STANDARD OPERATING PROCEDURE

WOUND CULTURES

1. Principle

The skin is subject to frequent irritation and injury. Infections at these sites are common
and may be caused by a variety of organisms, but are most commonly associated with
S. aureus, beta-hemolytic Streptococci, P. aeruginosa and enteric Gram negative bacilli.
The Gram smear of the specimen is important in evaluating culture results. The
presence of numerous pus cells with associated organisms indicates the likelihood of
infection caused by the organism seen. However, the presence of squamous epithelial
cells in the gram smear may indicate that the specimen is contaminated with superficial
cells, and therefore the organisms isolated may not represent the true pathogens
causing the infection.

Commensal skin flora includes a variety of organisms, predominantly, Corynebacterium

sp., Coagulase negative Staphylococcus, Micrococcus and nonpathogenic Neisseria sp.
These organisms may contaminate the specimen and compromise the culture results.
Care must therefore be taken to avoid contamination with superficial skin flora when
collecting these specimens.

2. Materials

a. Gram smear: clean glass slide and gram stain reagents

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

agar

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

d. Inoculating loops and swabs

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

3. Specimen

Specimens are collected using a clean, sterile swab and sent in transport medium. If
fluid or pus are sent this is placed in a sterile container with a screw cap lid.

If there is a delay in transport or processing the specimen should be refrigerated.

4. Quality Control (QC)

Check that the specimen has been correctly identified, and that the information on the
specimen and the accompanying requisition match.

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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

Processing of Specimens:

a. Direct Examination (Gram smear): Use a dry clean slide to prepare a smear, allow
the smear to dry, then fix and stain it. Note that the gram smear is prepared after the
plates have been inoculated.

Quantitate polymorphonuclear leukocytes (PMN’s), squamous epithelial cells and

types of organisms seen.

b. Culture

Media Inoculation and Incubation: Use the swab, or if pus is received dip a sterile
swab in the fluid and use this to inoculate the culture plates. Spread the inoculum in 4
quadrants to allow growth of isolated colonies.

Incubate the plates and examine after 24 hours. Isolate pathogens and identify using
standard tests as per the laboratory protocol. Perform AST testing on potential
pathogens. If there is no growth at 24 hours, re-incubate the plates for another 24
hours and re-examine for growth of pathogens.

7. Interpretation

Any growth of S. aureus, beta-hemolytic streptococci groups A, B, C and G and P.

aeruginosa is significant. The growth of any organism which was detected in the gram
smear in association with PMN’s is considered significant.

8. Reporting

Report the Gram smear result along with the culture.

A growth of 1 or 2 types of organisms other than skin commensals is significant if there

was >1+ pus cells seen on the original Gram stain. These organisms are reported along
with the AST results.

Growth of ≥ 3 types of coliforms or other Gram negative bacilli with normal skin
commensals is reported stating "commensal flora including mixed Gram negative
bacilli"; list any potential pathogens if present.

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9. Procedural Notes

The skin is colonized with normal flora such as coagulase negative staphylococci,
diphtheroids and alpha-haemolytic streptococci. The isolation of these organisms form
wounds is generally considered to be contamination. However, when isolated form
tissues and deep wounds especially those associated with prosthetic devices, these
organisms may be significant. Consult with the physician in these circumstances.

10. References

a. Clinical Microbiology Procedures Handbook, 3rd edition, 2010. ASM Press,

Washington. DC.

b. Manual of Clinical Microbiology, 8th edition, 2003. ASM Press, Washington. DC

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