CHAPTER 8

Microbiology

8.1 Specimen Collection, Media, and Methods

8.2 Enterobacteriaceae
8.3 Nonfermentative Bacilli
8.4 Miscellaneous and Fastidious Gram-Negative Rods
8.5 Gram-Positive and Gram-Negative Cocci
8.6 Aerobic Gram-Positive Rods, Spirochetes, Mycoplasmas and
Ureaplasmas, and Chlamydia
8.7 Anaerobic Bacteria
8.8 Mycobacteria
8.9 Mycology
8.10 Virology
8.11 Parasitology
8.12 Microbiology and Parasitology Problem-Solving
 8.1
Specimen Collection, Media, and Methods
1. The aseptic collection of blood cultures requires that the skin be cleansed with:
A. 2% iodine and then 70% alcohol solution
B. 70% alcohol and then 2% iodine or an iodophor
C. 70% alcohol and then 95% alcohol
D. 95% alcohol only
Microbiology/Apply knowledge of standard operating procedures/Specimen collection/1
2. When cleansing the skin with alcohol and then iodine for the collection of a blood
culture, the iodine (or iodophor) should remain intact on the skin for at least:
A. 10 seconds
B. 30 seconds
C. 60 seconds
D. 5 minutes
Microbiology/Apply knowledge of standard operating procedures/Specimen collection and
handling/1
3. What is the purpose of adding 0.025% to 0.050% sodium polyanethol sulfonate (SPS) to
nutrient broth media for the collection of blood cultures?
A. It inhibits phagocytosis and complement
B. It promotes formation of a blood clot
C. It enhances growth of anaerobes
D. It functions as a preservative
Microbiology/Apply knowledge of standard operating procedures/Media/1
4. A flexible calcium alginate nasopharyngeal swab is the collection device of choice for
recovery of which organism from the nasopharynx?
A. Staphylococcus aureus
B. Streptococcus pneumoniae
C. Corynebacterium diphtheriae
D. Bacteroides fragilis
Microbiology/Apply knowledge of standard operating procedure/Specimen collection and
handling/1
5. Semisolid transport media, such as Amies, Stuart, or Cary-Blair, are suitable for the
transport of swabs for culture of most pathogens except:
A. Neisseria gonorrhoeae
B. Enterobacteriaceae
C. Campylobacter fetus
D. Streptococcus pneumoniae
Microbiology/Select methods/Reagents/Media/Specimen collection and handling/2
 Answers to Questions 1–5
1. B To attain asepsis of the skin, 70% alcohol followed by 2% iodine is used for obtaining
blood cultures.
2. C The iodine should remain on the skin for 1 minute because instant antisepsis does not
occur when cleansing the skin for a blood culture.
3. A SPS is used in most commercial blood culture products because it functions as an
anticoagulant and prevents phagocytosis and complement activation. In addition, SPS
neutralizes aminoglycoside antibiotics. Addition of SPS may inhibit some Neisseria
and Peptostreptococcus, but this can be reversed with 1.2% gelatin.
4. C C. diphtheriae must be recovered from the deep layers of the pseudomembrane that
forms in the nasopharyngeal area. A flexible calcium alginate nasopharyngeal swab is
the best choice for collecting a specimen from the posterior nares and pharynx.
5. A Specimens for culture of N. gonorrhoeae are best if plated immediately or transported
in a medium containing activated charcoal to absorb inhibitory substances that hinder
their recovery from the specimens.

6. Select the method of choice for recovery of anaerobic bacteria from a deep abscess.
A. Cotton fiber swab of the abscess area
B. Skin snip of the surface tissue
C. Needle aspirate after surface decontamination
D. Swab of the scalpel used for débridement
Microbiology/Apply knowledge of standard operating procedures/Specimen collection and
handling/2
7. Select the primary and differential media of choice for recovery of most fecal pathogens.
A. MacConkey, blood, birdseed, and Campylobacter (Campy) agars
B. Hektoen, MacConkey, MacConkey-Sorbitol, Campy blood, colistin–nalidixic acid (CNA)
agars; Selenite-F broth (SEL)
C. CNA and Christensen urea agars and thioglycollate media
D. Blood, Campy, Mueller-Hinton agars, and thioglycollate media
Microbiology/Select methods/Reagents/Media/Stool culture/2
8. Select the media of choice for recovery of Vibrio cholerae from a stool specimen.
A. MacConkey agar and thioglycollate media
B. Thiosulfate–citrate–bile–sucrose (TCBS) agar and alkaline peptone water (APW) broth
C. Blood agar and SEL broth
D. CNA agar
Microbiology/Select methods/Reagents/Media/Stool culture/2
9. CNA agar is used primarily for the recovery of:
A. Neisseria species
B. Enterobacteriaceae
C. Pseudomonas aeruginosa
D. Staphylococcus aureus
Microbiology/Select methods/Reagents/Media/Gram-positive cocci/2
10. In the United States, most blood agar plates are prepared with 5% or 10% red blood
cells (RBCs) obtained from:
A. Sheep
B. Horses
C. Humans
D. Dogs
Microbiology/Select methods/Reagents/Media/Culture/1

Answers to Questions 6–10

6. C Anaerobic specimens are easily contaminated with organisms present on the skin or
mucosal surfaces when a swab is used. Needle aspiration of an abscess following
surface decontamination provides the least exposure to ambient oxygen.
7. B Hektoen agar selectively isolates pathogenic coliforms, especially Salmonella and
Shigella. MacConkey agar differentiates lactose fermenters from nonfermenters.
MacConkey with sorbitol isolates Escherichia coli 0157:H7. CNA agar contains
antibiotics that prohibit growth of gram-negative coliforms but not gram-positive
cocci. Campy blood agar contains the antibiotics cephalothin, trimethoprim,
vancomycin, polymyxin B, and amphotericin B to prevent growth of
Enterobacteriaceae, Pseudomonas spp., and fungi.
8. B TCBS agar is used to grow V. cholerae, which appear as yellow colonies as a result of
the use of both citrate and sucrose. APW is used as an enrichment broth and should be
subcultured to TCBS agar for further evaluation of Vibrio colonies.
9. D CNA agar inhibits the growth of gram-negative bacteria and is used to isolate gram-
positive cocci from specimens. This medium is especially useful for stool and wound
cultures because these may contain large numbers of gram-negative rods.
10. A Sheep RBCs are used in blood agar plates because they are readily available and less
inhibitory than cells of other species. The type of hemolysis is determined by the
source of RBCs. Sheep RBCs are chosen because of the characteristically clear
hemolysis produced by β-hemolytic streptococci, Staphylococcus, and other pathogens
producing β-hemolysins. Sheep blood does not support the growth of Haemophilus
haemolyticus, eliminating the possibility of confusing it with β-hemolytic streptococci
in throat cultures.

11. All of the following are appropriate when attempting to isolate N. gonorrhoeae from a
genital specimen except:
A. Transport the genital swab in charcoal transport medium
B. Plate the specimen directly on modified Thayer-Martin (MTM) medium
C. Plate the specimen directly on New York City or Martin-Lewis agar
D. Culture specimens in ambient oxygen at 37°C
Microbiology/Select methods/Reagents/Media/Culture/1
12. Chocolate agar and MTM agar are used for the recovery of:
A. Haemophilus spp. and Neisseria spp., respectively
B. Haemophilus spp. and N. gonorrhoeae, respectively
 C. Neisseria spp. and Streptococcus spp., respectively
D. Streptococcus spp. and Staphylococcus spp., respectively
Microbiology/Select methods/Reagents/Media/Stool culture/2
13. Cycloserine–cefoxitin–fructose agar (CCFA) is used for the recovery of:
A. Yersinia enterocolitica
B. Yersinia intermedia
C. Clostridium perfringens
D. Clostridium difficile
Microbiology/Select methods/Reagents/Media/Stool culture/1
14. Deoxycholate agar (DCA) is useful for the isolation of:
A. Enterobacteriaceae
B. Enterococcus spp.
C. Staphylococcus spp.
D. Neisseria spp.
Microbiology/Select methods/Reagents/Media/Stool culture/1
15. Xylose lysine deoxycholate (XLD) agar is a highly selective medium used for the
recovery of which bacteria?
A. Staphylococcus spp. from normal flora
B. Yersinia spp. that do not grow on Hektoen agar
C. Enterobacteriaceae from gastrointestinal specimens
D. Streptococcus spp. from stool cultures
Microbiology/Select methods/Reagents/Media/Stool culture/1

Answers to Questions 11–15

11. D MTM, New York City, and Martin-Lewis agars contain blood factors needed to
support the growth of N. gonorrhoeae as well as antibiotics that prevent growth of
normal genital flora. Cultures must be incubated in 3% to 7% carbon dioxide (CO2) at
35°C. Cultures should be held a minimum of 48 hours before being considered
negative.
12. B Chocolate agar provides X factor (hemin) and V factor (nicotinamide adenine
dinucleotide [NAD]) required for the growth of Haemophilus spp. Thayer-Martin
medium is a chocolate agar containing the antibiotics that permit isolation of N.
gonorrhoeae in specimens containing large numbers of gram-negative bacteria,
including commensal Neisseria spp.
13. D CCFA is used for recovery of C. difficile from stool cultures. Cycloserine and
cefoxitin inhibit growth of gram-negative coliforms in the stool specimen. C. difficile
ferments fructose, forming acid that, in the presence of neutral red, causes the colonies
to become yellow.
14. A DCA inhibits gram-positive organisms. N. gonorrhoeae and Neisseria meningitidis
are too fastidious to grow on DCA. Citrate and deoxycholate salts inhibit growth of
gram-positive bacteria. The media contain lactose and neutral red, allowing
differentiation of lactose fermenters (pink colonies) from nonfermenters (colorless).
15. C XLD agar is selective for gram-negative coliforms because of a high concentration
 (0.25%) of deoxycholate, which inhibits gram-positive bacteria. In addition, XLD is
differential for Shigella and Salmonella spp. The medium contains xylose, lactose, and
sucrose, which are fermented by most normal intestinal coliforms producing yellow
colonies. Shigella does not ferment the sugars and produces red (or clear) colonies.
Salmonella spp. ferment xylose; however, they also decarboxylate lysine in the
medium, causing production of ammonia. Therefore, Salmonella first appear yellow
but become red. Some Salmonella produce hydrogen sulfide (H2S) from sodium
thiosulfate and therefore appear as red colonies with black centers.

16. A sheep blood agar plate is used as a primary isolation medium when all of the
following organisms are to be recovered from a wound specimen except:
A. β-Hemolytic streptococci and coagulase-positive staphylococci
B. Haemophilus influenzae and Haemophilus parainfluenzae
C. Proteus spp. and Escherichia coli
D. Pseudomonas spp. and Acinetobacter spp.
Microbiology/Select methods/Reagents/Media/Wound culture/2
17. Prereduced and vitamin K1-supplemented blood agar plates are recommended isolation
media for:
A. Mycobacterium marinum and Mycobacterium avium intracellulare
B. Bacteroides, Peptostreptococcus, and Clostridium spp.
C. Proteus spp.
D. Enterococcus spp.
Microbiology/Select methods/Reagents/Media/Anaerobes/2
18. Which procedure(s) is (are) appropriate for the diagnosis of Chlamydia spp. infections
when using genital specimens?
A. Obtain urethral, cervical swabs and urine specimens placed in transport media for the
direct detection of antigen or nucleic acid and/or culture
B. Plate onto blood and chocolate agar
C. Inoculate into thioglycollate (THIO) broth
D. Plate onto MTM agar within 24 hours
Microbiology/Select methods/Reagents/Media/Virus culture/1
19. Specimens for virus culture should be transported in media containing:
A. Antibiotics and 5% sheep blood
B. Saline and 5% sheep blood
C. 22% bovine albumin
D. Antibiotics and protein nutrient
Microbiology/Select methods/Reagents/Media/Virus culture/1
20. Cerebrospinal fluid (CSF) should be cultured immediately, but if delayed, the specimen
should be:
A. Refrigerated at 4°C to 6°C
B. Frozen at –20°C
C. Stored at room temperature for no longer than 24 hours
D. Incubated at 37°C and cultured as soon as possible
Microbiology/Apply knowledge of standard operating procedures/Specimen collection and
transport/1
 Answers to Questions 16–20
16. B Both gram-positive cocci and gram-negative bacilli will grow on blood agar plates,
but the medium is used in conjunction with a selective medium, such as CNA agar, for
gram-positive cocci and MacConkey agar for gram-negative bacilli. H. influenzae
requires X and V factors, and H. parainfluenzae requires V factor; the primary
isolation medium for Haemophilus is chocolate agar.
17. B Anaerobic culture media can be prereduced before sterilization by boiling, saturation
with oxygen-free gas, and addition of cysteine or other thiol compounds. The final
oxidation reduction potential (Eh) of the medium should be approximately –150 mV to
minimize the effects of exposure of organisms to oxygen during inoculation.
18. A Chlamydiae are strict intracellular organisms and must be cultured using living cells
(e.g., cyclohexamide-treated McCoy cells). Direct smears can also be made at the time
of culture. Fluorescein-conjugated monoclonal antibodies may be used to identify the
organisms in infected cells. Cell cultures present limitations but are used if legal
situations (e.g., sexual abuse) are implied. Commercially available kits for antigen
detection and nucleic acid amplification and hybridization techniques, which have
been approved by the U.S. Food and Drug Administration (FDA), are not culture
methods but appear more reliable for the detection of infection in individuals who are
symptomatic and shedding large numbers of organisms.
19. D Media for transporting specimens for virus culture include Hanks balanced salt
solution with bovine albumin, Amies media, Stuart transport media, and Leibovitz-
Emory media. Media used for transporting specimens for viral culture are similar to
those for bacteria with the addition of a nutrient, such as fetal calf serum or albumin,
and antibiotics. Specimens should be refrigerated (not frozen) after being placed in the
transport media until the culture media can be inoculated.
20. D Fastidious organisms, such as Neisseria and Haemophilus, frequently isolated from
the CSF of patients with bacterial meningitis are preserved by placing the fluid in 3%
to 7% CO2 at 35°C to 37°C (or at room temperature for no longer than 30 minutes), if
the specimen cannot be cultured immediately.

21. The most sensitive method for the detection of β-lactamase in bacteria is by the use of:
A. Chromogenic cephalosporin
B. Penicillin
C. Oxidase
D. Chloramphenicol acetyltransferase
Microbiology/Select methods/Reagents/Media/Sensitivity testing/2
22. The breakpoint of an antimicrobial drug refers to:
A. The amount needed to cause bacteriostasis
B. A minimum inhibitory concentration (MIC) of 16 µg/mL or greater
C. An MIC of 64 µg/mL or greater
D. The optimal therapeutic level of drug that is achievable in serum
Microbiology/Apply principle of theory and practice related to laboratory
operations/Sensitivity testing/2
23. Which of the following variables may change the results of an MIC?
A. Inoculum size
B. Incubation time
C. Growth rate of the bacteria
D. All of these options
Microbiology/Apply knowledge to identify sources of error/Sensitivity testing/2
24. According to the Kirby-Bauer standard antimicrobial susceptibility testing method,
what should be done when interpreting the zone size of a motile, swarming organism,
such as a Proteus species?
A. The swarming area should be ignored
B. The results of the disk diffusion method are invalid
C. The swarming area should be measured as the growth boundary
D. The isolate should be retested after diluting to a 0.05 McFarland standard
Microbiology/Apply knowledge of standard operating procedures/Sensitivity testing/2
25. Which class of antibiotics is used for the treatment of serious gram-negative infections
as well as infections with Mycobacterium tuberculosis?
A. Cephalosporins
B. Penicillins
C. Tetracyclines
D. Aminoglycosides
Microbiology/Apply knowledge of fundamental biological characteristics/Antibiotics/1

Answers to Questions 21–25

21. A β-Lactamase production by bacteria that are resistant to penicillin and cephalosporin
is detected using one of these drugs as a substrate. Penicillin is hydrolyzed by β-
lactamase into acidic products that can be detected as a color change by a pH indicator.
In the iodometric method, a disk containing a penicillin–starch substrate turns blue
when a drop of iodine is added. The most sensitive method of detection is based on the
ability of the organism to hydrolyze the β-lactam ring of a chromogenic cephalosporin
(e.g., nitrocefin), which is used as the substrate. A positive test indicates the
organism’s resistance to penicillin, ampicillin, amoxicillin, carbenicillin, mezlocillin,
and piperacillin.
22. D The term breakpoint refers to an antimicrobial concentration in the serum associated
with optimal therapy using the customary dosing schedule. An organism is susceptible
if the MIC is at or below the breakpoint.
23. D In vitro testing of drugs is reliable if the method is standardized. In addition to the
first three variables, the type of media and the stability of antibiotics affect the results
of MIC testing and must be carefully controlled.
24. A A thin film of growth appearing in the zone area of inhibition around the
susceptibility disk should be ignored when swarming Proteus or other organisms are
encountered. Discontinuous, poor growth or tiny colonies near the end of the zone
should also be ignored.
25. D The aminoglycoside antibiotics are bactericidal agents that act by inhibiting protein
 synthesis. They show a low incidence of bacterial resistance but must be monitored
carefully because at high doses they can cause ototoxicity and nephrotoxicity. The
group includes amikacin, gentamicin, tobramycin, kanamycin, streptomycin, and
spectinomycin. These drugs are usually administered intravenously or intramuscularly
because they are poorly absorbed from the gastrointestinal tract.

26. Select the medium best suited for the recovery of Y. enterocolitica from a patient with
gastroenteritis.
A. Hektoen agar
B. Cefsulodin–irgasan–novobiocin (CIN) agar
C. Blood agar
D. Eosin methylene blue agar
Microbiology/Select methods/Reagents/Media/Gram-negative bacilli/2
27. A suspected case of plague requires which of the following procedures to confirm
Yersinia pestis?
A. Collection of multiple sets of blood culture specimens
B. Incubation of blood cultures at both 28°C and 35°C
C. Culture aspirates from bubos to MacConkey agar at room temperature
D. All of these options
Microbiology/Apply knowledge of procedures/Gram-negative bacilli/2
28. SITUATION: Abdominal pain, fever, vomiting, and nausea prompted an older male to
seek medical attention. A watery stool specimen producing no fecal leukocytes or
erythrocytes was cultured, and it grew a predominance of gram-negative fermentative
bacilli. The colonies were β-hemolytic on blood agar and cream colored on MacConkey
agar. The colonies were both oxidase and catalase positive. What is the most likely
identification?
A. Aeromonas hydrophilia
B. Escherichia coli
C. Salmonella spp.
D. Shigella spp.
Microbiology/Evaluate laboratory data to make identifications/Gram-negative bacilli/3
29. SITUATION: Several attendees of a medical conference in the Gulf coast area became
ill after frequenting a seafood restaurant. A presumptive identification of V. cholerae
was made after stool specimens from several subjects grew clear colonies on
MacConkey agar and yellow colonies on TCBS agar. Which key tests would help
eliminate Aeromonas and Plesiomonas spp.?
A. Mannitol fermentation, Na+ requirement
B. Oxidase, motility
C. Oxidase, nitrate
D. Hemolysis on blood agar, catalase
Microbiology/Select methods/Reagents/Media/Gram-negative bacilli/3
30. SITUATION: A group of elementary students became ill after eating undercooked
ground beef prepared in the school cafeteria. The suspected pathogen, E. coli serotype
0157:H7, is usually recovered using which of the following media?
A. Xylose lysine deoxycholate agar
B. MacConkey agar
 C. MacConkey agar with sorbitol
D. Hektoen agar
Microbiology/Select methods/Reagents/Media/Gram-negative bacilli/2

Answers to Questions 26–30

26. B CIN agar inhibits the growth of many organisms from the family Enterobacteriaceae.
Yersinia spp. are also recovered from MacConkey and Salmonella-Shigella agars. Y.
enterocolitica are seen as “bull’s eye” colonies on CIN agar after 48 hours at room
temperature.
27. D Y. pestis is on the list of agents of bioterrorism. Isolation and identification should be
performed in a facility with a Level II or higher biosafety rating. If there is a high risk
of aerosolizing the specimen during processing, procedures should be performed under
Level III biosafety conditions. Recovery of Y. pestis is highest if the specimen is
cultured within 2 hours of collection.
28. A The oxidase positive test result rules out the members of the Enterobacteriaceae
family. Colonies of A. hydrophilia and Plesiomonas spp. (both oxidase positive) might
be mistaken for Vibrio spp. because all three grow as clear colonies on MacConkey
agar, are β-hemolytic on blood agar, and are oxidase positive. Aeromonas spp. will
also grow on CNN agar and mimic Y. enterocolitica (a member of the
Enterobacteriaceae family and oxidase negative).
29. A All three organisms are positive for oxidase production and are motile. Plesiomonas
spp. do not grow on TCBS agar. Clear colonies on MacConkey agar and yellow
colonies on TCBS agar indicate Vibrio or Aeromonas spp. However, only Vibrio spp.
require Na+ (1% NaCl) in the medium for growth.
Vibrio Aeromonas Plesiomonas
Oxidase + + +
Na+ requirement + Neg Neg
Mannitol fermentation + + Neg
Growth onTCBS + + Neg
30. C E. coli 0157:H7 ferments lactose and, therefore, appears as dark pink colonies on
MacConkey agar. To differentiate E. coli 0157:H7 from normal fecal flora,
MacConkey agar with sorbitol is used. E. coli 0157:H7 does not ferment sorbitol, and
usually are colorless colonies.