Book

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
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
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

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
13. Cycloserine–cefoxitin–fructose agar (CCFA) is used for the recovery of:
A. Yersinia enterocolitica
B. Yersinia intermedia
C. Clostridium perfringens
D. Clostridium difficile
14. Deoxycholate agar (DCA) is useful for the isolation of:
A. Enterobacteriaceae
B. Enterococcus spp.
C. Staphylococcus spp.
D. Neisseria spp.
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

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
transport/1
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

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
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

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.
8.2
Enterobacteriaceae
1. Biochemically, Enterobacteriaceae species are gram-negative rods that:
A. Ferment glucose, reduce nitrate to nitrite, and are oxidase negative
B. Ferment glucose, produce indophenol oxidase, and form gas
C. Ferment lactose and reduce nitrite to nitrogen gas
D. Ferment lactose and produce indophenol oxidase
Microbiology/Apply knowledge of fundamental biological
characteristics/Biochemical/Gram-negative bacilli/1
2. The ortho-nitrophenyl-β-galactopyranoside (ONPG) test is most useful when
differentiating:
A. Salmonella spp. from Pseudomonas spp.
B. Shigella spp. from some strains of Escherichia coli
C. Klebsiella spp. from Enterobacter spp.
D. Proteus vulgaris from Salmonella spp.
Microbiology/Apply principles of basic laboratory procedures/Biochemical/2
3. The Voges-Proskauer (VP) test detects which end product of glucose fermentation?
A. Acetoin
B. Nitrite
C. Acetic acid
D. Hydrogen sulfide
4. At which pH does the methyl red (MR) test become positive?
A. 7.0
B. 6.5
C. 6.0
D. 4.5

1. A The family Enterobacteriaceae consists of more than 100 species and represents the
most commonly encountered isolates in clinical specimens. All Enterobacteriaceae
species ferment glucose, are nonsporulating, and are oxidase negative (except for
Plesiomonas shigelloides, recently added to the family but which is oxidase positive).
Most Enterobacteriaceae species are motile, but the genera Shigella and Klebsiella are
not.
2. B The ONPG test detects β-galactosidase activity and is most useful in distinguishing late
lactose fermenters from lactose nonfermenters. Some strains of E. coli are slow lactose
fermenters and may be confused with Shigella spp., which do not ferment lactose. E.
coli are ONPG positive, whereas Shigella spp. are ONPG negative.
3. A Acetoin or carbinol, an end product of glucose fermentation, is converted to diacetyl
after the addition of the VP reagents (β-naphthol and 40% potassium hydroxide
[KOH]). Diacetyl is seen as a red- to-pink–colored complex.
4. D Both MR and VP tests detect acid production from the fermentation of glucose.
However, a positive MR test result denotes a more complete catabolism of glucose to
highly acidic end products, such as formate and acetate, than occurs with organisms
that are VP positive only (e.g., Klebsiella pneumoniae).
5. A positive Simmons citrate test is seen as a:

A. Blue color in the medium after 24 hours of incubation at 35°C
B. Red color in the medium after 18 hours of incubation at 35°C
C. Yellow color in the medium after 24 hours of incubation at 35°C
D. Green color in the medium after 18 hours of incubation at 35°C
6. In the test for urease production, ammonia reacts to form which product?
A. Ammonium citrate
B. Ammonium carbonate
C. Ammonium oxalate
D. Ammonium nitrate
7. Which of the following reagents is added to detect the production of indole?
A. p-Dimethylaminobenzaldehyde
B. Bromcresol purple
C. Methyl red
D. Cytochrome oxidase
8. Decarboxylation of the amino acids lysine, ornithine, and arginine results in the
formation of:
A. Ammonia
B. Urea
C. Carbonate
D. Amines
9. Lysine iron agar (LIA) showing a purple slant and a blackened butt indicates:
A. Escherichia coli
B. Citrobacter spp.
C. Salmonella spp.
D. Proteus spp.
10. Putrescine is an alkaline amine product of which bacterial enzyme?
A. Arginine decarboxylase
B. Phenylalanine deaminase
C. Ornithine decarboxylase
D. Lysine decarboxylase

5. A The Simmons citrate test determines if an organism can utilize citrate as the sole source
of carbon. The medium turns blue, indicating the presence of alkaline products, such as
carbonate. Tubes are incubated a minimum of 24 hours at 35°C with a loose cap before
reading.
6. B The test for urease production is based on the ability of the colonies to hydrolyze urea
in Stuart broth or Christensen agar to form CO2 and ammonia. These products form
ammonium carbonate, resulting in alkalinization. This turns the pH indicator (phenol
red) pink at pH 8.0.
7. A The indole test detects the conversion of tryptophan (present in the media) to indole by
the enzyme tryptophanase. Indole is detected by the reaction with the aldehyde group
of p-dimethylaminobenzaldehyde (the active reagent in Kovac and Ehrlich reagents) in
acid, forming a red complex.
8. D Specific decarboxylases split dibasic amino acids (lysine, arginine, and ornithine),
forming alkaline amines. These products turn the pH indicators in the medium (cresol
red and bromcresol purple) from yellow to purple.
9. C LIA is used as an aid for the identification of Salmonella species. It contains
phenylalanine, lysine, glucose, thiosulfate, ferric ammonium citrate, and bromcresol
purple. Salmonella spp. produce H2S from thiosulfate. This reduces ferric ammonium
citrate, forming ferrous sulfate and causing the butt to blacken. Salmonella also
decarboxylate lysine to produce alkaline amines, giving the slant its purple color and
differentiating it from Citrobacter spp., which are lysine decarboxylase negative.
10. C Putrescine is the amine product of the decarboxylation of ornithine.
11. Which genera are positive for phenylalanine deaminase?

A. Enterobacter, Escherichia, and Salmonella
B. Morganella, Providencia, and Proteus
C. Klebsiella and Enterobacter
D. Proteus, Escherichia, and Shigella
12. Kligler iron agar (KIA) differs from triple-sugar iron agar (TSI) in the:
A. Ratio of lactose to glucose
B. Ability to detect H2S production
C. Use of sucrose in the medium
D. Color reaction denoting production of acid
Microbiology/Apply principles of basic laboratory
procedures/Methods/Reagents/Media/Gram-negative bacilli/2
13. The malonate test is most useful in differentiating which members of the
Enterobacteriaceae family?
A. Shigella
B. Proteus
C. Salmonella subgroups 2, 3 (the former Arizona)
D. Serratia
14. Which genera of the Enterobacteriaceae family are known to cause diarrhea and are
considered enteric pathogens?
A. Enterobacter, Klebsiella, Providencia, and Proteus
B. Escherichia, Salmonella, Shigella, and Yersinia
C. Pseudomonas, Moraxella, Acinetobacter, and Aeromonas
D. Enterobacter, Citrobacter, and Morganella
Microbiology/Apply knowledge of fundamental biological characteristics/Gram-negative
bacilli/1
15. An isolate of E. coli recovered from the stool of a patient with severe bloody diarrhea
should be tested for which sugar before sending it to a reference laboratory for
serotyping?
A. Sorbitol (fermentation)
B. Mannitol (oxidation)
C. Raffinose (fermentation)
D. Sucrose (fermentation)
Microbiology/Evaluate laboratory data to recognize health and disease states/Gram-
negative bacilli/3
16. Care must be taken when identifying biochemical isolates of Shigella because
serological cross reactions occur with:
A. Escherichia coli
B. Salmonella spp.
C. Pseudomonas spp.
D. Proteus spp.
bacilli/2

11. B Phenylalanine deaminase oxidatively deaminates phenylalanine, forming
phenylpyruvic acid. When a solution of ferric chloride is added, the iron reacts with
phenylpyruvic acid, forming a green-colored complex. Phenylalanine deaminase is
found in the genera Morganella, Providencia, and Proteus and is an excellent test to
determine if an organism belongs to this group. Rarely, isolates of Enterobacter may
be phenylalanine deaminase positive as well.
12. C Both KIA and TSI contain 10-fold more lactose than glucose, peptone, and phenol red
to detect acid production (turns yellow) and sodium thiosulfate and ferrous ammonium
sulfate to detect H2S production. However, TSI contains sucrose, and KIA does not.
Organisms fermenting either sucrose or lactose will turn the slant of the agar tube
yellow. Therefore, some organisms (e.g., many species of Cedecea, Citrobacter,
Edwardsiella, and Serratia) will produce a yellow slant on TSI but a red slant on KIA.
13. C The malonate test determines whether an organism can utilize sodium malonate as the
sole source of carbon. Malonate is broken down, forming alkaline metabolites that
raise the pH of the broth above 7.6. This causes bromthymol blue to turn from green to
deep blue (Prussian blue). E. coli, Shigella, and most Salmonella are malonate
negative, whereas Enterobacter and Salmonella (formerly Arizona) subgroups 2, 3a,
and 3b are positive. Proteus, Providencia, Serratia, and Yersinia are also malonate
negative.
14. B Escherichia, Salmonella, Shigella, and Yersinia are responsible for the majority of
enteric diarrhea cases attributable to the Enterobacteriaceae family.
15. A An isolate of E. coli (shiga-like producing toxin of E. coli [STEC]) recovered from a
stool culture in hemorrhagic colitis can be definitely identified only by serotyping. The
isolate is identified as E. coli by the usual biochemical reactions. The strain of E. coli
responsible for hemorrhagic colitis is O157:H7 and is usually negative for sorbitol
fermentation. Colonies of this strain of E. coli appear colorless on MacConkey agar
with sorbitol added.
16. A Serological confirmation of Shigella isolates is based on O antigen typing. If a
suspected Shigella spp. is serologically typed with polyvalent sera before it has been
correctly identified biochemically, a false-positive confirmation may occur with an
isolate that is E. coli (i.e., anaerogenic, non–gas-producing, lactose-negative or -
delayed, and nonmotile strains). These strains were formerly known as the
Alkalescens-Dispar serotype.
17. Which species of Shigella is most commonly associated with diarrheal disease in the
United States?
A. Shigella dysenteriae
B. Shigella flexneri
C. Shigella boydii
D. Shigella sonnei
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18. Which of the following tests best differentiates Shigella species from E. coli?
A. H2S, VP, citrate, and urease
B. Lactose, indole, ONPG, and motility
C. Hydrogen sulfide, MR, citrate, and urease
D. Gas, citrate, and VP
19. Which genera of Enterobacteriaceae are usually nonmotile at 36°C?
A. Shigella, Klebsiella, and Yersinia
B. Escherichia, Edwardsiella, and Enterobacter
C. Proteus, Providencia, and Salmonella
D. Serratia, Morganella, and Hafnia
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20. Fever, abdominal cramping, watery stools, and fluid and electrolyte loss preceded by
bloody stools 2 to 3 days before is characteristic of shigellosis but may also result from
infection with:
A. Campylobacter spp.
B. Salmonella spp.
C. Proteus spp.
D. Yersinia spp.
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21. Cold enrichment of feces (incubation at 4°C) in phosphate-buffered saline prior to
subculture onto enteric media enhances the recovery of:
A. Enterotoxigenic Escherichia coli
B. Salmonella paratyphi
C. Hafnia alvei
D. Yersinia enterocolitica
Microbiology/Apply principles of special procedures/Gram-negative bacilli/2

17. D The Shigella spp. are lactose nonfermenters that, for the most part, are biochemically
inert and are classified into serogroups A, B, C, and D as a result of their biochemical
similarity. S. sonnei is the species most often isolated from diarrhea cases in the United
States. It is more active biochemically than the other species because of ornithine
decarboxylase and β-galactosidase activity. These enzymes, found in most strains of S.
sonnei, distinguish it from other Shigella species.
18. B E. coli, when positive for lactose, indole, and ONPG, are usually motile. Shigella
species do not ferment lactose or produce indole, lack β-galactosidase, and are
nonmotile.
19. A Shigella spp. and Klebsiella spp. are, for the most part, nonmotile. Yersinia can be
motile at 22°C but is nonmotile at 36°C. Other members of Enterobacteriaceae that
have been isolated from human specimens and are usually nonmotile include
Leminorella, Rahnella, and Tatumella (considered unusual enteric pathogens).
20. A Shigella spp. and Campylobacter spp. are both causes of diarrhea, abdominal pain,
fever, and sometimes vomiting. Blood is present in the stools of patients infected with
Shigella as a result of invasion and penetration of the bowel by the organisms. Young
children may also exhibit bloody stools when infected with Campylobacter.
21. D Cold enrichment is especially useful when specimens contain large numbers of
normal flora that are sensitive to prolonged exposure to near-freezing temperature. In
addition to Yersinia, the technique has been used to enhance recovery of Listeria
monocytogenes from specimens containing other bacteria.
22. Which group of tests, along with colonial morphology on primary media, aids most in
the rapid identification of Enterobacteriaceae?
A. MR and VP, urease, and blood agar plate
B. Phenylalanine deaminase, urease, and CDC agar plate
C. Bacitracin, β-lactamase, and MacConkey agar plate
D. Indole, oxidase, MacConkey, and blood agar plates
23. A routine, complete stool culture procedure should include media for the isolation of E.
coli O157:H7 as well as:
A. Salmonella, Shigella, Yersinia, Campylobacter, and Staphylococcus aureus
B. Vibrio cholerae, Brucella, and Yersinia spp.
C. Staphylococcus aureus, group B streptococci, and group D streptococci
D. Clostridium difficile, Clostridium perfringens, and Yersinia spp.
24. Which group of tests best identifies the Morganella and Proteus genera?
A. Motility, urease, and phenylalanine deaminase
B. Malonate, glucose fermentation, and deoxyribonuclease (DNase)
C. Indole, oxidase, MR, and VP
D. Indole, citrate, and urease
25. Which group of tests best differentiates Enterobacter aerogenes from Edwardsiella
tarda?
A. Motility, citrate, and urease
B. H2S production, sucrose fermentation, indole, and VP
C. Lysine decarboxylase, urease, and arginine dihydrolase
D. Motility, H2S production, and DNase
26. Cronobacter sakazakii (formerly Enterobacter sakazakii) can best be differentiated from
Enterobacter cloacae by which of the following characteristics?
A. Yellow pigmentation and negative sorbitol fermentation
B. Pink pigmentation and positive arginine dihydrolase
C. Yellow pigmentation and positive urease
D. H2S production on TSI

22. D All Enterobacteriaceae species are oxidase negative (the exception being Plesiomonas
spp., which cluster with the genus Proteus). Through the use of nucleic acid–based
methods, oxidase positive Plesiomonas is now included in the family
Enterobacteriaceae. Because E. coli and Proteus spp. comprise the majority of the
organisms recovered from clinical specimens, they can be initially identified through
rapid testing without additional overnight testing. E. coli display a positive indole test,
and the colonial morphology on MacConkey agar is distinctive, showing flat, pink
(lactose-positive) colonies with a ring of bile precipitation. Proteus spp. swarm on
blood agar and are indole negative.
23. A V. cholerae and C. difficile are usually not included in a routine stool culture. If
Vibrio spp. are suspected, a special request should be included. Although MacConkey
agar will support the growth of Vibrio spp., normal enteric flora overgrow and occlude
these organisms. C. difficile culture requires special media (e.g., CCFA) that inhibit
other anaerobic flora and facultative anaerobic flora, and culture should be requested
specifically if symptoms warrant. MacConkey agar with sorbitol will allow the E. coli
O157:H7 to be recovered. Yersinia spp. can be detected on a regular MacConkey agar
plate.
24. A Morganella and Proteus spp. are motile, produce urease, and deaminate
phenylalanine.
24. B
Test E. aerogenes (% positive) E. tarda (% positive)
H2S 0 100
Sucrose >90 0
Indole <20 100
VP 100 0
Citrate 95 0
26. A C. sakazakii was formally called a yellow-pigmented E. cloacae and is best
differentiated from E. cloacae by sorbitol fermentation (95% positive for E. cloacae
and 0% for [E.] C. sakazakii). In addition, E. cloacae is usually positive for urease and
malonate (65% and 75%, respectively) and (E.) C. sakazakii is usually negative (1%
and less than 20%, respectively). Both are usually motile and arginine dihydrolase
positive.
27. Members of the genus Cedecea are best differentiated from Serratia spp. by which test
result?
A. Positive motility
B. Positive urease
C. Positive phenylalanine deaminase
D. Negative DNase
28. Which of the following organisms is often confused with the Salmonella species
biochemically and on plated media?
A. Escherichia coli
B. Citrobacter freundii
C. Enterobacter cloacae
D. Shigella dysenteriae
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29. A gram-negative rod is recovered from a catheterized urine sample from a nursing
home patient. The lactose-negative isolate tested positive for indole, urease, ornithine
decarboxylase, and phenylalanine deaminase and negative for H2S. The most probable
identification is:
A. Edwardsiella spp.
B. Morganella spp.
C. Ewingella spp.
D. Shigella spp.
30. Which single test best separates Klebsiella oxytoca from K. pneumoniae?
A. Urease
B. Sucrose
C. Citrate
D. Indole
31. Which of the following organisms, found in normal fecal flora, may be mistaken
biochemically for the genus Yersinia?
A. Klebsiella spp.
B. Proteus spp.
C. Escherichia coli
D. Enterobacter spp.
bacilli/2

27. D DNase is not produced by Cedecea spp. but is produced (along with proteinases) by
Serratia spp. Other key differential tests include lipase (positive for Cedecea, negative
for Serratia) and gelatin hydrolysis (negative for Cedecea, positive for Serratia).
28. B Biochemical differentiation is essential because Citrobacter isolates may give a false-
positive result on agglutination testing with Salmonella grouping sera. C. freundii
strains, like Salmonella spp., are usually H2S producers and may be confused with
Salmonella spp. unless the proper biochemical tests are utilized. C. freundii and
Salmonella spp. are adonitol, indole, and malonate negative. However, C. freundii is
KCN positive, whereas Salmonella spp. are KCN negative. (C. freundii are late lactose
fermenters and appear colorless at 24 hours on Mac).
29. B Morganella spp. are biochemically similar to Proteus spp., both being lactose
negative, motile, and positive for phenylalanine deaminase and urease. However,
Morganella spp. can be differentiated from Proteus spp. based on H2S, indole,
ornithine decarboxylase, and xylose fermentation. Ewingella spp. are usually positive
(70%) for lactose fermentation, whereas the other three genera are lactose negative.
30. D K. oxytoca and K. pneumoniae are almost identical biochemically except for the
ability to produce indole. Both organisms are nonmotile and usually test positive for
urease, sucrose, and citrate. However, K. oxytoca is indole positive, and K.
pneumoniae is indole negative.
31. B Proteus spp. are urease positive as are approximately 70% of Y. enterocolitica
isolates. Both organisms are lactose negative and motile. However, Yersinia is motile
at 22°C and not at 35°C (demonstrated by using motility media).
32. Why might it be necessary for both pink (lactose-positive) and colorless (lactose-
negative) colonies from an initial stool culture on MacConkey agar to be subcultured
and tested further for possible pathogens?
A. Most Shigella strains are lactose positive
B. Most Salmonella strains are maltose negative
C. Most Proteus spp. are lactose negative
D. Pathogenic Escherichia coli can be lactose positive or lactose negative
33. Which agar that is used for routine stool cultures is the medium of choice for the
isolation of Yersinia strains from stool specimens?
A. Salmonella–Shigella agar
B. Hektoen enteric agar
C. MacConkey agar
D. CNA agar
34. Which organism is sometimes mistaken for Salmonella and will agglutinate in
Salmonella polyvalent antiserum?
A. Citrobacter freundii strains
B. Proteus mirabilis strains
C. Shigella sonnei strains
D. Escherichia coli
bacilli/2
35. A bloody stool cultured from a 26-year-old woman after 3 days of severe diarrhea
showed the following results at 48 hours after being plated on the following media:
MacConkey agar: Little normal flora with many non–lactose-fermenting colonies
Hektoen enteric agar: Many blue-green colonies
Campylobacter blood agar and C. difficile agar: No growth
Clear colonies (from MacConkey agar): Negative for oxidase, indole, urease, motility, and
H2S
The most likely identification is:
A. Shigella spp.
B. Salmonella spp.
C. Proteus spp.
D. Escherichia coli

32. D Possible pathogenic strains of E. coli should be picked from MacConkey agar and
subcultured onto MacConkey agar with sorbitol. After subculture, these strains can be
serotyped or sent to a reference laboratory. Most E. coli normal flora ferment D-
sorbitol and appear pink to red on MacConkey–sorbitol agar. The E. coli strain
O157:H7 causes the enteric disease hemorrhagic colitis. It ferments D-sorbitol slowly
or not at all and appears as colorless colonies on MacConkey–sorbitol agar.
33. C CIN medium is the best agar for the isolation of Yersinia strains because it inhibits
growth of other coliforms, but it is not used routinely in clinical laboratories. Yersinia
spp. grow well on MacConkey agar incubated at 37°C, but the colonies are much
smaller than the other Enterobacteriaceae species; therefore, 25°C is the temperature
recommended for isolation. Some serotypes of Yersinia may be inhibited on more
selective media, such as Salmonella–Shigella or Hektoen. CNA agar inhibits the
growth of gram-negative bacteria.
34. A C. freundii and Salmonella spp. are H2S positive and indole, VP, and phenylalanine
deaminase negative. Biochemical characteristics that help to differentiate C. freundii
from Salmonella include lactose fermentation (50% of C. freundii are lactose positive,
whereas 100% of Salmonella are lactose negative) and urease production (70% of
Citrobacter are positive and greater than 99% of Salmonella are negative).
35. A Shigella is the most likely organism biochemically. E. coli are usually indole and
motility positive, and Proteus are motility and urease positive. Most Salmonella are
H2S positive. Shigella and Campylobacter cause bloody diarrhea because they invade
the epithelial cells of the large bowel; however, Campylobacter spp. do not grow on
MacConkey agar, and they are oxidase positive.
36. Which one of the following organisms (are) is generally positive for β-glycosidase
(utilizes lactose)?
A. Salmonella spp.
B. Shigella spp.
C. Proteus spp.
D. Escherichia coli
37. In the Kauffmann-White schema, the combined antigens used for serological
identification of the Salmonella spp. are:
A. O antigens
B. H antigens
C. Vi and H antigens
D. O, Vi, and H antigens
bacilli/1
38. The drugs of choice for treatment of infections with Enterobacteriaceae are:
A. Aminoglycosides, sulfamethoxazole-trimethoprim, third-generation cephalosporins
B. Ampicillin and nalidixic acid
C. Streptomycin and isoniazid (isonicotinylhydrazide [INH])
D. Chloramphenicol, ampicillin, and colistin
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39. The Shiga-like toxin (verotoxin) is produced mainly by which Enterobacteriaceae
species?
A. Klebsiella pneumoniae
B. Escherichia coli
C. Salmonella typhimurium
D. Enterobacter cloacae
bacilli/2

36. D Enterobacteriaceae species are grouped according to their ability to ferment lactose, a
β-galactoside. Salmonella, Shigella, Proteus, Providencia, and Morganella are usually
lactose nonfermenters. Others—including certain strains of E. coli, S. sonnei, H. alvei,
Serratia marcescens, and some Yersinia—appear to be lactose nonfermenters because
they lack the permease enzyme that actively transports lactose across the cell
membrane. However, true lactose nonfermenters do not possess β-galactosidase. The
test for β-galactosidase uses the substrate o-nitrophenyl-β-galactopyranoside. At an
alkaline pH, β-galactosidase hydrolyses the substrate, forming o-nitrophenol, which
turns the medium yellow.
37. D The Kaufmann-White schema groups Salmonellae on the basis of the somatic O
(heat-stable) antigens and subdivides them into serotypes based on their flagellar H
(heat-labile) antigens. The Vi (or K) antigen is a capsular polysaccharide that may be
removed by heating. In the United States, commercially available slide agglutination
tests utilize polyvalent antisera (A, B, C-1, C-2, D, E, and Vi) to group the Salmonella
spp. because 95% of isolated organisms belong to groups A through E with the Vi
antiserum identifying the capsular or “K” antigen of Salmonella serotype typhi.
Antiserum is also used against the flagella (“H”) antigens. If the Vi antigen is positive
and the O antigen is negative, the isolate must be heated in boiling water for 15
minutes to remove the capsule and retested with the antisera. There are over 2,200
serotypes of Salmonella.
38. A The drugs of choice for the Enterobacteriaceae species vary, and several genera
display patterns of resistance that aid in their identification. K. pneumoniae and
Citrobacter diversus are resistant to ampicillin and carbenicillin; most Enterobacter
spp. and Hafnia are resistant to ampicillin and cephalothin. Proteus, Morganella, and
Serratia are resistant to colistin. Providencia and Serratia are resistant to multiple
drugs. Several genera are resistant to chloramphenicol and most are resistant to
penicillin.
39. B Strains of E. coli that produce one or both of the Shiga-like toxins (SLT I and SLT II)
can cause bloody diarrhea (hemorrhagic colitis). In the United States, E. coli strain
O157:H7 is the serotype most often associated with hemorrhagic colitis.
40. Infections caused by Y. pestis are rare in the United States. Those cases that do occur
are most frequently located in which region?
A. New Mexico, Arizona, and California
B. Alaska, Oregon, and Utah
C. North and South Carolina and Virginia
D. Ohio, Michigan, and Indiana
bacilli/2
Answer to Question 40
40. A Approximately 15 cases of Y. pestis infection are confirmed in the United States
annually. Most originate in the Southwest. It is necessary to be aware of this regional
occurrence because untreated cases are associated with a mortality rate of
approximately 60%. Y. pestis is not fastidious and grows well on blood agar. It is
inactive biochemically, and this helps differentiate it from the other Enterobacteriaceae
species. Y. pestis is the only member of the Enterobacteriaceae family that infects
humans via an insect vector (animal transmission by a flea bite).
41. A leg culture from a nursing home patient grew gram-negative rods on MacConkey
agar as pink to dark pink oxidase-negative colonies. Given the following results, which is
the most likely organism?
TSI = A/A Indole = Neg MR = Neg
VP = + Citrate = + H2S = Neg
Urease = + Motility = Neg
Antibiotic susceptibility: Resistant to carbenicillin and ampicillin
A. Serratia marcescens
B. Proteus vulgaris
C. Enterobacter cloacae
D. Klebsiella pneumoniae
42. Four blood cultures were taken over a 24-hour period from a 20-year-old woman with
severe diarrhea. The cultures grew motile (room temperature), gram-negative rods. A
urine specimen obtained via catheterization also showed gram-negative rods (100,000
col/mL). Given the following results, which is the most likely organism?
TSI = A/A gas Indole = +
VP = Neg MR = +
H2S = Neg Citrate = Neg
Urease = Neg Lysine decarboxylase = +
(PD) Phenylalanine deaminase = Neg
A. Proteus vulgaris
B. Salmonella typhi
C. Yersinia enterocolitica
D. Escherichia coli
43. A stool culture from a 30-year-old man suffering from bloody mucoid diarrhea gave the
following results on differential enteric media:
MacConkey agar = clear colonies
XLD agar = clear colonies
Hektoen agar = green colonies
Salmonella–Shigella agar = small, clear colonies
Which tests are most appropriate for identification of this enteric pathogen?
A. TSI, motility, indole, urease, Shigella typing with polyvalent sera
B. TSI, motility, indole, lysine, Salmonella typing with polyvalent sera
C. TSI, indole, MR, VP, citrate
D. TSI, indole, MR, and urease
44. A leg-wound culture from a hospitalized 70-year-old man with diabetes grew motile,
lactose-negative colonies on MacConkey agar. Given the following biochemical reactions
at 24 hours, what is the most probable organism?
H2S (TSI) = Neg Indole = Neg
MR = Neg VP = +
DNase = + Citrate = +
Urease = Neg
(PD) Phenylalanine deaminase = Neg
Ornithine and lysine decarboxylase = +
Arginine decarboxylase = Neg
Gelatin hydrolysis = +
A. Proteus vulgaris
B. Serratia marcescens
C. Proteus mirabilis
D. Enterobacter cloacae

41. D K. pneumoniae and E. cloacae display similar IMViC (indole, MR, VP, and citrate)
reactions (00++) and TSI results. However, approximately 65% of E. cloacae strains
are urease positive compared with 98% of those of K. pneumoniae. Enterobacter spp.
are motile, and Klebsiella are nonmotile. The antibiotic pattern of resistance to
carbenicillin and ampicillin is characteristic of the nonmotile Klebsiella spp.
42. D Typically, the IMViC reactions for the organisms listed are:
E. coli: (++00) (PD = Neg)
S. typhi: (0+00)
Y. enterocolitica: (V+00)
P. vulgaris: (++00) (PD = +)
Note: Indole reaction is variable (V) for Y. enterocolitica.

43. A The most likely organism is a species of Shigella. Typically, Salmonella spp. produce
H2S-positive colonies that display black centers on the differential media (except on
MacConkey agar). The biochemical tests listed are necessary to differentiate Shigella
from E. coli because some E. coli strains cross-react with Shigella-typing sera. Shigella
spp. are one of the most common causes of bacterial diarrhea; group D (S. sonnei) and
group B (S. flexneri) are the species most often isolated.
44. B S. marcescens has been implicated in numerous nosocomial infections and is
recognized as an important pathogen with invasive properties. Gelatin hydrolysis and
DNase are positive for both the Proteus spp. and Serratia, but the negative urease and
phenylalanine deaminase are differential. E. cloacae does not produce DNase,
gelatinase, or lysine decarboxylase but will display red-pigmented colonies on
MacConkey agar.
45. Three blood culture specimens taken from a 30-year-old patient with cancer receiving
chemotherapy and admitted with a urinary tract infection (UTI) grew lactose-negative,
motile, gram-negative rods prior to antibiotic therapy. Given the following biochemical
reactions, which is the most likely organism?
H2S (TSI) = + Indole = + MR = +
VP = Neg Citrate = Neg Urease = +
DNase = + (PD) Phenylalanine deaminase = +
Gelatin hydrolysis = +
Ornithine decarboxylase = Neg
A. Proteus vulgaris
B. Proteus mirabilis
C. Serratia marcescens
46. Three consecutive stool cultures from a 25-year-old male patient produced scant
normal fecal flora on MacConkey and Hektoen agars. However, colonies on CIN agar
displayed “bull’s eye” colonies after 48 hours of incubation. The patient had been
suffering from enterocolitis with fever, diarrhea, and abdominal pain for 2 days. What
is the most likely identification of this gram-negative rod?
A. Escherichia coli
B. Proteus mirabilis
C. Yersinia enterocolitica
47. A 6-year-old female patient was admitted to the hospital following 2 days of severe
diarrhea. Cultures from three consecutive stool samples contained blood and mucus.
Patient history revealed that she had eaten a hamburger at a fast-food restaurant 3
days earlier. Which pathogen is most likely responsible for the following results?
Growth on:
XLD agar = Yellow colonies
HE agar = Yellow colonies
Mac agar = Light pink and dark pink colonies
Mac with sorbitol agar = Few dark pink and many colorless colonies
A. Salmonella spp.
B. Shigella spp.
C. Escherichia coli O157:H7
D. Yersinia enterocolitica

45. A Although P. mirabilis is more frequently recovered from patients with UTIs, P.
vulgaris is commonly recovered from immunosuppressed patients. P. mirabilis is
indole negative and ornithine decarboxylase positive but otherwise is very similar to P.
vulgaris.
46. C Most members of the Enterobacteriaceae family produce detectable growth on
MacConkey agar within 24 hours. Y. enterocolita produces non–lactose-fermenting
colonies on MacConkey agar, salmon-colored colonies on Hektoen agar, and yellow or
colorless colonies on XLD agar. If Y. enterocolitica is suspected, specialized agar
(CIN) is employed. The typical “bull’s eye” colonies, dark red with a translucent
border, can be confused with Aeromonas spp. that appear similarly on CIN agar. To
differentiate, an oxidase test must be performed because Yersinia spp. are oxidase
negative and Aeromonas spp. are oxidase positive.
47. C Inflammation with bleeding of the mucosa of the large intestine (hemorrhagic colitis)
is a result of an enterohemorrhagic E. coli (EHEC) infection associated with certain
serotypes, such as E. coli O157:H7. The source of the E. coli infection is from
ingestion of undercooked ground beef contaminated with fecal matter or drinking raw
milk.
48. Following a 2-week camping trip to the southwest United States, a 65-year-old male
patient was hospitalized with a high fever and an inflammatory swelling of the axilla
and groin lymph nodes. Several blood cultures were obtained, and cultures showed
growth of gram-negative rods resembling “closed safety pins.” The organism grew on
MacConkey agar showing non–lactose-fermenting colonies. Testing demonstrated a
nonmotile rod that was biochemically inert. What is the most likely pathogen identified?
A. Yersinia pestis
B. Klebsiella pneumoniae
C. Proteus vulgaris
D. Morganella morganii
49. The majority of clinical laboratories with a microbiology department should have the
capability of serotyping which pathogenic Enterobacteriaceae species?
A. Yersinia enterocolitica, Shigella spp.
B. Escherichia coli O157:H7, Salmonella spp., Shigella spp.
C. Yersinia pestis, Salmonella spp.
D. Edwardsiella spp., Salmonella spp.
Microbiology/Apply knowledge of standard operating procedures/Identification/2
50. Direct spread of pneumonic plague disease occurs by which route?
A. Fecal–oral route
B. Rat bite
C. Ingestion of contaminated tissue
D. Inhalation of contaminated airborne droplets
Microbiology/Apply knowledge of epidemiology of transmission/2
51. Which isolates of the Enterobacteriaceae family most commonly produce extended-
spectrum β-lactamase (ESBL)?
A. Escherichia coli and Klebsiella pneumoniae
B. Yersinia enterocolitica and Yersinia pestis
C. Morganella morganii and Proteus vulgaris
D. Salmonella typhi and Shigella sonnei
Microbiology/Apply knowledge of fundamental biological characteristics/Antibiotic
susceptibility/2

48. A Y. pestis is the cause of bubonic and pneumonic plague. Bubonic plague causes
swelling of the groin lymph nodes (bubos), whereas pneumonic plague involves the
lungs. The infection caused by bubonic plague may result in fulminant bacteremia that
is usually fatal. The transmission is from rodents (rats, ground squirrels, or prairie
dogs) to humans by the bite of fleas (vectors) or by ingestion of contaminated animal
tissues. Pneumonic plague is acquired via the airborne route when there is close
contact with other pneumonic plague victims.
49. B Preliminary serological grouping of the Salmonella spp. and Shigella spp. should be
performed because reliable commercial polyvalent antisera are available. Sorbitol-
negative (MacConkey agar with sorbitol) colonies of E. coli should be tested by using
commercially available antisera for somatic “O” antigen 157 and flagellar “H” antigen
7. However, Y. pestis isolates should be sent to a public health laboratory for testing
because clinical laboratories generally do not have the typing sera available.
50. D Bubonic plague involves an inflammatory swelling of the lymph nodes of the axilla
and groin, whereas pneumonic plague is associated with an airborne route involving
the lungs. Both infections are caused by the same member of the Enterobacteriaceae
family—Y. pestis.
51. A Point mutations occur in most members of the Enterobacteriaceae family that result in
production of a β-lactamase that hydrolyzes broad-spectrum antibiotics, such as the
cephalosporins, as well as penicillin and monobactam antibiotics. These are known as
ESBL producers. The most common ESBL organisms are K. pneumoniae and E. coli.
ESBL strains are detected by demonstrating their resistance to β-lactam antibiotics.
52. Additional methods of definitive identification for the Enterobacteriaceae family

include which of the following:
A. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-
TOF-MS)
B. Serodiagnosis
C. Nucleic acid–based studies
D. All of the above
Microbiology/Apply knowledge of identification methods/2
53. Which of the following Shigella spp. serotypes is the most often isolated in the United
States?
A. Serotype A (Shigella dysenteriae)
B. Serotype B (Shigella flexneri)
C. Serotype C (Shigella boydii)
D. Serotype D (Shigella sonnei)
Microbiology/Apply knowledge of stool pathogens/2
54. Which organism is transmitted by ingesting undercooked ground beef or raw milk
resulting in inflammation and bleeding of the mucosa of the large intestine (i.e.,
hemorrhagic colitis) which can also lead to hemolytic uremic syndrome (HUS) ?
A. Escherichia coli (STEC)—Shiga toxin
B. Escherichia coli (ETEC)—enterotoxigenic
C. Escherichia coli (EAEC)—enteroaggregative
D. Proteus mirabilis
Microbiology/Identification of characteristics/Gram-negative bacilli/2
55. This organism is transmitted by ingesting contaminated fresh water and/or shell fish,
resulting in watery, dysentery-like stools and chronic diarrhea. Culture on stool media
and initial testing show the following results:
MacConkey agar = Both lactose-fermenting and non–lactose-fermenting colonies
Sheep blood agar = Nonhemolytic, shiny, opaque, smooth, nonspreading
Oxidase = + DNase = Neg String test = Neg
The most likely preliminary identification is?
A. Proteus vulgaris
B. Escherichia coli
C. Salmonella spp.
D. Plesiomonas shigelloides
Microbiology/Evaluate test results to make identification/Gram-negative bacilli/2
Answer to Questions 52–55

52. D Serotyping for grouping of Salmonella spp., Shigella spp., and E.coli 0157:H7 using
commercially available slide agglutination test kits is done by most clinical
laboratories. Nucleic acid–based studies are usually performed in reference
laboratories if the demand is minimal in a clinical laboratory. MALDI-TOF MS is used
for the identification of Enterobacteriaceae species. Microbial genomic research
showed that many mass spectral peaks represented ribosomal proteins when compared
with 16S rRNA gene sequencing and could be used to identify clinically important
bacteria (Enterobacteriaceae) with the incorporation of mass spectral libraries and
software. One limitation has been identified in that MALDI-TOF MS cannot
differentiate between E. coli and Shigella spp. Species-level identification compared
with the reference method showed optimal performance in the case of many of the
Enterobacteriaceae.
53. D S. sonnei (Serotype D) is the most isolated species of Shigella in the United States.
Humans are the only reservoirs, and of the estimated 500,000 cases of shigellosis in the
United States per year, S. sonnei is responsible for 85% of the infections. It is primarily
a pediatric infection, with 60% seen in children under 10 years of age.
54. A E. coli (STEC) is the organism referred to as enterohemorrhagic E. coli, which
produces a toxin similar to the Shiga toxin produced by S. dysenteriae. Inflammation
and bleeding of the mucosa of the large intestine can lead to kidney failure and HUS.
HUS is characterized by low platelet counts and hemolytic anemia, leading to
transfusions and dialysis and death. The two most common serotypes are 0157:H7 and
0157:NM (nonmotile). Unlike in dysentery, the wet preps of the stools usually lack
white blood cells (WBCs).
55. D P. shigelloides, now considered a new member of the Enterobacteriaceae family,
causes gastrointestinal infections. Research shows that it may cross-react with Shigella
grouping antisera (Group D) causing misidentification. If an oxidase test is performed
first, this error can be avoided (Shigella = oxidase negative, whereas Plesiomonas =
oxidase positive).
8.3
Nonfermentative Bacilli
1. What are the most appropriate screening tests to presumptively differentiate and
identify the nonfermentative gram-negative bacilli (NFB) from the Enterobacteriaceae
species?
A. Catalase, decarboxylation of arginine, growth on blood agar
B. Motility, urease, morphology on blood agar
C. Oxidase, TSI, nitrate reduction, growth on MacConkey agar
D. Oxidase, indole, and growth on blood agar
Microbiology/Evaluate laboratory data to make identifications/NFB/2
2. Presumptive tests used for identification of the Pseudomonas spp. are:
A. Oxidase, oxidation–fermentation (OF) glucose (open), OF glucose (sealed), motility,
pigment production
B. Growth on blood agar plate (BAP) and eosin–methylene blue (EMB) agars, lysine
decarboxylation, catalase
C. Growth on MacConkey, EMB, and XLD agars and motility
D. Growth on mannitol salt agar and flagellar stain
3. Which tests are most appropriate to differentiate between Pseudomonas aeruginosa and
Pseudomonas putida?
A. Oxidase, motility, pyoverdin
B. Oxidase, motility, lactose
C. Oxidase, ONPG, DNase
D. Mannitol, nitrate reduction, growth at 42°C
4. Which test group best differentiates Acinetobacter spp. from P. aeruginosa?
A. Oxidase, motility, nitrate reduction
B. MacConkey growth, 37°C growth, catalase
C. Blood agar growth, oxidase, catalase
D. Oxidase, TSI, MacConkey growth

1. C NFB will grow on the slant of TSI or KIA, but they do not acidify the butt (glucose
fermentation), as do the Enterobacteriaceae. NFB can be cytochrome oxidase positive
or negative, but all the Enterobacteriaceae species, except Plesiomonas shigelloides,
are oxidase negative. Enterobacteriaceae species grow well on MacConkey agar and
reduce nitrate to nitrite, but NFB grow poorly or not at all on MacConkey and most do
not reduce nitrate. Nearly 70% of NFB recovered from clinical specimens are strains of
P. aeruginosa, Acinetobacter spp., or Stenotrophomonas maltophilia.
2. A The use of OF tubes helps to determine the presumption of a nonfermentative bacillus
(glucose oxidation positive and glucose fermentation negative). The positive
cytochrome oxidase test and pigment production indicate a possible Pseudomonas
species. Several NFB produce pigments that aid in species identification: P.
aeruginosa produces yellow pyoverdins (fluorescein) and/or pyocyanin (blue aqua
pigment). The characteristic grapelike odor of aminoacetophenone and growth at 42°C
are characteristic of P. aeruginosa.
3. D Both organisms are oxidase positive, motile, and produce pyoverdin. Both are negative
for ONPG and DNase. The differentiating tests are:
Test P. aeruginosa P. putida
Mannitol + Neg
Reduction of + Neg
nitrate to nitrite
42°C growth + Neg
4. A Acinetobacter spp. are nonmotile rods, which appear as coccobacillary forms in
clinical specimens. All are oxidase negative and catalase positive. P. aeruginosa
reduces nitrate to nitrite, whereas Acinetobacter spp. do not. The genus Acinetobacter
is divided into two groups: saccharolytic or glucose-oxidizing species and
asaccharolytic or nonglucose species.
5. In addition to motility, which test best differentiates Acinetobacter spp. and Alcaligenes
faecalis?
A. Triple sugar iron agar
B. Oxidase
C. Urease
D. Flagellar stain
Microbiology/Select methods/Reagents/Media/NFB/Identification/2
6. The most noted differences between P. aeruginosa and Stenotrophomonas maltophilia
are:
A. Oxidase, catalase, and TSI
B. Oxidase, catalase, and ONPG
C. Oxidase, 42°C growth, and polar tuft of flagella
D. Catalase, TSI, and pigment
7. Which nonfermentative bacillus is usually associated with a lung infection related to
cystic fibrosis (CF)?
A. Pseudomonas fluorescens
B. Pseudomonas aeruginosa
C. Pseudomonas putida
D. Burkholderia pseudomallei
Microbiology/Apply knowledge of fundamental biological characteristics/NFB/2
8. A nonfermenter recovered from an eye wound is oxidase positive, motile with polar
monotrichous flagella, and grows at 42°C. Colonies are dry, wrinkled or smooth, buff to
light brown, and are difficult to remove from the agar. In which DNA homology group
should this organism be placed?
A. Pseudomonas stutzeri
B. Pseudomonas fluorescens
9. Which organism is associated with immunodeficiency syndromes and melioidosis (a
glanders-like disease prevalent in Southeast Asia and northern Australia)?
A. Pseudomonas aeruginosa
B. Pseudomonas stutzeri

5. B The two genera, Acinetobacter and Alcaligenes, are very similar. Both use oxidation
for the metabolism of carbohydrate, with some strains being nonsaccharolytic. Both
grow well on MacConkey agar. However, Acinetobacter is nonmotile and oxidase
negative. Alcaligenes is motile by peritrichous flagella and oxidase positive.
6. C The two genera, Pseudomonas and Stenotrophomonas, are motile and grow well on
MacConkey agar. However, P. aeruginosa is oxidase positive and grows at 42°C but is
motile only by polar monotrichous flagella. S. maltophilia is oxidase negative, does not
grow at 42°C, and is motile by a polar tuft of flagella.
7. B P. aeruginosa is often recovered from the respiratory secretions of patients with CF. If
the patient is chronically infected with the mucoid strain of P. aeruginosa, the
biochemical identification is very difficult. The mucoid strain results from production
of large amounts of alginate, a polysaccharide that surrounds the cell.
8. A P. stutzeri produces dry, wrinkled colonies that are tough and adhere to the media as
well as smooth colonies. B. pseudomallei produces similar colony types but is
distinguished by biochemical tests and susceptibility to the polymyxins. The colonies
of P. stutzeri are buff to light brown because of the relatively high concentration of
cytochromes.
9. D B. pseudomallei produces wrinkled colonies resembling P. stutzeri. Infections are
usually asymptomatic and can be diagnosed only by serological methods. The
organism exists in soil and water in an area of latitude 20° north and south of the
equator (mainly in Thailand and Vietnam). Thousands of U.S. military personnel were
infected with these bacteria during the 1960s and 1970s. The disease may reactivate
many years after exposure and has been called the “Vietnamese time bomb.”
10. Which characteristics/biochemical tests are used to differentiate Burkholderia cepacia

from S. maltophilia?
A. Pigment on blood agar, oxidase, DNase
B. Pigment on MacConkey agar, flagellar stain, motility
C. Glucose, maltose, lysine decarboxylase
D. Triple-sugar iron agar, motility, oxidase
11. The following results were obtained from a pure culture of gram-negative rods
recovered from the pulmonary secretions of a 10-year-old cystic fibrosis patient with
pneumonia:
Oxidase = + Motility = +
Glucose OF (open) = + Gelatin hydrolysis = +
Pigment = Red Arginine dihydrolase = + (nonfluorescent)
Growth at 42°C = + Flagella = + (polar, monotrichous)
Which is the most likely organism?
A. Burkholderia pseudomallei
B. Pseudomonas stutzeri
C. Burkholderia cepacia
D. Pseudomonas aeruginosa
12. Alcaligenes faecalis (formerly A. odorans) is distinguished from Bordetella
bronchiseptica with which test?
A. Urease (rapid)
B. Oxidase
C. Growth on MacConkey agar
D. Motility
13. Chryseobacterium spp. are easily distinguished from Acinetobacter spp. by which of the
following two tests?
A. Oxidase, growth on MacConkey agar
B. Oxidase and OF (glucose)
C. TSI and urea hydrolysis
D. TSI and VP

10. A Both organisms produce yellowish pigment and have polar tuft flagella, but the
oxidase and DNase tests are differential.
Test B. cepacia S. maltophilia
Pigment on BAP Green-yellow Lavender-green
Oxidase + Neg
DNase Neg +
Motility + +
Glucose OF (open) + +
Maltose OF (open) + +
Lysine decarboxylase + +
11. D The oxidase test and red pigment (pyorubin), as well as growth at 42°C, distinguish P.
aeruginosa from the other nonfermenters listed, particularly B. cepacia, which is also
associated with CF.
12. A Alcaligenes spp. and Bordetella spp. (nonpertussis) are two organisms that are very
similar biochemically, but B. bronchiseptica is urease positive. Both organisms are
oxidase positive, grow on MacConkey agar, and are motile by peritrichous flagella. B.
bronchiseptica grows well on MacConkey agar, but other species of Bordetella are
fastidious gram-negative rods. B. bronchiseptica is the cause of mild respiratory
infections, whereas B. pertussis is the cause of whooping cough.
13. A Chryseobacterium spp. and Acinetobacter spp. often produce a yellow pigment on
blood or chocolate agar and are nonmotile. Acinetobacter spp. are oxidase negative,
grow on MacConkey agar, and are coccobacillary on the Gram-stained smear. In
contrast, Chryseobacterium spp. are oxidase positive, do not grow on MacConkey
agar, and are typically rod shaped. Elizabethkingia (formerly Chryseobacterium)
meningosepticum is highly pathogenic for premature infants. The organism is
transmitted to the neonate via the birth canal and is seen in outbreaks of neonatal units
as well as long-term care facilities.
14. A gram-negative coccobacillus was recovered on chocolate agar from the CSF of an
immunosuppressed patient. The organism was nonmotile and positive for indophenol
oxidase but failed to grow on MacConkey agar. The organism was highly susceptible to
penicillin. The most probable identification is:
A. Acinetobacter spp.
C. Pseudomonas stutzeri
D. Moraxella lacunata
15. Cetrimide agar is used as a selective isolation agar for which organism?
C. Moraxella spp.
D. Stenotrophomonas maltophilia
Microbiology/Select methods/Reagents/Media/NFB/Identification/2
16. A specimen from a 15-year-old female burn patient was cultured after débridement,
and the following results were obtained:
Oxidase = + Lysine decarboxylase = Neg
Catalase = + Motility = +
Ornithine decarboxylase = Neg Glucose = + for oxidation (open tube)
Arginine dihydrolase = + Maltose = Neg for oxidation (open tube)
Penicillin = Resistant Aminoglycosides = Susceptible
Colistin (Polymixin B) = Susceptible
These results indicate which of the following organisms?
B. Moraxella lacunata
D. Alcaligenes spp.
17. A yellow pigment–producing organism, growing on chocolate agar, testing oxidase
positive, nonmotile and does not grow on MacConkey agar was recovered from the
blood of a neonate. What is the most likely organism?
D. Elizabethkingia (formerly Chryseobacterium) meningosepticum
18. Which reagent(s) is (are) used to develop the red color indicative of a positive reaction
in the nitrate reduction test?
A. Sulfanilic acid and α-naphthylamine
B. Ehrlich and Kovac reagents
C. o-Nitrophenyl-β-D-galactopyranoside
D. Kovac reagent
Microbiology/Apply knowledge of biochemical reactions/Bacteria/1

14. D Moraxella spp. are oxidase positive and nonmotile, and this distinguishes them from
Acinetobacter spp. and most Pseudomonas spp. Moraxella spp. are highly sensitive to
penicillin, but Acinetobacter spp. and Pseudomonas spp. are penicillin resistant. M.
lacunata is implicated in infections involving immunosuppressed patients. M. lacunata
is usually associated with eye infections but can also be the cause of bacteremia and
respiratory infections in the immunosuppressed patient.
15. B Growth on Cetrimide (acetyl trimethyl ammonium bromide) agar is used for the
isolation and presumptive identification of P. aeruginosa. With the exception of P.
fluorescens, the other pseudomonads are inhibited along with related nonfermentative
bacteria. Colonies of P. aeruginosa appear as yellow-green to blue-green colonies on
Cetrimide agar.
16. C P. aeruginosa is a cause of a significant number of burn wound infections; these
organisms can exist in distilled water and underchlorinated water. Acinetobacter spp.
are oxidase negative and Moraxella spp. are highly susceptible to penicillin, and this
helps rule them out as possible causes.
17. D All species of Acinetobacter are oxidase negative, are nonmotile, and grow on
MacConkey agar. Elizabethkingia (formerly Chryseobacterium) spp. produce yellow
pigment (like some Acinetobacter) but are oxidase positive and do not grow well on
MacConkey agar. B. cepacia also produces a yellow pigment but is motile. P.
aeruginosa is motile and grows on Mac agar.
18. A In the nitrate test, nitrites formed by bacterial reduction of nitrates will diazotize
sulfanilic acid. The diazonium compound complexes with α-naphthylamine, forming a
red product. Media containing nitrates are used for the identification of nonfermenters.
When testing nonfermenters, it is wise to confirm a negative reaction using zinc dust.
The diazonium compound detects nitrite only, and the organism may have reduced
nitrates to nitrogen, ammonia, nitrous oxide, or hydroxylamine. Zinc ions reduce
residual nitrates in the media to nitrites. A red color produced after addition of zinc
indicates the presence of residual nitrates, confirming a true negative reaction. If a red
or pink color does not occur after adding zinc, then the organism reduced the nitrate to
a product other than nitrite, and the result is considered positive.
19. A culture from an intra-abdominal abscess produced orange-tan colonies on blood

agar that gave the following results:
Oxidase = + Nitrate reduction = +
KIA = Alk/Alk (H2S)+ Motility = + (single polar flagellum)
DNase = + Ornithine decarboxylase = +
Growth at 42°C = Neg MacConkey agar = NLF (non–lactose fermenter)
A. Shewanella putrefaciens
B. Acinetobacter spp.
D. Chryseobacterium spp.
20. Chryseobacterium spp. and B. cepacia are easily differentiated by which test?
A. Motility
B. OF glucose
C. Oxidase
D. Cetrimide agar
21. A 15-year-old female complained of a severe eye irritation after removing her soft
contact lenses. A swab specimen of the infected right eye was obtained by an
ophthalmologist, who ordered culture and sensitivity testing. The culture was plated on
blood agar and MacConkey agar. At 24 hours, growth of a gram-negative rod that
tested positive for cytochrome oxidase was noted. The Mueller-Hinton sensitivity plate
showed a bluish-green “lawn” of growth that proved highly resistant to most of the
antibiotics tested except amikacin, tobramycin, and ciprofloxacin. What is the most
likely identification?
A. Burkholderia cepacia
C. Stenotrophomonas maltophilia
D. Acinetobacter spp.
Microbiology/Apply knowledge of laboratory data to make identifications/GNNFB/3
22. Which of the listed Pseudomonas spp. is associated with the following virulence factors:
exotoxin A, endotoxins, proteolytic enzymes, antimicrobial resistance, and production
of alginate?
A. Pseudomonas fluorescens
B. Pseudomonas putida
Microbiology/Apply knowledge of virulence/Identification/GNNFB/2
23. A 20-year-old horse groomer exhibited a “glanders-like” infection. His history indicated
he had suffered several open wounds on his hands 2 weeks before the swelling of his
lymph nodes. A gram-negative rod was recovered from a blood culture that grew well
on blood and MacConkey agars. Most of the biochemical tests were negative, including
the cytochrome oxidase test. What is the most likely identification?
A. Burkholderia mallei
Microbiology/Apply epidemiology for ID/GNNFB/3

19. A S. putrefaciens produces abundant H2S on KIA or TSI. Shewanellae are the only
nonfermenters that produce H2S on these media.
20. A B. cepacia (93%) are weakly oxidase positive and motile. Chryseobacterium spp. are
oxidase positive but are nonmotile.
21. B P. aeruginosa is an opportunistic organism that is not part of the human normal flora.
Contact lens solution contamination, eye injury, or contact lens eye trauma are factors
that contribute to P. aeruginosa eye infections. The characteristic blue-green pigment
on Mueller-Hinton agar (pyocyanin pigment) produced by P. aeruginosa and the high
resistance to antibiotics aid in its identification.
22. D P. aeruginosa is highly resistant to many antimicrobial drugs as well as being one of
the most often cultured opportunistic organisms. This virulence factor allows for many
nosocomial infections, such as UTI, wounds (in burn patients), bacteremia, respiratory
tract infection, and CNS infection.
23. A B. mallei is rarely transmitted to humans. It is the causative agent of glanders in
mules, donkeys, and horses. It is not part of the human skin flora, and the most likely
transmission to humans is through broken skin.
24. A Vietnam War veteran presented with a “glanders-like” infection (melioidosis).

Several blood cultures produced gram-negative rods that were positive for cytochrome
oxidase, oxidized glucose and xylose, and grew at 42°C. What is the most likely
organism?
A. Stenotrophomonas maltophilia
B. Burkholderia pseudomallei
D. Acinetobacter spp.
Microbiology/Apply knowledge for identification/GNNFB/2
25. Cytochrome oxidase-positive, nonfermentative gram-negative bacilli were recovered
from the stool of a patient with CF. The isolates produced wet (mucoidy) light blue
colonies on tryptic soy agar. Which identification is most likely?
B. Pseudomonas putida
Microbiology/Apply knowledge for identification/NFGNB/2
26. Several postoperative hospitalized patients were colonized with gram-negative
coccobacilli growing on MacConkey agar. Specimens were obtained from blood, urine,
and wound sites. Testing revealed oxidase negative, nonmotile organisms. Which of the
following is the most likely cause of the nosocomial infections?
D. Pseudomonas putida
Microbiology/Apply knowledge for Identification/GNNFB/3
27. A nosocomial infection involving an 80-year-old female patient, recovering from
pneumonia, produced many oxidase-negative colonies on MacConkey agar. Further
testing results are:
Motility = + Maltose = + Resistant to most beta-lactams
Glucose = + Resistant to most aminoglycosides
B. Stenotrophomonas maltophilia
D. Burkholderia gladioli
Microbiology/Apply knowledge for Identification/GNNFB/3

24. B B. pseudomallei infections often produce abscesses in organs (liver, spleen, lungs) as
well as on the skin, in soft tissue, and in joints and bones. Vietnam War veterans
especially may harbor these organisms, which are limited to tropical and subtropical
environments (Southeast Asia and Australia). The organism may surface years later after
surviving in a latent state within phagocytes. There is a high prevalence of this organism
on the surfaces of rice paddies in northern Thailand.
25. D Patients with CF usually do not escape P. aeruginosa infections completely. P.
aeruginosa produces alginate that accounts for the “wet, mucoidy” appearance of
colonies. This overproduction of alginate is thought to cause the inhibition of
phagocytosis. The light blue color results from lower production of polyamine, and the
wet or mucoidy appearance is caused by overproduction of alginate. The result is chronic
infections in patients with CF with the “wet” form of P. aeruginosa.
26. A Acinetobacter spp. often colonize immunocompromised patients through intravenous
or urinary catheters. Acinetobacter spp. are nonmotile, oxidase negative, coccobacilli,
which grow well on MacConkey agar.
27. B S. maltophilia are oxidase negative, whereas all the other choices are oxidase positive
(B. gladioli are weakly oxidase positive).

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8.

Answers to Questions 6–10

Answers to Questions 11–15

Answers to Questions 21–25

Answers to Questions 26–30

Answers to Questions 1–4

5. A positive Simmons citrate test is seen as a:

Answers to Questions 5–10

11. Which genera are positive for phenylalanine deaminase?

Answers to Questions 11–16

Answers to Questions 17–21

Answers to Questions 22–26

Answers to Questions 27–31

Answers to Questions 32–35

Answers to Questions 36–39

Answers to Questions 41–44

Note: Indole reaction is variable (V) for Y. enterocolitica.

Answers to Questions 45–47

Answers to Questions 48–51

52. Additional methods of definitive identification for the Enterobacteriaceae family

Answer to Questions 52–55

Answers to Questions 1–4

Answers to Questions 5–9

10. Which characteristics/biochemical tests are used to differentiate Burkholderia cepacia

Answers to Questions 10–13

Answers to Questions 14–18

19. A culture from an intra-abdominal abscess produced orange-tan colonies on blood

Answers to Questions 19–23

24. A Vietnam War veteran presented with a “glanders-like” infection (melioidosis).

Answers to Questions 24–27

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