Appropriate Specimens For Anaerobic Cultures: (Phil J Microbiol Infect Dis 1998 27 (2) :71-73)

Appropriate Specimens for Anaerobic Cultures
Myrna Mendoza, M.D.
(Phil J Microbiol Infect Dis 1998; 27(2):71-73)
The clinician’s understanding of basic anaerobic bacteriology is critical in the

interpretation of an anaerobic culture result for the diagnosis and treatment of anaerobic infection.
Since anaerobes from part of the normal bacterial flora of the skin and mucous membrane, proper
selection and collection of clinical specimens for the laboratory diagnosis of an anaerobic
infection critical factors that will determine the clinical significance of the culture results.
Improved methods of collection, cultivation and identification of anaerobic bacteria have
broadened our knowledge of the various anaerobic genera and species and their role in human
disease.
BASIC CONSIDERATIONS
Anaerobes do not require oxygen for life or reproduction. Obligate anaerobes grow only
in the absence of oxygen. These anaerobes are oxygen sensitive and will die rapidly in an aerobic
environment.
There are microaerotolerant anaerobes that can tolerate 5% oxygen and aerotolerant ones
that can grow in air e.g. 20% oxygen. Few are facultative anaerobes that can grow in all
atmospheres. Examples of these types of anaerobic bacteria are summarized in Table 1.
Table 1. Classification of anaerobic bacteria
Most anaerobes of clinical significance belong to the moderate obligate anaerobe

category. These organisms are more tolerant to the toxic effects of oxygen than the strict obligate
anaerobes. They are however still killed by oxygen unless anaerobic conditions are maintained
properly during specimen collection and transport.
Anaerobes may also be classified as spore forming and non-spore forming bacilli or
cocci. Clostridium spp. are gram-positive spore forming bacilli and Sarcina spp. are gram-
positive spore forming cocci or bacilli. The endogenous anaerobes are found in the upper
respiratory tract, mouth, vagina, urethra, colon and skin. A listing of these endogenous floras is
found in Table 2. There are also exogenous anaerobes e.g. found in the environment, which can
cause disease in man, examples are Clostridium (tetani, botulinium and perfringens).
Anaerobic infections are typically mixed infections of two or more anaerobes or of
aerobes and anaerobes. Most genital and intraabdominal infections are polymicrobial infections.
Those involving the upper respiratory tract (e.g. sinusitis and oral/dental infections) are also
polymicrobial in nature. Pleuropulmonary infections are due to B. fragilis (20-25%),
Peptostreptococcus, Fusobacterium and others. In specimens from these clinical situations,
isolation of more than one anaerobic organism is common.
Table 2. Normal anaerobic flora that are commonly involved in human infections
SPECIMEN SELELECTION AND COLLECTION
The quality of specimen submitted to the laboratory for culture will ensure accuracy of
the clinical diagnosis. Proper specimen collection to avoid contamination with endogenous flora
should be observed. Appropriate sites for specimen collection will ensure representative sampling
of the disease process. In general, specimens that pass through areas with commensal flora are not
acceptable for anaerobic cultures. Examples are: coughed sputum, which becomes contaminated,
as it is passed through the upper respiratory tract and mouth and voided urine specimens, which
are contaminated by the urethral flora. The proper specimen to represent the lower respiratory
tract is therefore a transtracheal aspirate; this will bypass the flora of the upper respiratory tract
and mouth, or a lung aspirate. To sample urine, a suprapubic aspirate is the appropriate specimen
for anaerobic culture.
Acceptable Specimens
Specimens for anaerobic cultures are ideally biopsy samples or needle aspirates.
Anaerobic swabs are discouraged but sometimes cannot be avoided. Swabs are the least desirable
because of the small amount of the specimen and effect of drying. There is a greater chance of
contamination with normal microflora.
The accepted specimens for anaerobic processing are as follows:
Sites Acceptable specimen
CNS CSF, abscess, tissue
Dental/ENT Abscess, aspirates, tissues
Local abscess Needle aspirates
Pulmonary Transtracheal aspirates, lung aspirates, pleural fluid, tissue,
Protected bronchial washing
Abdominal Abscess aspirate, fluid and tissues
Urinary tract Suprapubic bladder aspirate
Genital tract Culdocentesis specimen, endometrial swabs
Ulcers/wounds Aspirate/swab pus from deep pockets or from under skin flaps
that have been decontaminated
Others Deep tissue or bone lesions, blood, bone marrow, synovial fluid,
tissues
Specimens that are normally sterile, such as blood, CSF and synovial fluid, should be
collected aseptically to prevent contamination by skin flora. In general, the best materials for
anaerobic cultures are obtained by needle aspiration and able tissue biopsy.
Unacceptable Specimens
Exudates, swabs from burns, wounds and skin abscesses are generally unacceptable for
anaerobic cultures. Cysts and abscess are contaminated with normal anaerobic flora. Gastric
contents, small bowel contents, feces, colo-cutaneous fistula and colostomy contents should not
be cultured for anaerobic bacteria. Voided and catheterized urine are contaminated with distal
urethral anaerobes and are therefore unacceptable for anaerobic cultures.
Respiratory specimens that are generally rejected for anaerobic cultures include nasal and
throat swabs, sputum and suction specimens; e.g. nasotracheal, tracheal and endotracheal
aspirates collected by suction and unprotected bronchial washing. These specimens are
contaminated with oral flora anaerobes.
HANDING AND TRANSPORT OF CLINICAL SPECIMENS
The basic principles to remember are proper collection of specimens to avoid

contamination with the normal microbial flora and prompt transport to the laboratory where
immediate processing is done. Interpreting anaerobic culture result should be easy if proper
collection and transport of the specimens have been assured.
Immediate delivery of the specimen is crucial in the isolation and identification of
anaerobes. Small tissue and specimens less than 2 ml should be delivered to the laboratory within
one hour after collection. Anaerobes in big tissues and large volume specimens can remain viable
for hours at room temperature provided it is kept moist in containers with tight fitting caps. If
delay is anticipated, an anaerobic transport system should be utilized especially for swabs and
small volume specimens.
There is now commercially available anaerobic transport media in vials and tubes with
anaerobic atmosphere (Port-a-cul) and bag or tube systems that act by removing molecular
oxygen (Biobag, Anaerobic pouch). Swabs can be placed in tubes with reduced transport media
and anaerobic atmosphere (Culturette, Port-a-cul). It is best that before actual collection of
specimen is done, the physician should ask for anaerobic transport tubes/vials with pre-reduced
media from the laboratory to ensure recovery of anaerobes.
Anaerobic transport tubes and/or devices should always be available at the OR and ER.
Specimens should be placed in leak-proof container with tight fitting caps. Of course, proper label
for identification with date and time of collection should accompany all specimens submitted for
culture. Put samples in room temperature while waiting for delivery to the laboratory. Some
anaerobes are killed by refrigeration.
Part of the initial processing is a direct specimen smear, which will determine quality of
specimens and provide the physician with rapid information for diagnosis and initial treatment. If
this is not routinely done in the laboratory, the physician should request for it. It will also help the
clinician to determine if additional better quality specimens should be collected. Gram strain of
direct smears from specimen will give the microbiologist an insight of the quantity and type of
organisms in the specimen.
EVALUATION AND INTERPRETATION OF RESULTS
The physician who collected the specimen can best evaluate the anaerobic culture result.
Interpretation of the result should be correlated with the clinical findings and how the specimen
was collected. Clinical signs suggesting possible infection with anaerobes include the following:
1. Foul smelling discharge
2. Infection in proximity to a mucosal surface
3. Gas in tissues
4. Negative aerobic cultures of specimens whose gram stains show organisms and
pus cells.
Trauma, vascular stasis and tissue necrosis provide favorable conditions for anaerobes to
multiply. In the presence of one or more of the above criteria, an anaerobic culture result will
most likely be significant. Result of mixed culture of anaerobic isolates is not uncommon and this
should not be dismissed as contaminants.
The gram stain result is helpful because bacteria present in the smear should be present in
the culture. Specimens from intraabdominal and genital infections usually yield polymicrobial
cultures of aerobes and anaerobes. Some aspirates/abscesses may contain more than one
anaerobe. These should all be corrected with the gram stain result.
The nature of the bacteria found can give clues to their importance in the disease process.
This will also guide the clinician on the proper management of the patient. In certain cases, when
interpretation of result may be difficult especially when a new name of an anaerobe was reported.
Dialogue with the microbiologist should be done.
REFERENCES
1. Baron EJ, Peterson LR, Finegold SM. Bailey and Scott's Diagnostic Microbiology 9th edition. Mosby-Yearbook, Inc. 1994.
2. Brooks GF, Butch JS, Ornston NC. Jamet, Melnick and Adelberg's Medical Microbiology 20th edition. Appleton and
Lange Scope Publication 1988.
3. Holden J. Collection and transport of clinical specimens for anaerobic culture. In H.D. Isenberg (ed.), Clinical Microbiolgy
Procedures Handbook, vol. 1, American Society for Microbiology, Washington, D.C.1996. p. 2.2.1-2.2.6
4. Koneban EW, Allen SD, Dowell VR. Color Atlas and Textbook of Diagnostic Microbiology 3rd edition. J.P. Lippincott
Co. 1993.

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Appropriate Specimens for Anaerobic Cultures

Myrna Mendoza, M.D.

(Phil J Microbiol Infect Dis 1998; 27(2):71-73)

The clinician’s understanding of basic anaerobic bacteriology is critical in the

Table 1. Classification of anaerobic bacteria

Most anaerobes of clinical significance belong to the moderate obligate anaerobe

SPECIMEN SELELECTION AND COLLECTION

HANDING AND TRANSPORT OF CLINICAL SPECIMENS

The basic principles to remember are proper collection of specimens to avoid

EVALUATION AND INTERPRETATION OF RESULTS

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