PROCESSING OF
ANAEROBIC CULTURES
MEDIA
CLINICAL SPECIMENS suitable for these cultures may include: bile, biopsy of endometrial tissue obtained with an endometrial suction curette, blood, bone marrow, cerebrospinal fluid, uterine contents (if collected using a protected swab)
DIRECT SMEAR is included if additional specimen
is sent in a sterile tube and if requested
INCUBATION
primary plates should be freshly prepared or used within 2 weeks of preparation
reduction of media in anaerobic environemnt eliminates dissolved oxygen but has no effect on the peroxides
anaerobic media should include a nonselective anaerobic blood agar
common anaerobic media: anaerobic blood agar, bacteroides bile esculin agar (BBE), Laked kanamycin-vancomycin (LKV), anaerobic phenylethyl alcohol agar (PEA), egg yolk agar (EYA), cycloserine cefoxitin fructose agar (CCFA), cooked meat broth, peptone–yeast extract–glucose broth (PYG), and thioglycollate broth
inoculated plates should be immediately incubated under anaerobic conditions at 35° to 37°C for 48 hours
cultures should not be exposed to oxygen until after the 48 hour incubation because of the anaerobe's sensitivity to oxygen
plates incubated in an anaerobic chamber can be examined at 24 hours without oxygen exposure for typical colonies of B. fragilis group or Clostridium perfringens
direct gram stain of clinical specimen
streak for isolation
anaerobic (BBE, LKV, PEA, BAP, CHOC, TIO, EYA)
examine individual colonies for distinctive morphologies
gram stain and subculture to BAP and CHOC
AEROTOLERANCE TEST: subculture organisms to a CHOC plate, incubate 37°C for 24 to 48 hours in 5% CO2 to detect slow growing aerobes
- gram-negative add kanomycin, vancomycin, or colistin disk in first quadrant, nitrate disk in second quadrant
- gram-positive cocci add nitrite and SPS
- gram-positive rod add nitrate
incubate anaerobically 37°C for 24 to 48 hours
SUBCULTURE COLONIES
CHOC to be incubated in CO2 for aerotolerance
BAP and CHOC to be incubated aerobically (purity plate)