A 52-year-old man who makes cheese from unpasteurized goat milk has had fever and night sweats for 3 weeks. One aerobic blood culture bottle flags positive on day 4, and the Gram stain shows tiny, faintly stained gram-negative coccobacilli. At 48 hours, pinpoint nonhemolytic colonies grow on sheep blood and chocolate agar, with no growth on MacConkey agar. The isolate is oxidase positive, catalase positive, nonmotile and urease positive within 30 minutes. The sentinel laboratory's best next step is to:

Keep work in a BSC and refer to the LRN lab

Report Bordetella bronchiseptica from the urease

Confirm the identity with an automated ID panel

Set up X and V factor tests for Haemophilus

Which results from a blood culture isolate should prompt referral for possible Yersinia pestis?

Oxidase-negative, non-motile gram-negative rod with bipolar staining

Lactose-fermenting gram-negative rod, indole positive

Oxidase-positive gram-negative rod, motile at 37 degrees C

Gram-negative coccobacillus needing X and V factors

An isolate that could not be ruled out as Brucella is being sent to a reference laboratory. How must it be classified for shipping?

A category A infectious substance in triple packaging

A category B biological substance in one container

An exempt human specimen at ambient temperature

Regulated medical waste sent by a licensed carrier

When a sentinel laboratory cannot rule out a select agent, the first step is to:

Telephone the state public health laboratory

Run the isolate on the mass spectrometry system

Hold the sealed plates until the next working day

Repeat the biochemicals to narrow the identification

Which pair of findings lets a sentinel laboratory rule out Bacillus anthracis for a large aerobic gram-positive rod?

Catalase positive and spore forming

BSL-3 pathogens and select agents

11 ASCP MLS practice questions on BSL-3 pathogens and select agents, part of Microbiology. The first 11 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A 52-year-old man who makes cheese from unpasteurized goat milk has had fever and night sweats for 3 weeks. One aerobic blood culture bottle flags positive on day 4, and the Gram stain shows tiny, faintly stained gram-negative coccobacilli. At 48 hours, pinpoint nonhemolytic colonies grow on sheep blood and chocolate agar, with no growth on MacConkey agar. The isolate is oxidase positive, catalase positive, nonmotile and urease positive within 30 minutes. The sentinel laboratory's best next step is to:
1. AKeep work in a BSC and refer to the LRN lab
2. BReport Bordetella bronchiseptica from the urease
3. CConfirm the identity with an automated ID panel
4. DSet up X and V factor tests for Haemophilus
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Answer: A · Keep work in a BSC and refer to the LRN lab
Tiny gram-negative coccobacilli that grow slowly on blood and chocolate agar but not on MacConkey agar, and are oxidase, catalase and rapidly urease positive, cannot be ruled out as Brucella, and the unpasteurized goat milk fits. Under the ASM sentinel guidelines, all further work stays in a class II biosafety cabinet, the state public health (LRN reference) laboratory is called, and the isolate is referred. Automated identification systems should not be used, because they create aerosols and can misidentify Brucella. Haemophilus does not grow on sheep blood agar, and Bordetella bronchiseptica is motile and grows on MacConkey agar. Staff who handled the culture outside a cabinet should be listed for an exposure assessment.
- B: B. bronchiseptica is motile and grows on MacConkey agar.
- C: Automated panels create aerosols and can misidentify Brucella.
- D: Haemophilus would not grow on sheep blood agar alone.
Question 2 · Medium
Which results from a blood culture isolate should prompt referral for possible Yersinia pestis?
1. ALactose-fermenting gram-negative rod, indole positive
2. BOxidase-positive gram-negative rod, motile at 37 degrees C
3. COxidase-negative, non-motile gram-negative rod with bipolar staining
4. DGram-negative coccobacillus needing X and V factors
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Answer: C · Oxidase-negative, non-motile gram-negative rod with bipolar staining
Y. pestis is a non-motile, oxidase-negative gram-negative rod that often shows bipolar safety-pin staining with Wright or Wayson stain, and it grows slowly, so automated systems may fail or mis-identify it. Those features in a patient with fever and a tender node should stop bench work and trigger referral. Motility, a positive oxidase or X and V dependence all point away from plague. Sentinel laboratories perform only the rule-out tests their protocol lists.
- A: Y. pestis does not ferment lactose and is indole negative.
- B: Y. pestis is oxidase negative and non-motile at every temperature.
- D: X and V factor dependence identifies Haemophilus influenzae.
Question 3 · Hard
An isolate that could not be ruled out as Brucella is being sent to a reference laboratory. How must it be classified for shipping?
1. AA category A infectious substance in triple packaging
2. BA category B biological substance in one container
3. CAn exempt human specimen at ambient temperature
4. DRegulated medical waste sent by a licensed carrier
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Answer: A · A category A infectious substance in triple packaging
Cultures of agents such as Brucella, Bacillus anthracis, Francisella tularensis and Yersinia pestis meet the definition of a category A infectious substance, shipped as UN 2814 in triple packaging by a trained, certified shipper. Category B, UN 3373, covers patient specimens and most routine diagnostic material. The receiving laboratory is telephoned before the shipment leaves. Transfer of a select agent also carries federal registration and reporting duties, which is why sentinel laboratories refer rather than retain such isolates.
- B: Category B covers diagnostic specimens, not cultures of these agents.
- C: An exempt specimen classification does not apply to a culture of a select agent.
- D: The isolate is being referred for testing, not discarded as waste.
Question 4 · Medium
When a sentinel laboratory cannot rule out a select agent, the first step is to:
1. ARun the isolate on the mass spectrometry system
2. BHold the sealed plates until the next working day
3. CTelephone the state public health laboratory
4. DRepeat the biochemicals to narrow the identification
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Answer: C · Telephone the state public health laboratory
The rule-out protocol ends in a telephone call: the state public health laboratory arranges referral to an LRN reference laboratory, and the clinician, infection control and the laboratory safety officer are informed. Further bench identification adds exposure risk and can give misleading answers. Commercial identification databases exclude or poorly represent these agents, so a card-based or mass spectrometry result cannot be relied on. The isolate is then packaged and shipped under category A rules.
- A: Identification databases are unreliable for these agents and the work is hazardous.
- B: Delay holds up the public health response; notification is immediate.
- D: Extra bench testing raises exposure risk and is not the sentinel laboratory's role.
Question 5 · Medium
Which pair of findings lets a sentinel laboratory rule out Bacillus anthracis for a large aerobic gram-positive rod?
1. ANon-hemolytic and non-motile
2. BBeta-hemolytic and motile
3. CGram positive and aerobic
4. DCatalase positive and spore forming
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Answer: B · Beta-hemolytic and motile
B. anthracis is non-hemolytic on sheep blood agar and non-motile, so an isolate that is beta-hemolytic or motile is ruled out and usually belongs to the B. cereus group. Catalase production, spore formation and aerobic growth are shared across the genus and exclude nothing. An isolate that cannot be ruled out is referred, with no further bench work. Colonies are tenacious and ground glass in appearance and should be handled in a biosafety cabinet.
- A: Those are the features of B. anthracis itself, so the agent cannot be ruled out.
- C: Both features apply to the whole genus.
- D: Catalase and spores are common to the genus and do not separate the species.
Question 6 · Medium
A tiny gram-negative coccobacillus from a lymph node aspirate grows on chocolate agar but not on sheep blood or MacConkey agar, and is oxidase negative. Which agent should be suspected?
1. AHaemophilus influenzae
2. BBrucella melitensis
3. CLegionella pneumophila
4. DFrancisella tularensis
Answer and explanation in LearnFree account
Question 7 · Hard
Large gram-positive rods from a painless skin eschar grow as non-hemolytic, non-motile colonies on sheep blood agar. What should the laboratory do?
1. ASeal the plates and refer the isolate to a reference laboratory
2. BSubculture to bicarbonate agar to look for a capsule
3. CTest penicillin susceptibility and report the result
4. DReport the isolate as a Bacillus cereus group contaminant
Answer and explanation in LearnFree account
Question 8 · Easy
How should a sentinel laboratory handle an isolate that may be Francisella tularensis?
1. AIn a chemical fume hood with the sash lowered
2. BOn the open bench with gloves and a laboratory coat
3. CIn a certified class II biosafety cabinet, using BSL-3 practices
4. DAt a laminar flow clean bench in the specimen area
Answer and explanation in LearnFree account
Question 9 · Easy
In the Laboratory Response Network, a hospital clinical microbiology laboratory functions as a:
1. ASentinel laboratory
2. BReference laboratory
3. CNational laboratory
4. DConfirmatory laboratory
Answer and explanation in LearnFree account
Question 10 · Hard
An oxidase-positive non-fermenter from the blood of a patient returning from Southeast Asia is resistant to gentamicin and colistin, and the automated system calls it Burkholderia cepacia. What is the next step?
1. ARepeat the automated identification from a fresh subculture
2. BSend the isolate for cystic fibrosis genotyping
3. CReport B. cepacia and add a comment about the travel history
4. DRefer for possible Burkholderia pseudomallei
Answer and explanation in LearnFree account
Question 11 · Hard
A technologist worked a blood culture isolate on the open bench, and it was later identified as Brucella melitensis. The appropriate action is:
1. ADisinfect the bench with bleach and take no further action
2. BWatch for symptoms and report only if fever develops
3. CDraw blood cultures from the technologist that day
4. DReport the exposure and refer staff for risk assessment and prophylaxis
Answer and explanation in LearnFree account

BSL-3 pathogens and select agents

Which results from a blood culture isolate should prompt referral for possible Yersinia pestis?

An isolate that could not be ruled out as Brucella is being sent to a reference laboratory. How must it be classified for shipping?

When a sentinel laboratory cannot rule out a select agent, the first step is to:

Which pair of findings lets a sentinel laboratory rule out Bacillus anthracis for a large aerobic gram-positive rod?

A tiny gram-negative coccobacillus from a lymph node aspirate grows on chocolate agar but not on sheep blood or MacConkey agar, and is oxidase negative. Which agent should be suspected?

Large gram-positive rods from a painless skin eschar grow as non-hemolytic, non-motile colonies on sheep blood agar. What should the laboratory do?

How should a sentinel laboratory handle an isolate that may be Francisella tularensis?

In the Laboratory Response Network, a hospital clinical microbiology laboratory functions as a:

An oxidase-positive non-fermenter from the blood of a patient returning from Southeast Asia is resistant to gentamicin and colistin, and the automated system calls it Burkholderia cepacia. What is the next step?

A technologist worked a blood culture isolate on the open bench, and it was later identified as Brucella melitensis. The appropriate action is: