STANDARD OPERATING PROCEDURE

SPUTUM CULTURES

1. Principle

Sputum cultures are performed to identify the causative agent of pneumonia and other
lower respiratory infections. Sputum cultures have a sensitivity of 40-60% for the
detection of a pathogen. This has been attributed to the fact that some of the respiratory
tract pathogens are difficult to grow e.g. M. pneumoniae and Chlamydia pneumoniae,
and also due to delay in collection and processing of the specimen. Therefore, it is
recommended that blood cultures be collected at the same time, if pneumonia is
suspected.

2. Materials

a. Clean glass slides and Gram stain reagents.

b. Media: Blood agar (BAP), Chocolate (CHOC), MacConkey (MAC), and Mueller-
Hinton agar.

c. Incubator at 35-37°C, with 5%CO2, or candle jar.

d. Inoculating loops, sterile swabs.

e. Reagents and discs for identification and susceptibility testing (AST) methods.

3. Specimen

Expectorated sputum in a sterile container. The specimen should be stored refrigerated

if there is a delay in transport to the laboratory. A delay in processing of >2 hours will
compromise the ability to isolate fastidious organisms such as S.pneumoniae and
H.influenzae.

Rejection criteria:

 Duplicate specimens collected on the same day

 24 hour collections

 Saliva

 Specimens contaminated with foreign substances e.g. food

4. Quality Control (QC)

Process the specimen as soon after receipt as possible. If there is a delay in processing
place the specimen in the refrigerator.

Check that the patient name and identifiers on the specimen match that on the
accompanying requisition.

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Ensure that all media and supplies used have passed the required QC and are used
within their expiry date.

5. Safety Precautions

Standard safety precautions for handling of patient specimens must be applied when
processing these specimens.

6. Procedure

Gram Smear

a. Select a purulent portion of the specimen and prepare and examine a Gram smear.

b. Enumerate the types of cells and bacteria seen.

c. Record results and assess suitability for culture.

d. If the specimen does not meet criteria for culture, reject the specimen and indicate
this on the report. Phone the ward to inform the nurse, and request a properly
collected specimen; document this on the report.

Culture

a. Select a purulent portion of the specimen and using a sterile swab inoculate a
portion on to BAP, CHOC and MAC plates. Streak the inoculum in 4 quadrants on
the plates in order to obtain isolated colonies. If Gram-positive diplococci were seen
in the gram smear an optochin disc may be placed in the 2nd quadrant of the BAP to
help in the early identification of S.pneumoniae.

b. Incubate the plates in 5% CO2 at 35-37°C.

c. Examine after 24 and 48 hours incubation.

7. Interpretation

a. Isolate and identify and perform antimicrobial susceptibility tests on any potential
pathogen present in significant numbers i.e. moderate to heavy growth in the 2nd
-4th quadrant, and /or lighter growth of a pathogen that was seen in the original
smear along with PMN’s.

b. Quantitate the pathogens and the normal flora.

Primary Pathogens: S.pneumoniae, H.influenzae, heavy or predominant growth of

S.aureus and gram-negative bacilli such as Klebsiella sp. or P.aeruginosa.

Normal respiratory flora such as Yeast, Viridans Gp. Streptococci and coagulase
negative staphylococci are not considered as pathogens from this site.

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8. Reporting

Report the gram smear result along with the culture result indicating the presence of
normal flora and any pathogen present; report the antibiotic susceptibility result on the
pathogens. If no pathogens are isolated report as “Normal Respiratory Flora isolated at
48 hours incubation”.

9. References

Clinical Microbiology Procedures Handbook, 3rd edition, 2007. ASM Press Washington
DC

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