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Respiratory cultures

16 min

Skills you'll build4 skills
  • Screen a sputum Gram stain for squamous cells before routine bacterial culture
  • Match a lower respiratory colony count to the threshold for its collection method
  • Select tests for Legionella, Mycoplasma, and other agents routine culture misses
  • Decide when a negative rapid strep antigen needs culture or NAAT backup

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

Try first

Try first

The screening Gram stain of an expectorated sputum shows about 40 squamous epithelial cells and 30 neutrophils per low-power field. The only request is routine bacterial culture. Your laboratory rejects routine sputum with more than 25 squamous epithelial cells per low-power field. What do you do?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

The sputum screening Gram stain

Sputum passes through the mouth, so a screening Gram stain checks it before routine bacterial culture. Squamous epithelial cells indicate saliva. A common policy rejects routine culture when the count is above 25 squamous cells per low-power field, records the reason and asks for a new specimen.1 Neutrophils help interpret the smear, and they do not determine acceptability.2

The screen covers routine bacterial culture only. Mycobacterial, fungal, Pneumocystis and Legionella requests are processed regardless of the squamous cell count, because the routine-culture screen does not apply to them.2,3

Quantitative lower respiratory cultures

Where a laboratory quantitates bronchoscopy and aspirate cultures, each specimen type has its own threshold.4

SpecimenCount supporting infection
Bronchoalveolar lavage (BAL)10⁴ CFU/mL or more
Protected specimen brush10³ CFU/mL or more
Endotracheal aspirate10⁵ CFU/mL or more

Convert the plate count first. CFU/mL = colonies ÷ volume plated (mL) × dilution factor. A count below the threshold gives less microbiologic support for infection. It does not prove colonization, and antimicrobials given before collection can lower it. The report gives the count, the organism and the laboratory's qualified comment.4

Organisms routine media do not grow

Routine respiratory media do not grow Legionella, Mycoplasma pneumoniae, Bordetella or chlamydiae. Each needs its own request.3,2

  • Legionella grows on buffered charcoal yeast extract (BCYE) agar in up to 14 days. The urinary antigen detects mainly L. pneumophila serogroup 1, so when suspicion stays high after a negative antigen, culture or a nucleic acid amplification test (NAAT) on a lower respiratory specimen is needed.3
  • M. pneumoniae is diagnosed by NAAT.2
  • Pertussis testing requires a posterior nasopharyngeal specimen.2

Group A streptococcus in the throat

Rapid antigen tests are specific and less sensitive than culture. A positive result needs no culture. A negative result in a child 3 years or older is backed up with culture or a validated molecular test. Adults do not need routine backup.5,6 A negative result from a validated NAAT used as the primary test generally needs no backup culture.6

Follow the bench demonstration

Sources for this step
References
  1. National Jewish Health, Advanced Diagnostic Laboratories. Respiratory culture: unacceptable conditions. Accessed September 27, 2026. https://www.nationaljewish.org/for-professionals/diagnostic-testing/advanced-diagnostic-laboratories/respiratory-culture
  2. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Karlowsky JA, Patel R, Pritt BS, eds. Manual of Clinical Microbiology. 13th ed. ASM Press; 2023.
  3. Centers for Disease Control and Prevention. Laboratory testing for Legionella. Accessed September 27, 2026. https://www.cdc.gov/legionella/php/laboratories/index.html
  4. Kalil AC, Metersky ML, Klompas M, et al. Management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis. 2016;63(5):e61-e111. doi:10.1093/cid/ciw353
  5. Shulman ST, Bisno AL, Clegg HW, et al. Clinical practice guideline for the diagnosis and management of group A streptococcal pharyngitis: 2012 update by the Infectious Diseases Society of America. Clin Infect Dis. 2012;55(10):e86-e102. doi:10.1093/cid/cis629
  6. Centers for Disease Control and Prevention. Clinical guidance for group A streptococcal pharyngitis. Accessed September 27, 2026. https://www.cdc.gov/group-a-strep/hcp/clinical-guidance/strep-throat.html

Watch one

Read each decision and its explanation.

An endotracheal aspirate arrives from a ventilated patient for quantitative culture.

  • Gram stain: many neutrophils, no squamous epithelial cells, many gram-negative rods.
  • Plate: 0.01 mL of a 1:100 dilution grows 24 colonies of Klebsiella pneumoniae.

How do you interpret the count?

  1. Read the specimen type: endotracheal aspirate, threshold 10⁵ CFU/mL.

    Each specimen type has its own threshold, so the specimen type on the label determines which threshold applies.

  2. Check the Gram stain: many neutrophils, no squamous cells and gram-negative rods that match the culture.

    The smear shows whether the specimen came from the lower airway and whether it matches the growth.

  3. Convert for the volume plated: 24 ÷ 0.01 mL = 2,400 CFU/mL in the diluted suspension.

    Each colony from a 0.01-mL inoculum represents 100 CFU/mL of the material plated.

  4. Apply the dilution: 2,400 × 100 = 240,000 = 2.4 × 10⁵ CFU/mL.

    The plated suspension was diluted 1:100, so the original fluid has 100 times the count.

  5. Compare: 2.4 × 10⁵ CFU/mL is at or above the 10⁵ CFU/mL aspirate threshold.

    The count is compared with the threshold for this specimen type only.

The aspirate contains 2.4 × 10⁵ CFU/mL of K. pneumoniae, which meets the 10⁵ CFU/mL threshold for an endotracheal aspirate. Report the count and the organism with the laboratory's interpretive comment.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

An expectorated sputum has more than 25 squamous epithelial cells per low-power field, which exceeds the laboratory's rejection criterion for routine culture. The request is for routine bacterial culture and Legionella culture. What happens to each request?

Incorrect. The squamous-cell screen measures salivary contamination for routine bacterial culture. A specimen for suspected legionellosis is processed despite apparent contamination, because Legionella recovery does not depend on it.

Incorrect. Squamous cells mark salivary contamination, so the routine culture is rejected. This policy sets no neutrophil minimum, and neutrophil numbers alone do not determine acceptability.

Correct. The squamous-cell screen measures salivary contamination for routine bacterial culture, and its rejection and reason are recorded. Mycobacterial, fungal, Pneumocystis, and Legionella requests are processed regardless of the squamous cell count, because the routine-culture screen does not apply to them.

Hint
  1. Consider which culture the squamous cell screen applies to.
  2. Consider whether Legionella recovery depends on the amount of saliva in the specimen.

Review Screening sputum for adequacy

Problem 2 of 3 · Hints available

A bronchoalveolar lavage (BAL) plate receives 0.1 mL of a 1:100 dilution and grows 15 colonies. What is the count in the original fluid?

Incorrect. Multiplying 15 by 100 applies the dilution and leaves out the 0.1-mL plated volume. Each colony on a 0.1-mL plate represents 10 CFU/mL of the diluted suspension.

Correct. The diluted suspension contains 15 ÷ 0.1 = 150 CFU/mL, and the original fluid contains 150 × 100 = 15,000 = 1.5 × 10⁴ CFU/mL, which reaches the 10⁴ CFU/mL threshold for BAL fluid. The count is reported with the laboratory's qualified comment and read with the Gram stain.

Incorrect. This treats the inoculum as 0.01 mL. With 0.1 mL plated, 15 ÷ 0.1 = 150 CFU/mL in the dilution, and 150 × 100 = 15,000 CFU/mL.

Hint
  1. Work in two steps: the volume plated first, then the dilution.
  2. Each colony from a 0.1-mL inoculum represents 10 CFU/mL of the diluted suspension.
  3. Compare the result with the threshold for BAL fluid.

Review Quantitative thresholds for lower respiratory cultures

Problem 3 of 3 · On your own

An 8-year-old's throat swab is negative on a rapid antigen test for group A streptococcus. The rapid test is the laboratory's only group A test on that swab. What happens next?

High specificity makes a positive result reliable. A negative result can still miss infection, because the test is less sensitive than culture.

Excluded group A streptococcus on a negative rapid antigen

Rapid antigen tests are specific but less sensitive than culture, so a negative result in a child 3 years or older is backed up with culture or a validated molecular test. Adults do not need routine backup, and a negative result from a validated NAAT used as the primary test generally needs no backup.

A negative rapid antigen in a child 3 years or older is backed up, because the test misses some infections. Culture on blood agar or a validated NAAT provides that backup result.

A repeat has the same sensitivity. The backup must be a more sensitive method.

A throat plate is read for beta-hemolytic streptococci and a few named organisms. Identifying every colony of the mixed flora adds cost without changing the report.

Review Detecting group A streptococcus

Use it

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

  • Desmond Stirling, 67, came in with severe pneumonia after a week at a spa hotel.
  • In the emergency department he produced a sputum for routine culture and Legionella culture. Its screen shows 35 squamous epithelial cells per low-power field.
  • His Legionella urinary antigen is negative.
  • He was intubated, and an endotracheal aspirate was sent for quantitative culture. Its plate received 0.01 mL of a 1:10 dilution and grew 42 colonies of Staphylococcus aureus.
Decision 1 of 3

What happens to the two requests on the sputum?

The routine culture fails the screen and is rejected with its reason. The Legionella culture is plated on BCYE agar.

The squamous-cell screen applies to routine culture only. Legionella recovery does not depend on it, and rejecting that request discards a specimen collected before intubation.

Rejected a Legionella request because of squamous cells

The squamous-cell screen measures salivary contamination for routine bacterial culture. Mycobacterial, fungal, Pneumocystis, and Legionella requests are processed regardless of the squamous-cell count, so rejecting them along with the routine culture discards a specimen the screen does not apply to.

Severity does not change the effect of salivary contamination on a routine culture. Oral flora would still overgrow the plates.

Review Screening sputum for adequacy

Decision 2 of 3

How is the aspirate count reported?

The 10⁴ CFU/mL figure is the BAL threshold. An endotracheal aspirate is compared with the 10⁵ CFU/mL threshold.

Used the BAL threshold for an endotracheal aspirate

Thresholds differ by specimen, with 10⁴ CFU/mL for bronchoalveolar lavage (BAL), 10³ CFU/mL for a protected brush, and 10⁵ CFU/mL for an endotracheal aspirate. Judging an aspirate count of 6.3 × 10⁴ CFU/mL against the BAL threshold calls it significant, although it is below the 10⁵ CFU/mL aspirate threshold.

42 ÷ 0.01 mL = 4,200 CFU/mL, and 4,200 × 10 = 42,000 = 4.2 × 10⁴ CFU/mL. That is below 10⁵ CFU/mL, so the qualified comment says the count gives less microbiologic support for infection.

A count below the threshold does not establish colonization. The organism and count are reported with the qualified comment.

This omits the 1:10 dilution. The original fluid contains 10 times the count of the diluted suspension.

Review Quantitative thresholds for lower respiratory cultures

Decision 3 of 3

The clinician asks whether the negative urinary antigen and the routine growth rule out Legionella. What does the laboratory say?

Routine media cannot grow Legionella, and the antigen detects mainly serogroup 1. Neither result excludes the organism.

Excluded atypical agents on a negative routine culture

Routine respiratory media do not recover Legionella, Mycoplasma pneumoniae, Bordetella, or chlamydiae, so a negative routine culture cannot rule them out. Reporting it as excluding atypical pneumonia leaves the specific culture, antigen test, or NAAT undone.

The antigen misses other species and serogroups, and routine plates do not grow the organism. Culture on BCYE detects all species and serogroups, and a NAAT on a lower respiratory specimen is not limited to serogroup 1.

A repeat antigen has the same limitation. It still detects mainly L. pneumophila serogroup 1.

Review Pathogens missed by routine culture

Each decision depends on which test was requested. The squamous cell screen and the aspirate threshold apply to routine bacterial culture. The Legionella request needed its own medium and was not affected by either.

For reference

Sources checked