Micro 8.1 Specimen Collection, Media, and Methods

Question 1

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

Answer 1

B. 70% alcohol and then 2% iodine or an iodophor

Question 2

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

Answer 2

C. 60 seconds

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

Question 3

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

Answer 3

A. It inhibits phagocytosis and complement

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.

Question 4

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

Answer 4

C. Corynebacterium diphtheriae

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.

Question 5

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

Answer 5

A. Neisseria gonorrhoeae

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.

Question 6

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

Answer 6

C. Needle aspirate after surface decontamination

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.

Question 7

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

Answer 7

B. Hektoen, MacConkey, MacConkey-Sorbitol, Campy blood, colistin–nalidixic acid (CNA) agars; Selenite-F broth (SEL)

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.

Question 8

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

Answer 8

B. Thiosulfate–citrate–bile–sucrose (TCBS) agar and alkaline peptone water (APW) broth

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.

Question 9

9. CNA agar is used primarily for the recovery of:

A. Neisseria species
B. Enterobacteriaceae
C. Pseudomonas aeruginosa
D. Staphylococcus aureus

Answer 9

D. Staphylococcus aureus

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.

Question 10

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

Answer 10

A. Sheep

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.

Question 11

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

Answer 11

D. Culture specimens in ambient oxygen at 37°C

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.

Question 12

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

Answer 12

B. Haemophilus spp. and N. gonorrhoeae, respectively

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.

Question 13

13. Cycloserine–cefoxitin–fructose agar (CCFA) is used for the recovery of:

A. Yersinia enterocolitica
B. Yersinia intermedia
C. Clostridium perfringens
D. Clostridium difficile

Answer 13

D. Clostridium difficile

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.

Question 14

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

A. Enterobacteriaceae
B. Enterococcus spp.
C. Staphylococcus spp.
D. Neisseria spp.

Answer 14

A. Enterobacteriaceae

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)

Question 15

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

Answer 15

C. Enterobacteriaceae from gastrointestinal specimens

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

Question 16

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.

Answer 16

B. Haemophilus influenzae and Haemophilus parainfluenzae

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.

Question 17

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.

Answer 17

B. Bacteroides, Peptostreptococcus, and Clostridium spp.

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

Question 18

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

Answer 18

A. Obtain urethral, cervical swabs and urine specimens placed in transport media for the direct detection of antigen or nucleic acid and/or culture

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

Question 19

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

Answer 19

D. Antibiotics and protein nutrient

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.

Question 20

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

Answer 20

D. Incubated at 37°C and cultured as soon as possible

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.

Question 21

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

Answer 21

A. Chromogenic cephalosporin

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.

Question 22

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

Answer 22

D. The optimal therapeutic level of drug that is achievable in serum

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.

Question 23

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

Answer 23

D. All of these options

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.

Question 24

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

Answer 24

A. The swarming area should be ignored

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.

Question 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

Answer 25

D. Aminoglycosides

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.

Question 26

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

Answer 26

B. Cefsulodin–irgasan–novobiocin (CIN) agar

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.

Question 27

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

Answer 27

D. All of these options

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.

Question 28

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.

Answer 28

A. Aeromonas hydrophilia

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).

Question 29

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

Answer 29

A. Mannitol fermentation, Na+ requirement

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.

Question 30

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

Answer 30

C. MacConkey agar with sorbitol

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.