Standard Operating Procedure

Subject Culture: Wounds

References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicade (CMS) and or COLA.

Applicable To
Employees of the Gundersen Health System Laboratory, Gundersen St. Joseph Hospital Laboratory,
Gundersen Boscobel Area Hospital, Gundersen Palmer Lutheran Hospital, Gundersen Moundview, and
Gundersen Tri-County Hospital Laboratories.

Detail
This document establishes the procedure for the culture and identification of potential pathogens from
wounds.

PRINCIPLE:
Microorganisms residing on the skin and mucous membranes of humans, as well as microorganisms in
the environment, can cause infections if they enter normally sterile tissue through breaks in the skin or
normally intact mucous membranes. Because virulence mechanisms are not always necessary for such
organisms to cause disease, virtually any species can be involved. Interpretation of culture results should
be based on gram stain criteria. Extensive laboratory testing should be done only after consultation with
the clinician.

CLINICAL SIGNIFICANCE:
In appropriately collected specimens the presence of one of the primary agents of skin and tissue
infections may indicate the need for antimicrobial therapy. Since skin and soft tissue infections and
abscesses can be polymicrobial, empiric treatment is often broad in spectrum, and there is little need to
identify and perform antimicrobial susceptibility testing on all isolates. Moreover, the accumulation of
inflammatory cells and resultant collection of pus is a hallmark of local infection. Evidence of this
process can be documented by the presence of white blood cells in the Gram stain. Therefore the
quality of a wound specimen can be assessed by Gram stain, which should be used to guide the extent
of microbiology work up. The presence of epithelial cells indicates contamination of the specimen with
skin or mucous membrane microbiota, therefore compromising the significance of the culture results.

General Considerations
1. In all cases, efforts should be made to collect specimens prior to initiation of antimicrobial
therapy, and only from wounds that are clinically infected, deteriorating or that fail to heal over
a long period of time.

Page 1 of 7
 Standard Operating Procedure

2. Proper skin disinfection, preferably with alcohol, prior to specimen collection and prompt
transport to the lab is necessary. Specimens delayed in transport or containing mixed skin
and/or colonizing organisms yield misleading information and waste valuable laboratory
resources.
3. Sampling of viable infected tissue, rather than superficial debris or necrotic tissue.
4. Tissue samples or aspirated material are optimal for isolation of infecting organisms. Specimens
obtained surgically are procured at great expense and are not without risk to the patient;
therefore, for optimal evaluation, enough material should be collected to allow proper,
microbiologic examination of the specimen.
5. Aspirates or deep tissue are preferred to swabs. When swabs must be sent, be sure the swab is
well soaked with the tissue fluid.

SPECIMEN:
Refer to Lab-1215 Microbiology Specimen Collection and Transport.

Specimen types
1. Tissue obtained during sterile surgical procedures is often the highest yield specimen from a
work for various reasons. The skin surface and surgical areas are disinfected, preferably with
alcohol which is allowed to dry for one to two minutes, before collection, and the specimen is
obtained by an invasive technique. Depending upon the source of the tissue, in most cases,
most normal microbiota are not absent, per se.
2. Aspirated material from an abscess or deep wound, obtained to prevent contamination by
surface or mucosal microogranism, is the next best specimen type. Uninvolved skin or mucous
membrane should be thoroughly disinfected with alcohol before aspiration.
3. Pus and exudates obtained from an infected site during surgery can be aspirated into a syringe
directly through a plastic catheter or without a needle to avoid puncturing an organ. This
method can be used during surgery when there is sufficient volume to allow the fluid to flow
into the container.
4. Swab specimens are the least desirable because they hold the least volume and are most
subject to contamination. Before a swab is applied, skin surrounding the infected site should be
disinfected with alcohol that is allowed to dry, as thoroughly as for an aspiration. If the wound
or sinus opening is small, a ‘mini-tip’ swab should be used.

Transport
1. Tissue and bone should be placed in a sterile container and transported rapidly to the laboratory
to prevent drying. DO NOT PLACE IN ALCOHOL OR FORMALIN.
2. Material aspirated with a needle and syringe should be transferred to a sterile container and
promptly transported to the laboratory. If aspirated material is small, or if anaerobic culture is
ordered, a syringe with the needle removed and a blunt red stopper in place should be labeled
correctly and hand carried to the lab.
3. ESwab specimens should be transported to the lab as soon as possible for processing. If there is
a delay in transport swabs are stable at room temperature (20-25°C) or refrigerated (4-8°C) for
up to 48hrs. Neisseria gonorrhea cultures collected on ESwabs are stable at room temperature
(20-25°C) or refrigerated (4-8°C) for up to 24hrs.

REAGENTS AND MATERIALS:

Page 2 of 7
 Standard Operating Procedure

Sterile Scalpel
Mortar and pestle
Beadmill tubes
Sterile forceps
Pasteur pipettes
Sheep Blood Agar Plates, (BAP) BBL
Chocolate Agar Plates, (CHOC) BBL
Mac Conkey (MAC), BBL
Phenyl Ethyl Alcohol Agar Plate, (PEA) BBL
Thioglycollate Broth, (THIO) NOT setup when the specimen is sent on a swab.
Thayer-Martin agar Plates, (TM) OR JEMBEC
Glass slides

EQUIPMENT / INSTRUMENTATION: N/A

QUALITY CONTROL:
Refer to Lab-3257 User Quality Assurance Procedure for Culture Media.

Implementation
1. Tissue and bone:
a. Aseptically put the piece of tissue in a sterile mortar.
b. Homogenize the tissue
i. For small, delicate pieces of tissue homogenize with a sterile scalpel and/or
grinding with a pestle.
ii. For larger pieces of tissue and bone specimens homogenize by Beadmil. See
Lab 3024 Beadmill Tissue Processing.
c. Use a Pasteur pipette to inoculate the homogenized specimen to media.
i. (Go to step 3)
2. Culturette swab (Fisherfinest Stuarts media) specimens go to step 3. ESwab specimens
should be plated by using a sterile pipette to inoculate plates with two drops of Amies
media to each plate. The flocked swab provided in the ESwab system may also be used to
inoculate media but must be dipped into the liquid Amies media in between each plate.
3. Inoculate the following media:
1-THIO Broth. Inoculate first. NOT setup when the specimen is sent on a swab.
1-BAP
1-CHOC
1 MAC
1-PEA
1 slide for Gram stain
4. Add a JEMBEC plate to all specimens from genital sources. NOTE: Physicians should not be
ordering generic genital cultures. Requests should be targeted towards specific suspected
pathogens (example: GC, Staph aureus for toxic shock syndrome)
5. Streak the plates out with an inoculating loop for isolation.
6. Place the thio broth in the non-CO2 incubator. Incubate other plates in the 35oC CO2
incubator or candle jar.
7. Gram stain the slide. Read Gram stain and enter the results into the Epic.

Page 3 of 7
 Standard Operating Procedure

8. Examine all plates and THIO broth after 18 hours incubation. Discard the plates after 48
hours if negative, but hold THIO broth for 7 full days. Check daily and perform a Gram stain
before discarding.
9. Specimens sent on swab; culture plates are held three days.

PROCEDURAL NOTES:
NOTE*: If PEA is used, organisms from the PEA plate must be subbed to a BAP before doing a VITEK II
identification and sensitivity.

CALCULATIONS: N/A

INTERPRETATION:

Gram stain:
1. A STAT Gram stain will be done immediately and preliminary results will be entered into the
computer. A non-STAT will be done as soon as possible and the report will be entered into the
computer. See Lab 3279 Gram Stain and Lab 0130 Critical Lab Values, Lab Reporting Protocol for
more details.
a. The Gram stain will be used in evaluation of the culture. Good-quality open wound
specimens are defined as having polymorphonuclear leukocytes (PMNs) in the direct
smear or a history of diabetes or immune-compromised condition. Record the
relative number of WBCs, epithelial cells, and bacterial/fungal morphotypes. Note:
a poor-quality specimen shows numerous epithelial cells and no PMNs on gram
stain.
General Culture Reporting and Interpretation:
1. Read plates and broth daily.
2. For cultures of lymph nodes and lungs, work in a biological safety cabinet, since some pathogens
found in these specimens are hazardous (e.g. Fransicella, Mycobacterium, Brucella).
3. Generally identify up to three microorganisms if any of the following is true:
a. PMNs present in the direct smear.
b. The specimen was collected from a normally sterile site
c. The specimen was of good quality (e.g. no to small# epithelial cells present)
d. The organism was seen on the direct smear.
4. Perform only minimal testing to indicate the type of microbiota present for noninvasively
collected specimens with any of the following:
a. Moderate to large numbers epithelial cells present on the smear
b. No evidence of infection of the smear (e.g. No WBC Seen) and no clinical information
accompanying the specimen to indicate an infection.
c. >=3 organisms growing in the culture. See exceptions for specific organisms that are
always reported.
5. The following organisms are usually considered significant even in low numbers or in mixed
cultures:
a. Organisms that grow only on chocolate: ex. Haemophilus influenzae, N. gonorrhoeae,
Francisella tularensis, Kingella
b. Identify Streptococcus pyogenes, Streptococcus dysgalactiae ssp equisimilis or
Streptococcus agalactiae.

Page 4 of 7
 Standard Operating Procedure

c. Staphylococci
i. S. aureus;
I. Perform AST from invasively collected specimens and from others if the
Gram stain indicates a good-quality specimen and an infectious process
with this organism (e.g. WBCs, lack of epi’s, morphology seen in smear).
ii. Coagulase Negative Staphylococci:
I. When present, perform AST only if they are the only organisms isolated
from invasively collected specimens, if they are associated with WBCs in
the direct smear, or if they are isolated from multiple cultures. Report
as normal skin flora if found in mixed cultures in any amount from
superficial wound specimens or if mod-lg# epi’s seen in Gram stain.
d. Viridans streptococci and enterococci
i. Identify at lease to the genus level from surgically, invasively collected
specimens where the organism is the single or predominant pathogen and the
Gram stain indicates infection (WBCs).
ii. Include in normal microbiota if found in mixed cultures and not predominant.
iii. If determined to be a significant isolate or if indicated by infection control,
perform AST on enterococci. Perform AST on enterococci only if isolate is from
a normally sterile site in pure or predominant culture.
e. Gram positive rods
i. Identify other Gram-positive rods if they are numerous and are associated with
WBCs in smear or if isolated from multiple cultures. Otherwise include these in
skin flora. If specimen is from a normally sterile site or biopsy, rule out Listeria,
Erysipelothrix, Bacillus cereus, B. anthracis, Arcanobacterium, Corynebacterium
diphtheriae, C. ulcerans, Nocardia, and Actinomyces.
f. Include yeasts as part of normal microbiota unless predominant or numerous. Except
for specimens from normally sterile sites, generally ID to species level.
g. Enteric Gram-negative rods (Enterobacteriaceae):
i. If only 1-2 species present or predominant and are indicated in smear (with
WBCs) identify and perform AST.
I. Note: For specimens from the abdominal cavity, the aerobic plates may
contain only a few E. coli organisms, but the smear appears to represent
mixed morphologies. In such cases, do not set up AST until the
anaerobic culture can be interpreted. Potentially, the anaerobic
microbiota may be the significant, predominant pathogen(s).
ii. If enteric bacilli are in small amounts or not predominant, or if >2 species are
present with no predominant strain, report as “mixed gastrointestinal flora.”
I. Rule out fecal pathogens (Salmonella, Shigella, Yersinia,
Campylobacter).
II. Generally, save a representative plate in cabinet for 7 days in case work
up is requested.
iii. ID and AST on multiple morphologies of enteric Gram-negative rods only upon
special request after consultation with Infectious Disease.
h. Non-Enterobacteriaceae:
i. Rule out organisms that are always considered pathogenic (e.g. Brucella,
Francisella, Haemophilus, Pasteurella). Generally, these organisms will not grow

Page 5 of 7
 Standard Operating Procedure

on MAC. Some are common in bite wounds. Work under the biological safety
hood if select agents are suspected.
ii. Identify obvious Pseudomonas aeruginosa (ox+, metallic sheen, grape odor) and
Stenotrophomonas maltophilia (yellow with bluish border growth, oxidase
negative). If in pure culture or significant amounts and smear suggests an
infectious process perform AST.
iii. Identify oxidase +, indole + organisms with are likely to be Aeromonas or Vibrio.
Also examine for pigmented negative rods Sphingobacterium and
Chromobacterium violaceum.
iv. Identify and perform AST on other negative rods (Pseudomonas other than
aeruginosa, Acinetobacter, and other glucose-non-fermenting Gram-negative
rods) if obtained from a normally sterile site or is indicated by smear.
v. Perform ID and AST on any organism as directed by Infectious Disease
consultant, or by direction of Medical Director.

All positive culture plates (save representative plates) will be held for 7 days (either at room temp or
refrigerated) after culture is completed for additional work if requested by a physician.

LIMITATIONS:
1. The microbiologist plays a critical role in the treatment of wound infections because
practitioners often consider the report from the laboratory as definitive proof of infection.
Providing inappropriate identifications and susceptibility results can prompt unnecessary
treatment.
2. The results of wound, abscess, and tissue cultures will only be as valuable as the quality of the
specimen submitted, transport, and expedient processing.
3. The presence of WBCs is an indication of an inflammatory or infectious process, while the
presence of epithelial cells may indicate surface contamination of the specimen. Specimens
containing numerous epithelial cells yield culture results of questionable accuracy in the
diagnosis of the infectious process, and one can consider rejection of these specimens for
culture
4. If a patient is immunocompromised or has poor vascular supply, inflammatory cells may not be
present in the specimen as a guide to the extent of workup of the cultures.
5. Low levels of organisms or fastidious organisms that grow poorly on the direct plates may be
missed in culture
6. Antibiotics administered prior to sample collection may negatively affect the recovery of the
organisms associated with the infection
7. Many wound infectious are polymicrobial, and the isolation of an organism in culture may or
may not correlate with infection of the wound.
8. Unusual diagnoses and treatment considerations may alter the usually policies of the laboratory
in workup of organisms and reporting AST
9. The lack of isolation of a pathogen does not necessarily mean that the laboratory was unable to
detect the agent. Other inflammatory diseases can have the same presentations as infectious
processes, including the presence of WBCs on the Gram stain.

REVIEW AND CHANGES:

Page 6 of 7
 Standard Operating Procedure

This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Medical Director or designated
person. Changes require retyping document or form and review by the Medical Director.

REFERENCES:
1. Carson, JA (ed). 2016. Wound/Abscess and Soft Tissue Cultures: Aerobic Bacteriology. Section
3.13.1.1-3.13.1.20. In Leber, A. L.(ed). Clinical Microbiology Procedures Handbook, 4th ed, vol 1.
ASM Press, Washington, DC. 2016.
2. BD ESwab package insert 2016.
3. Jorgensen JH et al. (ed). 2015. Manual of Clinical Microbiology, 11th ed.

Page 7 of 7