tract infections. Similarly, 
the vast majority of mixed bacterial infections with anaer-
obes are caused by organisms that are members of the normal 
microbiota.
The relative numbers of specific organisms found in a 
culture are important when members of the normal micro-
biota are the cause of infection. When numerous gram- 
negative rods of species such as Klebsiella pneumoniae are 
found mixed with a few normal nasopharyngeal bacteria in a 
sputum culture, the gram-negative rods are strongly suspect 
as the cause of pneumonia because large numbers of gram-
negative rods are not normally found in sputum or in the 
nasopharyngeal microbiota; the organisms should be iden-
tified and reported. In contrast, abdominal abscesses com-
monly contain a normal distribution of aerobic, facultatively 
anaerobic, and obligately anaerobic organisms, representa-
tive of the gastrointestinal microbiota. In such cases, iden-
tification of all species present is not warranted; instead, it 
is appropriate to report “normal gastrointestinal microbiota.”
Yeasts in small numbers are commonly part of the nor-
mal microbial microbiota. However, other fungi are not 
normally present and therefore should be identified and 
reported. Viruses usually are not part of the normal micro-
biota as detected in diagnostic microbiology laboratories, but 
can be found in otherwise healthy individuals, presumably as 
asymptomatic infections. Latent viruses such as herpes sim-
plex or CMV, or live vaccine viruses such as poliovirus can be 
detected in asymptomatic cases. In some parts of the world, 
stool specimens commonly yield evidence of parasitic infec-
tion without symptoms present. Therefore, the clinical pre-
sentation of infectious disease illness along with the relative 
number of potentially pathogenic organisms is important in 
establishing the correct diagnosis.
Members of the normal microbiota that are most com-
monly present in patient specimens and that may be reported 
as “normal microbiota” are discussed in Chapter 10.
LABORATORY AIDS IN THE SELECTION 
OF ANTIMICROBIAL THERAPY
The antimicrobial drug used initially in the treatment of an 
infection is chosen on the basis of clinical impression after the 
clinician is convinced that an infection exists and has made 
a tentative etiologic diagnosis on clinical grounds. On the 
basis of this “best guess,” a drug that is likely to be effective 
against the suspect agent(s) can be selected (see Chapter 28). 
Before this drug is administered, specimens are obtained 
for laboratory detection of the causative agent. The results 
of these examinations may allow for narrowing of antibiot-
ics to targeted therapy (as opposed to broad gram-positive 
and gram-negative coverage for sepsis). The identification of 
certain microorganisms that are uniformly drug-susceptible 
eliminates the necessity for further testing and permits the 
selection of optimally effective drugs based on the organism’s 
known susceptibility profile. When the organism resistance 
profile is varied, tests for drug susceptibility of isolated micro-
organisms will guideoptimal drug choice (see Chapter 28). 
Disk diffusion susceptibility tests measure the ability of 
bacteria to grow on the surface of an agar plate in the pres-
ence of paper disks containing antibiotic drug. The drug 
diffuses out into the surrounding agar, inhibiting bacterial 
growth in a circular area surrounding the disk. The diameter 
of this zone of growth inhibition is measured, and correlates 
with the susceptibility of the isolate being tested. The choice 
of drugs to be included in a routine susceptibility test battery 
should be based on the susceptibility patterns of isolates in 
the laboratory, the type of infection (community-acquired 
or nosocomial), the source of the infection, and cost-
effectiveness analysis for the patient population. The Clinical 
and Laboratory Standards Institute (CLSI) (Wayne, PA) pro-
vides recommendations for which agents to test based on the 
organism recovered and the specimen type, and interpretive 
criteria (susceptible, intermediate, or resistant) based on the 
measured zone size.
The sizes of zones of growth inhibition vary with the 
pharmacologic characteristics of different drugs. Thus, the 
zone size of one drug cannot be compared to the zone size 
of another drug acting on the same organism. However, for 
any one drug the zone size can be compared to a standard, 
provided that media, inoculum size, and other conditions are 
carefully controlled. This makes it possible to define for each 
drug a diameter of inhibition zone that distinguishes suscep-
tible from intermediate or resistant strains.
The disk test measures the ability of drugs to inhibit the 
growth of bacteria in vitro. The results correlate reasonably 
well with therapeutic response in those disease processes in 
vivo when body defenses can eliminate infectious microor-
ganisms, but may be less well correlated with response in 
immunocompromised patients. The selection of appropri-
ate antibiotic therapy depends on clinical as well as bacterial 
factors, such as use of bactericidal rather than bacteriostatic 
drugs for endocarditis, or drugs that will penetrate the 
blood–brain barrier for central nervous system infections 
(see Chapter 28). Minimum inhibitory concentration (MIC) 
tests measure the ability of organism to grow in broth culture 
in the presence of various dilutions of antibiotics. It measures 
more exactly the concentration of an antibiotic necessary to 
inhibit growth of a standardized inoculum under defined 
conditions. A semiautomated microdilution method is used 
in which defined amounts of drug are dissolved in a mea-
sured small volume of broth and inoculated with a standard-
ized number of microorganisms. The end point, or MIC, is 
considered the last broth cup (lowest concentration of drug) 
remaining clear, ie, free from microbial growth. The MIC 
provides a better estimate of the probable amount of drug 
necessary to inhibit growth in vivo and thus helps in gaug-
ing the dosage regimen necessary for the patient. Guidelines 
available from CLSI provide interpretive criteria, defining 
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CHAPTER 47 Principles of Diagnostic Medical Microbiology 755
strains as resistant, intermediate, or susceptible to a certain 
drug based on the MIC.
The MIC only shows that bacterial growth is inhibited at 
that drug concentration; there may still be viable bacteria that 
can recover when the drug is removed. Bactericidal effects can 
be estimated by subculturing the clear broth from MIC test-
ing onto antibiotic-free solid media. The result, eg, a reduction 
of colony-forming units by 99.9% below that of the control, is 
called the minimal bactericidal concentration (MBC).
Because empiric therapy must often be given before the 
results of antimicrobial susceptibility tests are available, it is 
recommended by CLSI that laboratories publish an antibio-
gram annually that contains the results of susceptibility test-
ing in aggregate for particular organism–drug combinations. 
For example, it may be important to know the most active 
β-lactam antimicrobial agent targeted against Pseudomonas 
aeruginosa among intensive care unit patients in a particu-
lar hospital. This allows the best therapy to be chosen based 
on clinical suspicion of the infecting organism and known 
locally circulating strains.
The selection of a bactericidal drug or drug combination 
for each patient can be guided by specialized laboratory tests. 
Such tests measure either the rate of killing (time-kill assay) 
or the proportion of the microbial population that is killed 
in a fixed time by patient serum (serum bactericidal testing). 
Synergy testing measures the ability of drugs to enhance bac-
terial killing when present in combination; drugs showing 
synergy may be more effective when given together to treat 
infection. Few clinical laboratories perform this type of spe-
cialized susceptibility testing.
DIAGNOSIS OF INFECTION BY 
ANATOMIC SITE
Wounds, Tissues, Bones, Abscesses, 
and Fluids
Microscopic study of smears and culture of specimens from 
wounds or abscesses often gives early and important indica-
tions of the nature of the infecting organism and thus helps in 
the choice of antimicrobial drugs. Specimens from diagnostic 
tissue biopsies should be submitted for microbiologic as well 
as histologic examination. Such specimens for bacteriologic 
examination are submitted fresh, without fixatives or disin-
fectants, and are cultured by a variety of methods.
The pus in closed, undrained soft tissue abscesses fre-
quently contains only one organism as the infecting agent; 
most commonly staphylococci, streptococci, or enteric gram-
negative rods. The same is true in acute osteomyelitis, where 
the organisms can often be cultured from blood before the 
infection has become chronic. Multiple microorganisms are 
frequently encountered in abdominal abscesses and abscesses 
contiguous with mucosal surfaces as well as in open wounds. 
When deep suppurating lesions, such as chronic osteomy-
elitis, drain onto exterior surfaces through a sinus or fistula, 
the microbiota of the surface through which the lesion drains 
must not be mistaken for that of the deep lesion. Instead, 
specimens should be aspirated from the primary infection 
through uninfected tissue.
Bacteriologic examination of pus from closed or deep 
lesions must include culture by anaerobic methods. Anaero-
bic bacteria (Bacteroides, Fusobacteria, etc) sometimes play 
an essential causative role, and mixtures of aerobes and 
anaerobes are often present.
The methods used for cultures must be suitable for the 
semiquantitative recovery of common bacteria and also for 
recovery of specialized microorganisms, including myco-
bacteria and fungi. Eroded skin and mucous membranes are 
frequently the sites of yeast or fungus infections. Candida, 
Aspergillus, and other yeasts or fungi can be seen microscopi-
cally in smears or scrapings from suspicious areas and can be 
grown in cultures. Treatment of a specimen with KOH and 
calcofluor white greatly enhances the observation of yeasts 
and molds in the specimen.
Exudates that have collected in the pleural, peritoneal, 
pericardial, or synovial spaces must be aspirated with asep-
tic technique. If the material is frankly purulent, smears and 
cultures are made directly. If the fluid is clear, it can be centri-
fuged and the sediment used for stained smears and cultures. 
The culture method used must be suitable for the growth 
of organisms suspected on clinical grounds—for example, 
mycobacteria, anaerobic organisms—as well as the commonly 
encountered pyogenic bacteria. Some fluid specimens clot, 
and culture of an anticoagulated specimen may be necessary. 
The following chemistry and hematology results are sugges-
tive of infection: specific gravity greater than 1.018, protein 
content greater than 3 g/dL (often resulting in clotting), and 
white cell counts greater than 500–1000/μL. Polymorpho-
nuclear leukocytes(PMNs) predominate in acute untreated 
pyogenic infections; lymphocytes or monocytes predominate 
in chronic infections. Transudates resulting from neoplastic 
growth may grossly resemble infectious exudates by appear-
ing bloody or purulent and by clotting on standing. Cytologic 
study of smears or of sections of centrifuged cells may dem-
onstrate the neoplastic nature of the process.
Blood
Since bacteremia frequently portends life-threatening illness, 
its early detection is essential. Blood culture is the single most 
important procedure to detect systemic infection due to bac-
teria. It provides valuable information for the management of 
febrile, acutely ill patients with or without localizing symptoms 
and signs and is essential in any patient in whom infective endo-
carditis is suspected even if the patient does not appear acutely 
or severely ill. In addition to its diagnostic significance, recovery 
of an infectious agent from the blood provides invaluable aid in 
determining antimicrobial therapy. Every effort should there-
fore be made to isolate the causative organisms in bacteremia.
In healthy persons, properly obtained blood specimens 
are sterile. Although microorganisms from the normal 
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respiratory and gastrointestinal microbiota occasionally enter 
the blood, they are rapidly removed by the reticuloendothe-
lial system. These transients rarely affect the interpretation of 
blood culture results. If a blood culture yields microorgan-
isms, this fact is of great clinical significance provided that 
contamination can be excluded. Contamination of blood cul-
tures with normal skin microbiota is most commonly due to 
errors in the blood collection procedure. Therefore, proper 
technique in performing a blood culture is essential.
The following rules, rigidly applied, yield reliable results:
1. Use strict aseptic technique. Wear gloves—they do not 
have to be sterile.
2. Apply a tourniquet and locate a fixed vein by touch. Release 
the tourniquet while the skin is being prepared.
3. Prepare the skin for venipuncture by cleansing it vigor-
ously with 70–95% isopropyl alcohol. Using 2% tincture 
of iodine or 2% chlorhexidine, start at the venipuncture 
site and cleanse the skin in concentric circles of increas-
ing diameter. Allow the antiseptic preparation to dry for 
at least 30 seconds. Do not touch the skin after it has been 
prepared.
4. Reapply the tourniquet, perform venipuncture, and (for 
adults) withdraw approximately 20 mL of blood.
5. Add the blood to aerobic and anaerobic blood culture 
bottles.
6. Properly label and promptly transport the specimens to 
the laboratory.
Several factors determine whether blood cultures will 
yield positive results: the volume of blood cultured, the dilu-
tion of blood in the culture medium, the use of both aerobic 
and anaerobic culture media, and the duration of incubation. 
For adults, 20 mL per culture is usually obtained, and half is 
placed in an aerobic blood culture bottle and half in an anaer-
obic one, with one pair of bottles comprising a single blood 
culture. Commercial manufacturers of blood culture sys-
tems optimize the broth composition, volume, and antibiotic 
neutralizing agents used (activated charcoal or resin beads). 
Automated blood culture systems use a variety of methods 
to detect positive cultures. These automated methods allow 
frequent monitoring of the cultures—as often as every few 
minutes—and earlier detection of positive ones. The media in 
the automated blood culture systems are so enriched and the 
detection systems so sensitive that blood cultures using the 
automated systems do not need to be processed for more than 
5 days. In general, subcultures are indicated only when the 
machine indicates that the culture is positive. Manual blood 
culture systems are obsolete and are likely to be used only in 
laboratories in developing countries that lack the resources 
to purchase automated blood culturing systems. In manual 
systems, the blood culture bottles are examined two or three 
times a day for the first 2 days and daily thereafter for 1 week. 
In the manual method, blind subcultures of all the blood cul-
ture bottles on days 2 and 7 may be necessary.
The number of blood specimens that should be drawn 
for cultures and the period of time over which this is done 
depend in part on the severity of the clinical illness. In 
hyperacute infections, eg, gram-negative sepsis with shock or 
staphylococcal sepsis, it is appropriate to obtain a minimum 
of two blood cultures from different anatomic sites, prefer-
ably through peripheral venipuncture. More recent literature 
has suggested that three to four blood cultures may be neces-
sary. In other bacteremic infections, eg, subacute endocardi-
tis, three blood specimens should be obtained over 24 hours. 
A total of three blood cultures yields the infecting bacteria 
in more than 95% of bacteremic patients. If the initial three 
cultures are negative and occult abscess, fever of unexplained 
origin, or some other obscure infection is suspected, addi-
tional blood specimens should be cultured when possible 
before antimicrobial therapy is started.
It is necessary to determine the significance of a posi-
tive blood culture. The following criteria may be help-
ful in differentiating “true positives” from contaminated 
specimens:
1. Growth of the same organism in repeated cultures 
obtained at different times from separate anatomic sites 
strongly suggests true bacteremia.
2. Growth of different organisms in different culture bottles 
suggests contamination but occasionally may follow clini-
cal problems such as wound sepsis or ruptured bowel.
3. Growth of normal skin microbiota, eg, coagulase-negative 
staphylococci, diphtheroids (corynebacteria and propioni-
bacteria), or anaerobic gram-positive cocci, in only one of 
several cultures suggests contamination. Growth of such 
organisms in more than one culture or from specimens 
from a high-risk patient, such as an immunocompromised 
bone marrow transplant recipient, enhances the likeli-
hood that clinically significant bacteremia exists.
4. Organisms such as viridans streptococci or enterococci 
are likely to grow in blood cultures from patients sus-
pected to have endocarditis, and gram-negative rods such 
as E coli are likely to grow in blood cultures from patients 
with clinical gram-negative sepsis. Therefore, when such 
“expected” organisms are found, they are more apt to be 
etiologically significant.
The following are the bacterial species most commonly 
recovered in positive blood cultures: staphylococci, includ-
ing S aureus; viridans streptococci; enterococci, including 
Enterococcus faecalis; gram-negative enteric bacteria, includ-
ing E coli and K pneumoniae; P aeruginosa; pneumococci; and 
Haemophilus influenzae. Candida species, other yeasts, and 
some dimorphic fungi such as H capsulatum grow in blood 
cultures, but many fungi are rarely, if ever, isolated from 
blood. CMV and HSV can occasionally be cultured from 
blood, but most viruses and rickettsiae and chlamydiae are 
not cultured from blood. Parasitic protozoa and helminths 
do not grow in blood cultures.
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CHAPTER 47 Principles of Diagnostic Medical Microbiology 757
In most types of bacteremia, examination of direct blood 
smears is not useful. Diligent examination of Gram-stained 
smears of the buffy coat from anticoagulated blood will 
occasionally show bacteria in patients with S aureus infec-
tion, clostridial sepsis, or relapsing fever. In some microbial 
infections (eg, anthrax, plague, relapsing fever, rickettsiosis, 
leptospirosis, spirillosis, psittacosis), inoculation of blood 
into animals may give positive results more readily than does 
culture. In practicality, this is never donein clinical labora-
tories and diagnosis may be made by alternate means such as 
serology or nucleic acid amplification tests.
Urine
Bacteriologic examination of the urine is done mainly when 
signs or symptoms point to urinary tract infection, renal 
insufficiency, or hypertension. It should always be done 
in persons with suspected systemic infection or fever of 
unknown origin. It is desirable for women in the first trimes-
ter of pregnancy to be assessed for asymptomatic bacteriuria.
Urine secreted in the kidney is sterile unless the kidney 
is infected. Uncontaminated bladder urine is also normally 
sterile. The urethra, however, contains a normal microbiota, 
so that normal voided urine contains small numbers of bac-
teria. Because it is necessary to distinguish contaminating 
organisms from etiologically important organisms, only 
quantitative urine examination can yield meaningful results.
The following steps are essential in proper urine 
examination.
A. Proper Collection of Specimen
Proper collection of the specimen is the single most impor-
tant step in a urine culture and the most difficult. Satisfactory 
specimens from females are problematic.
1. Have at hand a sterile, screw-cap specimen container 
and two to three gauze sponges soaked with nonbacte-
riostatic saline (antibacterial soaps for cleansing are not 
recommended).
2. Spread the labia with two fingers and keep them spread 
during the cleansing and collection process. Wipe the 
urethra area once from front to back with each of the 
saline gauzes.
3. Start the urine stream and, using the urine cup, collect a 
midstream specimen. Properly label the cup.
The same method is used to collect specimens from males; 
the foreskin should be kept retracted in uncircumcised males.
Catheterization carries a risk of introducing microor-
ganisms into the bladder, but it is sometimes unavoidable. 
Separate specimens from the right and left kidneys and ure-
ters can be obtained by the urologist using a catheter at cys-
toscopy. When an indwelling catheter and closed collection 
system are in place, urine should be obtained by sterile aspi-
ration of the catheter with needle and syringe, not from the 
collection bag. To resolve diagnostic problems, urine can be 
aspirated aseptically directly from the full bladder by means 
of suprapubic puncture of the abdominal wall. This proce-
dure is usually done in infants.
For most examinations, 0.5 mL of ureteral urine or 5 mL 
of voided urine is sufficient. Because many types of microor-
ganisms multiply rapidly in urine at room or body temper-
ature, urine specimens must be delivered to the laboratory 
rapidly or refrigerated not longer than overnight. Alterna-
tively, transport tubes that contain boric acid may be used if 
specimens cannot be refrigerated.
B. Microscopic Examination
Much can be learned from simple microscopic examina-
tion of urine. A drop of fresh uncentrifuged urine placed 
on a slide, covered with a coverglass, and examined with 
restricted light intensity under the high-dry objective of an 
ordinary clinical microscope can reveal leukocytes, epithelial 
cells, and bacteria if more than 105/mL are present. Finding at 
least 105 organisms per milliliter in a properly collected and 
examined urine specimen is strong evidence of active urinary 
tract infection. A Gram-stained smear of uncentrifuged mid-
stream urine that shows gram-negative rods is diagnostic of a 
urinary tract infection.
Brief centrifugation of urine readily sediments pus cells, 
which may carry along bacteria and thus may help in micro-
scopic diagnosis of infection. The presence of other formed 
elements in the sediments—or the presence of proteinuria—is 
of little direct aid in the specific identification of active uri-
nary tract infection. Pus cells may be present without bacteria, 
and, conversely, bacteriuria may be present without pyuria. 
The presence of many squamous epithelial cells, lactobacilli, 
or mixed flora on culture suggests improper urine collection.
Some urine dipsticks contain leukocyte esterase and 
nitrite, measurements of polymorphonuclear cells and bacte-
ria, respectively, in the urine. Positive reactions are strongly 
suggestive of bacterial urinary tract infection, while negative 
reactions for both indicate a low likelihood of urinary tract 
infection, except for neonates and immunocompromised 
patients.
C. Culture
Culture of the urine, to be meaningful, must be performed 
quantitatively. Properly collected urine is cultured in mea-
sured amounts on solid media, and the colonies that appear 
after incubation are counted to indicate the number of bacteria 
per milliliter. The usual procedure is to spread 0.001–0.05 mL 
of undiluted urine on blood agar plates and other solid media 
for quantitative culture. All media are incubated overnight 
at 37°C; growth density is then compared with photographs 
of different densities of growth for similar bacteria, yielding 
semiquantitative data.
In active pyelonephritis, the number of bacteria 
in urine collected by ureteral catheter is relatively low. 
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758 SECTION VII Diagnostic Medical Microbiology and Clinical Correlation
While accumulating in the bladder, bacteria multiply rapidly 
and soon reach numbers in excess of 105/mL—far more than 
could occur as a result of contamination by urethral or skin 
microbiota or from the air. Therefore, it is generally agreed 
that if more than 105 colonies/mL are cultivated from a prop-
erly collected and properly cultured urine specimen, this 
constitutes strong evidence of active urinary tract infection. 
The presence of 105 bacteria or more of the same type per mil-
liliter in two consecutive specimens establishes a diagnosis 
of active infection of the urinary tract with 95% certainty. If 
fewer bacteria are cultivated, repeated examination of urine 
is indicated to establish the presence of infection.
The presence of fewer than 104 bacteria per milliliter, 
including several different types of bacteria, suggests that 
organisms come from the normal microbiota and are con-
taminants, usually from an improperly collected specimen. 
The presence of 104/mL of a single type of enteric gram-nega-
tive rod is strongly suggestive of urinary tract infection, espe-
cially in men. Occasionally, young women with acute dysuria 
and urinary tract infection will have 102–103/mL. If cultures 
are negative but clinical signs of urinary tract infection are 
present, “urethral syndrome,” ureteral obstruction, tuber-
culosis of the bladder, gonococcal infection, or other disease 
must be considered.
Cerebrospinal Fluid
Meningitis ranks high among medical emergencies, and 
early, rapid, and precise diagnosis is essential. Diagnosis of 
meningitis depends on maintaining a high index of suspi-
cion, securing adequate specimens properly, and examining 
the specimens promptly. Because the risk of death or irrevers-
ible damage is great unless treatment is started immediately, 
there is rarely a second chance to obtain pretreatment speci-
mens, which are essential for specific etiologic diagnosis and 
optimal management.
The most urgent diagnostic issue is the differentiation 
of acute purulent bacterial meningitis from “aseptic” and 
granulomatous meningitis. The immediate decision is usu-
ally based on the cell count, the glucose concentration and 
protein content of CSF, and the results of microscopic search 
for microorganisms (see Case 1, Chapter 48). The initial 
impression is modified by the results of culture, serologic 
tests, nucleic acid amplification tests, and other laboratory 
procedures. In evaluating the results of CSF glucose deter-
minations, the simultaneous blood glucose level must be con-
sidered. In some central nervous system neoplasms, the CSF 
glucose level is low.
A. Specimens
As soon as infection of the central nervous system is suspected, 
blood samples are taken for culture, and CSF is obtained.