Anaerobic culture (overview)

Principle

• Obligate anaerobes lack or have limited enzymes (e.g. catalase, superoxide dismutase) to handle oxygen and grow only in reduced conditions.
• Specimens, transport and incubation must protect organisms from oxygen exposure.

Specimen

• Acceptable: aspirates (pus, fluids), tissue biopsies, blood, deep wounds collected with a needle and syringe or in anaerobic transport media.
• Unacceptable: throat or nasopharyngeal swabs, expectorated sputum, voided urine, stool (except C. difficile), vaginal swabs and superficial wound swabs, because normal flora contains anaerobes.
• Transport at room temperature; do not refrigerate.

Reagents and equipment

• Pre-reduced anaerobically sterilized media: anaerobic blood agar (e.g. Brucella or CDC with hemin and vitamin K), phenylethyl alcohol (PEA) agar, kanamycin–vancomycin laked blood (KVLB), Bacteroides bile esculin (BBE), and thioglycolate or chopped meat broth.
• Anaerobic chamber, jar or pouch with gas mixture of about 5–10% H2, 5–10% CO2 and 80–90% N2; palladium catalyst; indicator (methylene blue or resazurin, colorless when anaerobic).

Procedure

• Process promptly; Gram stain the specimen.
• Incubate plates at 35–37 °C; jars are usually not opened before 48 h, and cultures are held about 5–7 days.
• Aerotolerance test each colony type: subculture to anaerobic blood agar and to aerobic/CO2 media.
• Identify with special potency disks (vancomycin 5 µg, kanamycin 1 mg, colistin 10 µg), biochemicals or MALDI-TOF.

Quality control and pitfalls

• Check the indicator for each run and include a control organism (e.g. Clostridium perfringens or Bacteroides fragilis).
• Oxygen exposure during transport or processing is the major cause of false negatives.

Clinical use

• Abscesses, aspiration pneumonia, intra-abdominal and gynecologic infections, gas gangrene and actinomycosis; clues include foul odor, gas in tissue and sulfur granules.