Sputum and tracheal aspirate

Sputum and tracheal aspirates from intubated patients are cultured in an attempt to diagnose pneumonia which is divided into four broad categories:

  • Community acquired pneumonia (CAP)
  • Nosocomial or Hospital acquired pneumonia (HAP) and the important subset ventilator-associated pneumonia (VAP)
  • Aspiration pneumonia
  • Pneumonia in immunocompromised patients (e.g. HIV, transplant patients)

The most common organisms causing CAP include Streptococcus pneumoniae, Mycoplasma pneumoniae, Respiratory viruses, Chlamydophila (formerly Chlamydia) pneumoniae and Haemophilus influenzae. HAP is more commonly caused by enterobacteriaciae, non-fermenting Gram – negative bacilli (e.g. Pseudomonas aeruginosa) and Staphylococcus aureus. Aspiration pneumonia is usually caused by a mixture of oral aerobes and anaerobes.

In addition to the organisms noted above, unusual agents such as Pneumocystis jiroveci, Cryptococcus neoformans and mycelial fungi (e.g. Aspergillus spp.) may be found in immunocompromised patients.

Dimorphic fungi Histoplasmosa capsulatum and Blastomyces dermatiditis are to be considered in patients who have traveled to endemic areas especially if also immunocompromised.

See Bacterial pathogens of the Respiratory tract and CNS from notes section.

ET tube tips are not processed


Direct Examination

Flush small volumes of endotracheal tube (ETT) secretions from suction tubing of neonates with a small volume of sterile trypticase soy broth (TSB) into a sterile container before processing as an un-screened sputum specimen.

An ETT tip from an adult is an unacceptable specimen and will not be processed. Notify the ward immediately and issue a report stating “ETT tips are not acceptable for culture”.

Requests for “STAT” acid-fast bacilli (AFB) staining must be approved by the lead technologist or microbiologist before processing in-house.

Gram Stain
Expectorated sputum is always contaminated to some degree with oropharyngeal organisms. Because culture of mouth contents (saliva) is not useful in the diagnosis of lung infections, a microscopic screening process is used to guide culture and interpretation.

Do not screen samples from immunocompromised patients including cystic fibrosis patients.

Do not screen any endotracheal tube aspirates or suctioned samples.

If ONLY Mycobacterium tuberculosis (TB) or fungus culture is requested process without screening. (If fungus is requested as one of several test requests, regular screening is appropriate.)

Screening Procedure
Select the most purulent portion of the specimen for Gram staining and culture. Scan the smear under low power (10X objective) and examine for epithelial cells and WBCs.

Rejection Criteria

  • Greater than 10 epithelial cells / low power field – Discard culture plates without examining.

  • Less than 10 epithelial cells / low power field – Examine and report, with quantitation, routine Gram stain results (see Reporting section tab above). Continue incubation of culture plates.

Note: If the number of WBCs is 10 times the number of epithelial cells and there is 3+ or 4+ of a single morphotype of bacteria, accept the specimen and interpret the culture.

Approved requests for STAT acid fast stain
Direct smear from an un-concentrated specimen.

Fungus requests
Prepare smear for KOH Calcofluor. Read only if specific request for fungi.


Blood Agar (BA) CO2 35°C x 48 hours
MacConkey Agar (MAC) O2 35°C x 48 hours
Chocolate Agar (CHOC) CO2 35°C x 48 hours

In fungus specifically requested add:

Sabouraud Brain Heart Infusion Agar (SABHI) O2 30°C x 4 weeks
SABHI with Chloramphenicol (SABHI-C) O2 30°C x 4 weeks

If B. cepacia is requested or specimen is from a patient with Cystic Fibrosis, add:

*Burkholderia cepacia* Agar (BC) O2 35°C x 3 days

Keep the BA, CHOC and MAC plates CO2 35°C x 3 days

If Nocardia is requested, add:

Vancomycin Colistin Nalidixic acid (VCN) CO2 35°C x 5 days

Interpretation Examine the plates after 24 and 48 hours incubation.

Identify any amount of filamentous fungus or Cryptococcus neoformans

Identify all Probable respiratory pathogens if there is a moderate to heavy growth (>2+).

Identify all Possible respiratory pathogens if there is a moderate to heavy growth (>2+) growth AND if predominant (i.e. amount of pathogen growth greater than that of normal oral flora).

Identify all Probable and Possible respiratory pathogens if there is a light growth (2+) AND predominant (i.e. amount of pathogen growth greater than that of normal oral flora) AND if any amount of WBCs are seen in gram stain.

If heavy growth of normal oral flora and heavy growth of coliforms, do not identify the coliforms and report as heavy growth of normal oral flora only.

If there is a question regarding the significance of an isolate, consult the lead technologist or microbiologist.

Probable respiratory pathogens

  • Streptococcus pneumoniae
  • Moraxella catarrhalis
  • Hemophilus influenzae
  • Group A streptococcus
  • Pseudomonas aeruginosa
  • Burkholderia cepacia
  • Nocardia spp.
  • Filamentous fungus
  • Cryptococcus neoformans

Possible respiratory pathogens

  • Staphylococcus aureus
  • Enterobacteriaciae (if more than 2 types consult lead technologist or microbiologist before workup)

These two organisms are commonly found in lower respiratory specimens in small amounts in association with normal oral flora. Report only normal oral flora.

  • Yeast not Cryptococcus neoformans
  • Group B, C and G streptococcus
  • Other gram negative bacilli (not listed above) of single morphological type

For cystic fibrosis patients
Report any amount of B. cepacia.


Gram Stain

Rejected Sputum Report
Greater than 10 squamous epithelial cells per low power field. “Microscopic examination shows many squamous epithelial cells indicating excessive oropharyngeal contamination. Specimen not adequate for culture.”

Acceptable Sputum Report
Report with quantitation:

  • Presence or absence of pus cells
  • Presence or absence of squamous epithelial cells
  • Presence of predominant respiratory pathogens (amount greater than that of normal oral flora)
  • Presence of “mixed oropharyngeal bacteria”
  • “No bacteria seen”

If yeast is predominant organism seen report with quantitation. If yeast is not the predominant organism, report the Gram stain as usual and do not identify the presence of yeast.


Negative Report
Report with quantitation “Normal oral flora” or “No growth”.
“No B. cepacia isolated” if B. cepacia culture is requested.

Positive Report
Quantitate and report significant isolates with appropriate sensitivities. Report with quantitation “Normal oral flora” if also present.
“Filamentous fungus isolated, identification to follow” (DO NOT quantitate) if appropriate.

References P.R. Murray, E.J. Baron, M.A. Pfaller, R.H. Yolken. 2003. Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C.

H.D. Izenberg. 2003. Respiratory Tract Cultures, – in Clinical Microbiology Procedures Handbook, 2nd ed. Vol.1 ASM Press, Washington, D.C.