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CSF and sterile body fluids

16 min

Skills you'll build3 skills
  • Process CSF for culture at once and keep it at room temperature
  • Read a CSF molecular panel against its target list and continue the culture
  • Split a sterile body fluid between culture bottles, a Gram stain, and plates

At the bench: Select specimen preparation, media, and incubation for a culture

Try first

Try first

A lumbar puncture sends four numbered CSF tubes to the laboratory. Which tube goes to microbiology for Gram stain and bacterial culture?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

CSF processing

Suspected meningitis is an emergency. Process CSF on arrival, read the Gram stain without delay, and call any organisms seen under the urgent-notification procedure.1

  • Which tube. A later tube, commonly the second or third, goes for Gram stain and culture, because the first contains the most skin flora. A single tube goes to microbiology regardless of what else was ordered.1
  • How much. Gram stain and bacterial culture need 0.5 to 1.0 mL. Mycobacterial and fungal cultures target 5 to 10 mL. When volume is short, the laboratory and the ordering clinician decide together which tests go first.1
  • What temperature. Room temperature, because cold injures N. meningitidis. Only a small aliquot for viral culture may be refrigerated briefly.1
  • Concentrate. With 1 mL or more, centrifuge and leave about 0.5 mL of sediment for the smear and the culture. A cytocentrifuged smear shows about 100 times more organisms than a direct smear.2,3

Rapid tests and culture

A multiplex meningitis panel detects bacterial, viral and fungal targets faster than culture. It detects only the targets on its list. A negative panel does not exclude organisms that are not on the panel, and a positive panel is called at once.1 The culture continues either way. It recovers organisms outside the panel and supplies the isolate for susceptibility testing.1 Shunt infections come mostly from skin flora, such as coagulase-negative staphylococci, S. aureus, Corynebacterium and Cutibacterium.3 For suspected cryptococcal meningitis, the capsular antigen test is the preferred rapid test.1

Other sterile fluids

Pleural, peritoneal, pericardial and joint fluids arrive by needle aspiration in a sterile container.1

  • 1 to 5 mL is enough for most bacteria, and 10 to 15 mL is preferred for mycobacteria or fungi.
  • For peritoneal dialysis peritonitis, 5 to 10 mL of effluent into each of an aerobic and an anaerobic blood-culture bottle is the preferred method.4
  • A bottle gives no direct Gram stain and obscures the original organism load and mix. Bottles are paired with a separate specimen for the smear and plates.1
  • Peritonitis from a gut perforation brings a mixed flora that broth can mask. It needs an anaerobic-transport specimen for Gram stain and plates.1

The fluid is centrifuged, and the sediment is used for both the Gram stain and the culture.1

Follow the bench demonstration

Sources for this step
References
  1. Miller JM, Binnicker MJ, Campbell S, et al. Guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2024 update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. Published online March 5, 2024. doi:10.1093/cid/ciae104
  2. Tunkel AR, Hartman BJ, Kaplan SL, et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis. 2004;39(9):1267-1284. doi:10.1086/425368
  3. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Karlowsky JA, Patel R, Pritt BS, eds. Manual of Clinical Microbiology. 13th ed. ASM Press; 2023.
  4. Li PK-T, Chow KM, Cho Y, et al. ISPD peritonitis guideline recommendations: 2022 update on prevention and treatment. Perit Dial Int. 2022;42(2):110-153. doi:10.1177/08968608221080586

Watch one

Read each decision and its explanation.

Leon Staines, 58, has a ventriculoperitoneal shunt and a fever. CSF from the shunt reservoir reaches the laboratory at 14:20.

  • Cytocentrifuged Gram stain: few neutrophils, rare gram-positive cocci in clusters.
  • Multiplex meningitis panel: no targets detected. The laboratory's panel lists no staphylococci among its targets.
  • Culture: plated and incubating.

How do you read the panel?

  1. Start with the Gram stain: cocci in clusters in CSF are called at once under the urgent-notification procedure.

    Organisms seen in CSF are urgent regardless of any other test result.

  2. Read the panel against its target list: staphylococci are not on it, so the negative result does not apply to the cocci seen.

    A panel detects only the organisms on its target list.

  3. Consider the source: shunt infections come mostly from skin flora, and coagulase-negative staphylococci and S. aureus both form clusters.

    The specimen source determines which organisms are expected.

  4. Continue incubating the culture and work up what grows.

    Culture recovers organisms outside the panel and supplies the isolate for susceptibility testing.

  5. Report the panel as not detected for its listed targets, with the culture pending.

    The report states which targets the panel covers.

The negative panel does not cover the organisms on the smear. Call the Gram stain now, report the panel for its listed targets only, and identify the cocci and test their susceptibility from the culture.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A CSF specimen for Gram stain and bacterial culture cannot be processed for a short time. How is it kept until processing?

Incorrect. Cold injures sensitive organisms such as Neisseria meningitidis. Only a small aliquot intended for viral culture may be refrigerated briefly.

Incorrect. CSF for bacterial culture stays at room temperature regardless of the delay, because cold injures N. meningitidis. Time-based refrigeration rules apply to specimens such as urine, stool, and respiratory samples.

Correct. Cold injures sensitive organisms such as N. meningitidis, so CSF for bacterial and fungal culture stays at room temperature during a brief unavoidable delay.

Hint
  1. Think about which organism in CSF is most sensitive to handling.
  2. Ask what a delay at each temperature does to that organism.

Review Cerebrospinal fluid collection and routing

Problem 2 of 3 · Hints available

Peritoneal fluid from suspected secondary bacterial peritonitis after a bowel perforation arrives in blood-culture bottles only. What does the laboratory request?

Correct. Broth enrichment can mask clinically important members of a mixed gut flora. Bottles alone also allow no direct Gram stain and no assessment of the original organism load and mix. The laboratory requests a conventional anaerobic-transport specimen for Gram stain and plated media.

Incorrect. Bottles are the preferred technique for suspected peritoneal dialysis peritonitis. Secondary peritonitis from a gut source involves a mixed flora that broth enrichment can mask.

Incorrect. Additional bottles add more broth enrichment. They still allow no direct Gram stain and no assessment of the original organism load and mix.

Hint
  1. Picture the flora that leaks from a perforated bowel.
  2. Ask what a direct smear and plates show about a mixed flora.

Review Collection and transport

Problem 3 of 3 · On your own

A CSF meningitis panel detects Streptococcus pneumoniae, and the result has been called. The ordering team asks you to cancel the CSF culture, since the organism is known. What do you do?

The panel identifies the organism and gives no full susceptibility result. Without the culture, no isolate is available for testing.

The panel result is already verified and called, and a repeat adds no new information.

Susceptibility testing needs a viable isolate, and pneumococcal susceptibility can change the treatment. The culture also recovers any organism outside the panel.

Review Rapid and adjunct cerebrospinal fluid tests

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Two specimens reach the microbiology bench at 22:00.
  • Tomasz Allcott, 34: one tube of CSF, 1.5 mL. The orders are Gram stain, bacterial culture, fungal culture and mycobacterial culture. The courier asks whether it can go in the refrigerator until morning.
  • Gloria Draper, 59, on peritoneal dialysis with cloudy effluent: an aerobic and an anaerobic blood-culture bottle, each inoculated at the bedside with 8 mL of effluent. No other container came with them.
Decision 1 of 3

What happens to Mr. Allcott's CSF tonight?

Cold injures N. meningitidis, and delay reduces recovery. CSF for culture is processed at once.

Refrigerated CSF intended for routine bacterial culture

Cold injures sensitive organisms such as Neisseria meningitidis, so refrigerated CSF can lose the pathogen and give a false-negative culture. CSF for bacterial and fungal culture is kept at room temperature, and only a small aliquot for viral culture may be refrigerated briefly.

CSF is an emergency specimen, kept at room temperature and processed on arrival.

Every culture loses organisms with delay. The whole workup starts on arrival.

Review Cerebrospinal fluid collection and routing

Decision 2 of 3

The 1.5 mL cannot support every order. What do you do?

Mycobacterial and fungal cultures target 5 to 10 mL. Even shares leave every test short.

The laboratory and the clinician decide together when volume is limited. The Gram stain and bacterial culture need only 0.5 to 1.0 mL.

A lumbar puncture cannot be repeated at will, and this CSF is irreplaceable. It is worked up with the tests the clinician prioritizes.

Review Receipt, prioritization, and rejection

Decision 3 of 3

What do you do with Ms. Draper's bottles?

Bottles are the preferred method for dialysis peritonitis. A separate specimen adds the direct Gram stain and plated culture that bottles cannot give.

Bottles alone give no direct Gram stain and no view of the organism load and mix. The smear is the first result the clinician receives.

Accepted bottle-only fluid when a smear was needed

Bottle inoculation improves recovery of low-burden organisms. Broth from a bottle cannot give a direct Gram stain and hides the original burden and mix of organisms. In secondary peritonitis from a gut source, broth enrichment can mask important members of the mixed flora. A specimen sent only in bottles loses the direct Gram stain and the plated culture.

Bedside bottle inoculation improves recovery for dialysis peritonitis. Rejecting them discards the best culture.

Review Collection and transport

Each specimen is handled according to the container it arrived in. Mr. Allcott's single small tube is irreplaceable, so it is processed at room temperature on arrival and its tests are prioritized with the clinician. Ms. Draper's bottles support recovery and give no smear, so a separate specimen completes the workup.

For reference

Sources checked