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The sentinel laboratory and select agents

16 min

  • Stop routine identification and contain a suspected select agent
  • Run sentinel rule-out tests for B. anthracis, Brucella, and F. tularensis
  • Report and refer an isolate that cannot be ruled out as a select agent

Read the full reference

Try first

Try first

A blood culture grows a large gram-positive rod. At 18 hours its colonies on sheep blood agar are nonhemolytic, flat and ground glass, with comma-shaped projections at the edge. The next step on the routine bench is MALDI-TOF. What do you do?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Recognize, rule out, refer

A sentinel laboratory is the front line of the Laboratory Response Network (LRN). Its job with a suspected biothreat agent is narrow. It recognizes the organism, tries to rule it out with a few conventional tests, and refers anything it cannot exclude. Identification and susceptibility testing belong to the LRN reference laboratory.1,2

Containment starts with suspicion

Primary specimens are handled with biosafety level 2 (BSL-2) practices. Once one of these organisms is suspected, all work moves into a certified Class II biological safety cabinet under BSL-3 practices:1,2

  • Plates are taped shut and never opened to smell.
  • Catalase is done by the tube method and motility in semisolid medium, both inside the cabinet.
  • MALDI-TOF and automated panels are not used. The suspension aerosolizes, and the databases misidentify these agents.

A system result such as "Bacillus cereus group" still needs the rule-out, because the group contains B. anthracis.2

The rule-out tests

Suspected agentCannot be ruled out whenRuled out by
B. anthracisLarge gram-positive rod, nonhemolytic, catalase positive, nonmotileBeta-hemolysis at 24 hours, or a negative catalase
Brucellosis-causing BrucellaTiny gram-negative coccobacillus, no satellite growth, oxidase, catalase and urease positiveMacConkey colonies over 0.5 mm at 24 hours, mucoid colonies, or motility
Francisella tularensisTiny, faint gram-negative coccobacillus, scant growth on blood agar, growth on chocolate, no satellite growth, oxidase negative, catalase weak or negative, beta-lactamase positiveAny reaction outside that pattern
Yersinia pestisGram-negative rod, catalase positive, oxidase, indole and urease negativeAny reaction outside that pattern

A failed rule-out leads to referral and names no species. A nonmotile, nonhemolytic Bacillus can be B. megaterium, and only the reference laboratory confirms B. anthracis.2,3,4,5

Report and refer

When an agent cannot be excluded, several things happen at once:1,3,5

  1. Tell the physician that the organism cannot be ruled out and the isolate has been referred.
  2. Telephone the LRN reference laboratory immediately.
  3. Notify public health, infection prevention and the infectious disease service.
  4. Secure the isolate and the original specimens.
  5. Ship nothing without prior arrangement. The isolate travels as a Category A infectious substance, packed by trained, certified staff.6
References
  1. Association of Public Health Laboratories. Recognize. Rule-Out. Refer. Biothreat Agent Bench Cards for the Sentinel Laboratory. APHL; 2018. Accessed September 27, 2026. https://www.aphl.org/aboutAPHL/publications/Documents/2018_BiothreatAgents_SentinelLab_BenchCards_PRINT.pdf
  2. American Society for Microbiology, Association of Public Health Laboratories. Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases: Bacillus anthracis and Bacillus cereus biovar anthracis. Revised November 2024. Accessed September 27, 2026. https://asm.org/guideline/anthrax-bacillus-anthracis
  3. American Society for Microbiology, Association of Public Health Laboratories. Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases: brucellosis-causing Brucella species (BBS). Revised August 2025. Accessed September 27, 2026. https://asm.org/guideline/brucellosis-causing-brucella-species-bbs-sentinel
  4. American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases: Francisella tularensis. Revised March 2016. Accessed September 27, 2026. https://asm.org/getContentAsset/1d7c754e-e9ac-479c-843e-70cd3b9b17ca/dfc3d011-8f63-43f6-9ed8-4b444333a1d0/tularemia?language=en-US
  5. American Society for Microbiology. Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases: Yersinia pestis. Revised March 2016. Accessed September 27, 2026. https://asm.org/getContentAsset/508b784f-bdf7-4beb-8088-8a649c150d65/dfc3d011-8f63-43f6-9ed8-4b444333a1d0/Y-pestis-fixed-figures
  6. American Society for Microbiology, Association of Public Health Laboratories. Sentinel level clinical laboratory guidelines for suspected agents of bioterrorism and emerging infectious diseases: packing and shipping infectious substances. ASM; 2021. Accessed September 27, 2026. https://asm.org/guideline/packing-and-shipping-infectious-substances

Watch one

A blood culture bottle flags on day 4 in a man who makes goat cheese from his own herd's unpasteurized milk. Work is already inside the cabinet under BSL-3 practices.

  • Gram stain: tiny, faintly staining gram-negative coccobacilli.
  • Growth: pinpoint on blood and chocolate agar at 24 hours, 0.5 to 1 mm at 48 hours. Colonies are white, nonhemolytic and not mucoid. MacConkey shows no growth at 24 hours.
  • No satellite growth around the Staphylococcus aureus streak.
  • Oxidase positive. Catalase positive by the tube method.
  • Urease positive on Christensen's agar at 2 hours. Tube motility negative.

Can a brucellosis-causing Brucella species be ruled out?

  1. Read the Gram stain. A tiny gram-negative coccobacillus puts Brucella and Francisella on the list.

    The Gram stain sets which rule-out applies.

  2. Read the growth. The colonies grew on blood agar and match Brucella.

    Slow, pinpoint growth on blood and chocolate agar with nothing on MacConkey is the Brucella pattern.

  3. Check satellite growth. There is none, so Haemophilus is unlikely.

    On blood agar, Haemophilus grows only as satellites around a staphylococcal streak.

  4. Read oxidase and catalase. Both are positive, which fits Brucella.

    Brucella is oxidase and catalase positive, and Francisella is oxidase negative.

  5. Read urease. It is positive.

    Urease is the last positive reaction in the Brucella rule-out.

  6. Look for an excluding finding. MacConkey shows no growth, the colonies are not mucoid, and motility is negative.

    Any one excluding finding would rule Brucella out.

A brucellosis-causing Brucella species cannot be ruled out. The isolate is referred to the LRN reference laboratory, and the laboratory reports that it cannot be excluded.

Your turn

Problem 1 of 3

A large gram-positive rod from a blood culture is nonhemolytic and nonmotile in semisolid medium. Before those results, MALDI-TOF had reported it as Bacillus cereus group. What does the MALDI-TOF result mean here?

Correct. A result naming Bacillus cereus group contains B. anthracis within it, so it is not a rule-out. Secure the isolate, move all further work into the cabinet under BSL-3 practices, and begin the conventional rule-out and referral process. MALDI-TOF and automated systems are not used on a suspected isolate, because preparing the suspension aerosolizes, and because the databases misidentify these organisms.

Incorrect. Databases misidentify B. anthracis as B. cereus, and a nonmotile, nonhemolytic large gram-positive rod fits B. anthracis, so the work moves into the cabinet.

Incorrect. A sentinel laboratory leaves susceptibility testing on a suspected biothreat agent to the reference laboratory, which does the definitive work.

Hint
  1. Ask which species sit inside the group the instrument named.
  2. Compare the hemolysis and motility results with the B. anthracis rule-out.

Review Containment and bench practice

Problem 2 of 3

A sentinel laboratory cannot rule out Yersinia pestis from a lymph-node aspirate isolate. How is the result reported and handled?

Correct. The physician is told that the organism cannot be excluded and that the isolate has been referred. The LRN reference laboratory is telephoned immediately, public health and infection prevention are notified, the original specimens and cultures are secured, and nothing is shipped without prior arrangement, as a Category A infectious substance sent by a trained, certified shipper.

Incorrect. The sentinel laboratory takes the identification no further and sets up no susceptibility testing. Confirmation belongs to the reference laboratory.

Incorrect. The physician is told now that the organism cannot be excluded and has been referred, so the infectious disease service and infection prevention can act while confirmation is pending.

Hint
  1. The sentinel laboratory's job ends at rule-out and referral.
  2. Think about who needs to act while confirmation is pending.

Review Notification, referral, and escalation

Problem 3 of 3

A large gram-positive rod from a wound culture is nonhemolytic at 24 hours, catalase positive by the tube method and nonmotile in semisolid medium. What is the result of the sentinel rule-out?

The rule-out cannot identify B. anthracis. Other species give the same pattern, and only the reference laboratory confirms it.

Treated a failed rule-out as an identification

A sentinel rule-out either excludes an agent or refers the isolate to the reference laboratory for identification. A nonmotile, nonhemolytic Bacillus can be B. megaterium, and only the reference laboratory confirms B. anthracis. The report says the agent cannot be excluded and the isolate was referred.

Every finding matches B. anthracis. A rule-out needs beta-hemolysis or a negative catalase.

B. megaterium can look like this, and so can B. anthracis. The pattern names no species.

Treated a failed rule-out as an identification

A sentinel rule-out either excludes an agent or refers the isolate to the reference laboratory for identification. A nonmotile, nonhemolytic Bacillus can be B. megaterium, and only the reference laboratory confirms B. anthracis. The report says the agent cannot be excluded and the isolate was referred.

Nonhemolytic, catalase positive and nonmotile is the pattern that cannot be ruled out. The reference laboratory completes the identification.

Review Bacillus anthracis and Bacillus cereus biovar anthracis

Use it

  • Hollis Brandt (MRN 4410287), 64, hunts rabbits every fall and skins them himself.
  • He has an ulcer on his hand and a tender axillary lymph node. The node aspirate went to culture.
  • At 48 hours: scant growth on blood agar, gray-white 1-mm colonies on chocolate agar, and no growth on MacConkey.
  • Gram stain of the colonies: tiny, very faint gram-negative coccobacilli.
  • Yesterday a technologist opened the plates on the open bench and did a slide catalase.
Decision 1 of 3

What happens first?

The picture fits Francisella tularensis. Work moves into the cabinet, and yesterday's open-bench work is an exposure to evaluate at once.

Automated systems aerosolize the suspension and can misidentify F. tularensis as Haemophilus influenzae. Only the conventional rule-out tests are done.

Kept identifying a suspected select agent

Automated panels and MALDI-TOF need a concentrated suspension, which aerosolizes, and their databases misidentify these agents, as with B. anthracis reported as B. cereus. Continuing routine identification exposes staff and can release a wrong species. A suspected isolate moves into the biological safety cabinet under BSL-3 practices for conventional rule-out tests.

Earlier exposure does not justify more of it. Every step from now on happens in the cabinet.

Review Containment and bench practice

Decision 2 of 3

In the cabinet the isolate shows no satellite growth, is oxidase negative and catalase weakly positive, and is beta-lactamase positive. What is the result?

Scant growth on blood agar, growth on chocolate, no satellites, oxidase negative, weak catalase and beta-lactamase positive is the full pattern that cannot be ruled out.

On blood agar, H. influenzae grows as satellites around a staphylococcal streak. This isolate shows none.

Brucella is oxidase positive and grows well on blood agar. This isolate is oxidase negative.

A rule-out that fails refers the isolate. Only the reference laboratory identifies F. tularensis.

Treated a failed rule-out as an identification

A sentinel rule-out either excludes an agent or refers the isolate to the reference laboratory for identification. A nonmotile, nonhemolytic Bacillus can be B. megaterium, and only the reference laboratory confirms B. anthracis. The report says the agent cannot be excluded and the isolate was referred.

Review Bacillus anthracis and Bacillus cereus biovar anthracis

Decision 3 of 3

How is the result reported?

The physician, infection prevention and the infectious disease service need to act now. The report goes out while confirmation is pending.

Delayed the report until the reference laboratory confirmed

The physician is told at once that the agent cannot be excluded and the isolate was referred, so infection prevention and the infectious disease service can act. The Laboratory Response Network (LRN) reference laboratory is called immediately, public health is notified, and nothing ships without prior arrangement.

The sentinel laboratory neither identifies the agent nor tests its susceptibility. Both belong to the reference laboratory.

The physician hears at once that the agent cannot be excluded. The reference laboratory is called immediately and directs what to send.

Review Notification, referral, and escalation

The clue that settles this case is scant growth on blood agar with good growth on chocolate. That growth pattern put Francisella on the list before any reaction was read, and it should have moved the plates into the cabinet on the first day. Mr. Brandt's physician now hears that the agent cannot be excluded, and the reference laboratory finishes the work.

Keep

Sources checked