ASCP MLS Microbiology

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

B. In order to attain asepsis of the skin, 70% alcohol followed by 2% iodine is used for obtaining blood

cultures.

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 sec

B. 30 sec

C. 60 sec

D. 5 min

C. The iodine should remain on the skin for 1 min because instant antisepsis does not occur when cleansing the skin for a blood culture.

What is the purpose of adding 0.025%-0.050% sodium polyanetholsulfonate (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

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.

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

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.

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

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.

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

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.

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, Campy, colistin-nalidixic acid (CNA) agars

C. CNA and Christensen urea agars and thioglycollate media

D. Blood, Campy, Mueller-Hinton agars, and thioglycollate media

B. Hektoen agar selectively isolates pathogenic coliforms, especially Salmonella and Shigella. MacConkey agar differentiates lactose fermenters from nonfermenters. CNA agar contains antibiotics that prohibit growth of gram-negative coliforms but not gram-positive cocci. Campy agar contains the antibiotics cephalothin, trimethoprim, vancomycin, polymyxin B, and amphotericin B to prevent growth of Enterobacteriaceae, Pseudomonas spp., and fungi.

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 selenite-F (SEL) broth

D. CNA agar

B. TCBS agar is used to grow Vibrio 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.

Colistin-nalidixic acid agar (CNA) is used primarily for the recovery of:

A. Neisseria species

B. Enterobacteriaceae

C. Pseudomonas aeruginosa

D. Staphylococcus aureus

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.

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

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.

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 on modified Thayer-Martin (MTM) medium

C. Plate the specimen on New York City or Martin-Lewis agar

D. Culture specimens in ambient oxygen at 37°C

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%-7% CO2 at 35°C. Cultures should be held a minimum of 48 hours before being considered negative.

Chocolate agar and modified Thayer-Martin 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

B. Chocolate agar provides X factor (hemin) and V factor (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.

Cycloserine-cefoxitin-fructose agar (CCFA) is used for the recovery of:

A. Yersinia enterocolitica

B. Yersinia intermedia

C. Clostridium perfringens

D. Clostridium difficile

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.

Deoxycholate agar (DCA) is useful for the isolation of:

A. Enterobacteriaceae

B. Enterococcus spp.

C. Staphylococcus spp.

D. Neisseria spp.

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).

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

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.

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.

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.

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.

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.

Which procedure is appropriate for culture of genital specimens in order to recover Chlamydia spp.?

A. Inoculate cycloheximide-treated McCoy cells

B. Plate onto blood and chocolate agar

C. Inoculate into thioglycollate (THIO) broth

D. Plate onto modified Thayer-Martin agar within 24 hours

A. Chlamydiae are strict intracellular organisms and must be cultured using living cells. Direct smears can also be made at the time of culture. Staining cells with iodine may reveal the characteristic reddish-brown inclusions sometimes seen in Chlamydia infections. Fluorescein-conjugated monoclonal antibodies may be used to identify the organisms in infected cells.

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 nutrient

D. Media for transporting specimens for virus culture include Hanks balanced salt solution with bovine albumin, 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 after being placed in the transport media until the culture media can be inoculated.

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

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%-7% CO2 at 35°C-37°C (or at room temperature for no longer than 30 min), if the specimen cannot be cultured immediately.

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

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. A loop

of β-lactamase-positive organisms applied to the

center of the blue spot will reduce the iodine to iodide,

causing the area to clear. The most sensitive method

of detection is based upon the ability of the organism

to hydrolyze the β-lactam ring of a chromogenic

cephalosporin.

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. A MIC of 64 μg/mL or greater

D. The level of drug that is achievable in serum

D. The 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.

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

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.

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

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.

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

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.

Select the medium best suited for the recovery of Yersinia enterocolitica from a patient with gastroenteritis.

A. Hektoen agar

B. Cefsulodin-Irgasan-Novobiocin (CIN) agar

C. Blood agar

D. Eosinmethylene blue agar

B. CIN agar inhibits the growth of many other

organisms from the family Enterobacteriaceae.

Yersinia spp. are also recovered from MacConkey

and Salmonella-Shigella agars.

A suspected case of plague requires which of the following procedures in order to confirm Yersinia pestis?

A. Collection of multiple sets of blood cultures

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

D. Yersinia 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.

SITUATION: Abdominal pain, fever, vomiting, and nausea prompted an elderly male to seek medical attention. A watery stool specimen

producing no fecal leukocytes or erythrocytes was cultured and grew a predominance of gram-negative fermentative bacilli. The colonies were beta-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.

A. The oxidase positive test result rules out the

members of the Enterobacteriaceae family. Colonies

of Aeromonas hydrophilia and Plesiomonas spp.

might be mistaken for Vibrio spp. since all three

grow as clear colonies on MacConkey agar, are beta

hemolytic on blood agar, and are oxidase positive.

SITUATION: Several attendees of a medical conference in the Gulf coast area became ill after frequenting a seafood restaurant. A presumptive identification of Vibrio cholera 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

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: Oxidase +, Na+ Requirement, Mannitol fermentation +, Growth on TCBS +

Aeromonas: Oxidase +, Na+ Neg, Mannitol fermentation +, Growth on TCBS +

Plesiomonas: Oxidase +, Na+ Neg, Mannitol fermentation Neg, Growth on TCBS Neg

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. XLD agar

B. MacConkey agar

C. MacConkey agar with sorbitol

D. Hektoen agar

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.

Biochemically, the Enterobacteriaceae 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

A. The family Enterobacteriaceae consists of more than 100 species and represents the most commonly encountered isolates in clinical specimens. All Enterobacteriaceae ferment glucose and are oxidase negative and nonsporulating. Most Enterobacteriaceae are motile, but the genera Shigella and Klebsiella are not.

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.

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 while

Shigella spp. are ONPG negative.

The Voges-Proskauer (VP) test detects which end product of glucose fermentation?

A. Acetoin

B. Nitrite

C. Acetic acid

D. Hydrogen sulfide

A. Acetoin or carbinol, an end product of glucose

fermentation, is converted to diacetyl after the

addition of the VP reagents (α-naphthol and 40%

potassium hydroxide [KOH]). Diacetyl is seen as a

red- to pink-colored complex.

At which pH does the methyl red (MR) test become positive?

A. 7.0

B. 6.5

C. 6.0

D. 4.5

D. Both MR and VP tests detect acid production from

the fermentation of glucose. However, a positive MR

test 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).

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

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.

In the test for urease production, ammonia reacts to form which product?

A. Ammonium citrate

B. Ammonium carbonate

C. Ammonium oxalate

D. Ammonium nitrate

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.

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

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's and Ehrlich's reagents) in acid, forming a

red complex.

Decarboxylation of the amino acids lysine, ornithine, and arginine results in the formation of:

A. Ammonia

B. Urea

C. CO2

D. Amines

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.

Lysine iron agar (LIA) showing a purple slant and a blackened butt indicates:

A. E. coli

B. Citrobacter spp.

C. Salmonella spp.

D. Proteus spp.

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 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.

Putrescine is an alkaline amine product of which bacterial enzyme?

A. Arginine decarboxylase

B. Phenylalanine deaminase

C. Ornithine decarboxylase

D. Lysine decarboxylase

C. Putrescine is the amine product of the

decarboxylation of ornithine.

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

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.

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

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.

The malonate test is most useful in differentiating which members of the Enterobacteriaceae?

A. Shigella

B. Proteus

C. Salmonella subgroups 2, 3 (the former Arizona)

D. Serratia

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.

Which genera of the Enterobacteriaceae 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

B. Escherichia, Salmonella, Shigella, and Yersinia are

responsible for the majority of enteric diarrhea cases

attributable to the Enterobacteriaceae family.

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)

A. An isolate of E. coli 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.

Care must be taken when identifying biochemical isolates of Shigella because serological cross reactions occur with:

A. E. coli

B. Salmonella spp.

C. Pseudomonas spp.

D. Proteus spp.

A. Serological confirmation of Shigella isolates is based

upon 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.

Which species of Shigella is most commonly associated with diarrheal disease in the United States?

A. S. dysenteriae

B. S. flexneri

C. S. boydii

D. S. sonnei

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 owing to ornithine decarboxylase and β-galactosidase activity. These enzymes, found in most strains of S. sonnei, distinguish it from other Shigella species.

Which of the following tests best differentiates Shigella species from E. coli?

A. Hydrogen sulfide, VP, citrate, and urease

B. Lactose, indole, ONPG, and motility

C. Hydrogen sulfide, MR, citrate, and urease

D. Gas, citrate, and VP

B. E. coli, positive for lactose, indole, and ONPG are

usually motile. Shigella species do not ferment

lactose or produce indole, lack β-galactosidase,

and are nonmotile.

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

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 the Enterobacteriaceae that have been isolated from human specimens and are usually nonmotile include Leminorella, Rahnella, and Tatumella.

Fever, abdominal cramping, watery stools, and fluid and electrolyte loss preceded by bloody stools 2-3 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.

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. Young children may also exhibit bloody stools when infected with Campylobacter.

Cold enrichment of feces (incubation at 4°C) in phosphate-buffered saline prior to subculture onto enteric media enhances the recovery of:

A. Enterotoxigenic E. coli

B. Salmonella paratyphi

C. Hafnia alvei

D. Y. enterocolitica

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.

Which group of tests, along with colonial morphology on primary media, aids most in the rapid identification of the 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

D. All Enterobacteriaceae are oxidase negative. Because E. coli and Proteus spp. comprise a 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.

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. S. aureus, group B streptococci, and group D streptococci

D. Clostridium difficile, Clostridium perfringens, and Yersinia spp.

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 (CCFA) that inhibit other anaerobic flora and facultative anaerobic flora and 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.

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

A. Morganella and Proteus spp. are motile, produce

urease, and deaminate phenylalanine.

Which group of tests best differentiates Enterobacter aerogenes from Edwardsiella tarda?

A. Motility, citrate, and urease

B. Hydrogen sulfide (H2S) production, sucrose fermentation, indole, and VP

C. Lysine decarboxylase, urease, and arginine dihydrolase

D. Motility, H2S production, and DNase

B.

E. aerogenes (% positive) | E. tarda (% positive)

H2S: 0 | 100

Sucrose: >90 | 0

Indole: <20 | 100

VP: 100 | 0

Citrate: 95 | 0

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

A. E. sakazakii is 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. sakazakii). In addition, E. cloacae is usually positive for urease and malonate (65% and 75%, respectively) and E. sakazakii is usually negative (1% and < 20%, respectively). Both species are usually motile and arginine dihydrolase positive.

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

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).

Which of the following organisms is often confused with the Salmonella species biochemically and on plated media?

A. E. coli

B. Citrobacter freundii

C. Enterobacter cloacae

D. Shigella dysenteriae

B. Biochemical differentiation is essential because

Citrobacter isolates may give a false-positive

agglutination test 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.

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.

B. Morganella are biochemically similar to Proteus spp.,

both being lactose negative, motile, and positive for

phenylalanine deaminase and urease. However,

Morganella can be differentiated from Proteus spp.

based upon H2S, indole, ornithine decarboxylase,

and xylose fermentation. Ewingella spp. are usually

positive (70%) for lactose fermentation, whereas

the other three genera are lactose negative.

Which single test best separates Klebsiella oxytoca from K. pneumoniae?

A. Urease

B. Sucrose

C. Citrate

D. Indole

D. K. oxytoca and K. pneumoniae are almost identical

biochemically except for the ability to produce

indole. Both organisms are usually positive for urease,

sucrose, and citrate. However, K. oxytoca is indole

positive and K. pneumoniae is indole negative.

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. E. coli

D. Enterobacter spp.

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 using

motility media).

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 E. coli can be lactose positive or lactose negative

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.

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

C. Cefsulodin-irgasan-novobiocin (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; 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.

Which organism is sometimes mistaken for Salmonella and will agglutinate in Salmonella polyvalent antiserum?

A. C. freundii strains

B. Proteus mirabilis strains

C. S. sonnei strains

D. E. coli

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).

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) tested negative for oxidase, indole, urease, motility, and H2S

The most likely identification is:

A. Shigella spp.

B. Salmonella spp.

C. Proteus spp.

D. E. coli

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.

Which of the following organisms are generally positive for β-galactosidase?

A. Salmonella spp.

B. Shigella spp.

C. Proteus spp.

D. E. coli

D. Enterobacteriaceae 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, Hafnia 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.

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

D. The Kaufmann-White schema groups the 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. There are over 2,200 serotypes of Salmonella.

The drugs of choice for treatment of infections with Enterobacteriaceae are:

A. Aminoglycosides, trimethoprim-sulfamethoxazole, third-generation cephalosporins

B. Ampicillin and nalidixic acid

C. Streptomycin and isoniazid

D. Chloramphenicol, ampicillin, and colistin

A. The drugs of choice for the Enterobacteriaceae 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.

The Shiga-like toxin (verotoxin) is produced mainly by which Enterobacteriaceae?

A. Klebsiella pneumoniae

B. E. coli

C. Salmonella typhimurium

D. Enterobacter cloacae

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.

Infections caused by Yersinia 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

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, which helps to differentiate

it from other Enterobacteriaceae.

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

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 for Klebsiella.

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 by 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 = +

Phenylalanine deaminase = Neg

A. Proteus vulgaris

B. Salmonella typhi

C. Yersinia enterocolitica

D. E. coli

D. Typically, the IMViC reactions for the organisms

listed are:

E. coli (++00)

S. typhi (0+00)

Y. enterocolitica (V+00)

P. vulgaris (++00)

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

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.

A leg-wound culture from a hospitalized 70-year-old diabetic man 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

Phenylalanine deaminase = Neg

Ornithine and lysine decarboxylase = +

Arginine decarboxylase = Neg

Gelatin hydrolysis = +

A. Proteus vulgaris

B. Serratia marcescens

C. Proteus mirabilis

D. Enterobacter cloacae

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.

Three blood cultures taken from a 30-year-old cancer patient receiving chemotherapy and admitted with a urinary tract infection 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 = +

Phenylalanine deaminase = +

Gelatin hydrolysis = +

Ornithine decarboxylase = Neg

A. Proteus vulgaris

B. Proteus mirabilis

C. Serratia marcescens

D. Klebsiella pneumoniae

A. Although P. mirabilis is more frequently recovered from patients with urinary tract infections, 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.

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 (cefsulodin-irgasan-novobiocin) displayed "bulls-eye" colonies after 48 hours 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. E. coli

B. Proteus mirabilis

C. Yersinia enterocolitica

D. Klebsiella pneumoniae

C. Most members of the Enterobacteriaceae family

produce detectable growth on MacConkey agar

within 24 hours. Yersinia enterocolita produces

non-lactose-fermenting colonies on MacConkey

agar, salmon-colored colonies on Hektoen agar, and

yellow or colorless colonies on XLD agar. If Yersinia

enterocolitica is suspected, specialized agar (CIN) is

employed. The typical bulls-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,

since Yersinia spp. are oxidase negative and

Aeromonas spp. are oxidase positive.

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 a hamburger lunch 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. E. coli O157:H7

D. Yersinia enterocolitica

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.

Following a 2-week camping trip to the Southwest (US), 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, resulting in growth of gram-negative rods resembling "closed safety pins." The organism grew on MacConkey's agar showing non-lactose-fermenting colonies. Testing demonstrated a nonmotile rod that was biochemically inert. What is the most likely

identification?

A. Yersinia pestis

B. Klebsiella pneumoniae

C. Proteus vulgaris

D. Morganella morganii

A. Yersinia 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.

The majority of clinical laboratories with a microbiology department should have the capability of serotyping which pathogenic Enterobacteriaceae?

A. Yersinia enterocolitica, Shigella spp.

B. E. coli O157:H7, Salmonella spp., Shigella spp.

C. Yersinia pestis, Salmonella spp.

D. Edwardsiella spp., Salmonella spp.

B. Preliminary serological grouping of the Salmonella

spp. and Shigella spp. should be performed, since

reliable commercial polyvalent antisera are available.

Sorbitol-negative (MacConkey agar with sorbitol)

colonies of E. coli should be tested using commercially

available antisera for somatic "O" antigen 157 and

flagellar "H" antigen 7. However, Yersinia pestis isolates

should be sent to a public health laboratory for

testing, since clinical laboratories generally do not

have the typing sera available.

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

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, Yersinia pestis.

Which isolates of the Enterobacteriaceae family most commonly produce extended-spectrum β-lactamase (ESBL)?

A. E. coli and Klebsiella pneumoniae

B. Yersinia enterocolitica and Yersinia pestis

C. Morganella morganii and Proteus vulgaris

D. Salmonella typhi and Shigella sonnei

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 Klebsiella pneumonia and E. coli. ESBL

strains are detected by demonstrating their resistance

to β-lactam antibiotics.

What are the most appropriate screening tests to presumptively differentiate and identify the nonfermentative gram-negative bacilli (NFB) from the Enterobacteriaceae?

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

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 are oxidase negative. The Enterobacteriaceae grow well on MacConkey agar and reduce nitrate to nitrite, but the NFB grow poorly or not at all and most do not reduce nitrate. Nearly 70% of the NFB recovered from clinical specimens are:

Strains of Psuedomonas aeruginosa

Acinetobacter baumannii

Stenotrophomonas maltophilia

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

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 as well as growth at 42°C are characteristics of P. aeruginosa.

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

D. Both organisms are oxidase positive, motile, and

produce pyoverdin. Both are negative for ONPG and DNase. The differentiating tests are:

P. aeruginosa | P. putida

Mannitol: + | Neg

Reduce NO3 to NO2: + | Neg

42°C growth: + | Neg

Which test group best differentiates Acinetobacter baumannii from P. aeruginosa?

A. Oxidase, motility, NO3 reduction

B. MacConkey growth, 37°C growth, catalase

C. Blood agar growth, oxidase, catalase

D. Oxidase, TSI, MacConkey growth

A. Acinetobacter spp. are nonmotile rods that appear as

coccobacillary forms from clinical specimens. All are

oxidase negative and catalase positive. P. aeruginosa

reduces NO3 to NO2, while A. baumannii does not.

In addition to motility, which test best differentiates Acinetobacter spp. and Alcaligenes spp.?

A. TSI

B. Oxidase

C. Catalase

D. Flagellar stain

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.

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

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.

Which Pseudomonas is usually associated with a lung infection related to cystic fibrosis?

A. P. fluorescens

B. P. aeruginosa

C. P. putida

D. Burkholderia pseudomallei

B. P. aeruginosa is often recovered from the respiratory

secretions of cystic fibrosis patients. 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.

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 in color, and are difficult to remove from the agar. In which DNA homology group should this organism be placed?

A. Pseudomonas stutzeri

B. Pseudomonas fluorescens

C. Pseudomonas alcaligenes

D. Pseudomonas diminuta

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.

Which organism is associated with immunodeficiency syndromes and melioidosis (a glanders-like disease in Southeast Asia and northern Australia)?

A. Pseudomonas aeruginosa

B. Pseudomonas stutzeri

C. Pseudomonas putida

D. Burkholderia pseudomallei

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."

Which biochemical tests are needed 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. TSI, motility, oxidase

A. Both organisms produce yellowish pigment and have

polar tuft flagella, but the oxidase and DNase tests are

differential.

B. cepacia | S. maltophilia

Pigment on BAP: Green-yellow | Lavender-green

Oxidase: + | Neg

DNase: Neg | +

Motility: + | +

Glucose OF (open): + | +

Maltose OF (open): + | +

Lysine decarboxylase: + | +

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 (nonfluorescent)

Arginine dihydrolase = +

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

D. The oxidase test and red pigment (pyorubin), as well

as growth at 42°C, distinguish P. aeruginosa from the

other pseudomonads listed, particularly B. cepacia,

which is also associated with cystic fibrosis.

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

A. Alcaligenes and Bordetella are genera belonging to

the Alcaligenaceae family. The two organisms 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.

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

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 stain smear. In contrast,

Chryseobacterium spp. are oxidase positive, do not

grow on MacConkey agar, and are typically rod

shaped. Chryseobacterium meningosepticum is highly

pathogenic for premature infants.

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.

B. Pseudomonas aeruginosa

C. Pseudomonas stutzeri

D. Moraxella lacunata

D. Moraxella spp. are oxidase positive and nonmotile,

which 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.

Cetrimide agar is used as a selective isolation agar for which organism?

A. Acinetobacter spp.

B. Pseudomonas aeruginosa

C. Moraxella spp.

D. Stenotrophomonas maltophilia

B. Cetrimide (acetyl trimethyl ammonium bromide)

agar is used for the isolation and identification of

P. aeruginosa. With the exception of P. fluorescens, the

other pseudomonads are inhibited along with related

nonfermentative bacteria.

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 (open tube) = + for oxidation

Arginine dihydrolase = +

Maltose (open tube) = Neg for oxidation

Penicillin = Resistant

Aminoglycosides = Susceptible

Colistin (Polymixin B) = Susceptible

These results indicate which of the following organisms?

A. Acinetobacter baumannii

B. Moraxella lacunata

C. Pseudomonas aeruginosa

D. Acinetobacter lwoffii

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, ruling them

out as possible causes.

A yellow pigment-producing organism that is oxidase positive, nonmotile, and does not grow on MacConkey agar is:

A. Acinetobacter baumannii

B. Acinetobacter lwoffii

C. Burkholderia cepacia

D. Chryseobacterium meningosepticum

D. All species of Acinetobacter are oxidase negative and grow on MacConkey agar. Chryseobacterium spp.

produce yellow pigment (like Acinetobacter) but are

oxidase positive and do not grow on MacConkey

agar. B. cepacia also produces a yellow pigment

but is motile.

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's and Kovac's reagents

C. o-Nitrophenyl-β-D-galactopyranoside

D. Kovac's reagent

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 the 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 test is considered positive.

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

The most likely identification is:

A. Shewanella putrefaciens

B. Acinetobacter spp.

C. Pseudomonas aeruginosa

D. Chryseobacterium spp.

A. S. putrefaciens produces abundant H2S on KIA or TSI. Shewanellae are the only nonfermenters that produce H2S on these media.