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1.2.11 Microbiological study.




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This article is from the Food Preserving FAQ, by Eric Decker ericnospam@getcomputing.com with numerous contributions by others.

1.2.11 Microbiological study.

Stock culture and spore harvesting. A stock culture of C. sporogenes
PA 3679 American Type Culture Collection (ATCC) 7955 maintained in
cooked meat medium (Difco Laboratories, Inc.) was used in this study.
The culture was obtained from the Food Microbiology Laboratory culture
collection of Kansas State University (Manhattan, KS). One-tenth
milliliter of the stock culture was transferred to a tube containing
10 ml of sterile cooked meat medium (Difco) and the tube was placed
in an anaerobic jar with GasPak Plus (BBL Microbiology Systems,
Cockeysville, MO) and incubated at 37 C for 18 to 20 h. This procedure
was repeated three times to insure active growth of viable vegetative
cells of C. sporo genes PA 3679. After the third incubation, a loopful
of the suspension was streaked onto Tryptose Sulfite Cycloserine (TSC)
agar plates (1). The TSC agar consisted of Shahidi-Ferguson-Perfringens
agar base (Difco) supplemented with 8% (vol/vol) filter-sterilized
solution of D-Cycloserine (400 j.tglml final concentration). Streaked
TSC agar plates were placed in an anaerobic jar, which was set as
described previously and incubated at 37 C for 18 to 20 h. Following
incubation, an isolated colony, typically round, black and smooth, 0.5
to 1.0 mm in diameter, was tested biochemically using the diagnostic
kit of RapID ANA II system (Innovative Diagnostic Systems, Inc.,
Norcross, GA). One isolated colony was also transferred to 10 ml of
fresh cooked meat medium in a test tube, which was vortexed for 5 s
(high speed). The suspension was then transferred (2 ml/tube) to five
tubes containing 10 ml fresh cooked meat medium. These tubes were
incubated anaerobically at 37 C for 18 to 20 h then the tubes were
removed and kept in refrigerator at 40C as stock culture. With the
purified stock culture, spores were harvested according to the method
described by Vareltzis et al. (11), in which the stock culture is added
to fluid thioglycollate medium (FTG), heated, cooled and then incubated
overnight. The FTG medium is then added to the sporulation medium and
incubated again for 10 days rather than 7 days as done by Vareltzis et
al. (11), since preliminary studies indicated it resulted in higher
recovery of spores. Spores were harvested by a series of centrifugation
and resuspension steps and kept in freezer. Prior to use they were
thawed for 1 to 2 h at room temperature.

Spore titer determination and inoculum preparation. After thawing,
spores were homogenized by shaking the bottle up and down about 5 to
8 times. One milliliter of this homogenized spore suspension was then
serially diluted in 99 ml sterile phosphate buffer to obtain the desired
inoculum level (ca. 106 colony forming units (CFU)Iml) to be used for the
inoculated group. The spore titer was determined by serially diluting 1
ml of spores in phosphate buffer. Each dilution was then tested in
duplicate using Fungs Double Tube (FDT) method (1). Presence of black
colonies in the FDT indicated germination of spores in the plating
medium (TSC agar) and such colonies were referred to as viable spores of
C. sporo genes PA 3679. One black colony was picked with a needle and
transferred in a buffer solution. This solution was then used to identify
the colony with a RapID ANA II diagnostic kit (Innovative Diagnostic
Systems). Count of viable spores of C. sporogenes PA 3679 was determined
during a preliminary study by two detection methods, direct agar
plating and FDT, using TSC agar and brain heart infusion agar (BHI)
supplemented with 0.05% ferric ammonium citrate and 0.1% sodium sulfite
(Difco). The preliminary study showed that TSC agar in the FDT system
recovered higher numbers of C. sporogenes PA 3679 and was, therefore,
used for enumeration of the organism in this research. If present in the
FDT, Clostridium perfringens would also show black colonies, which within
the same incubation period are much larger than those formed by C.
sporo genes PA 3679 (<1 mm in diameter). Regardless of size, any black
colony in the FDT using TSC agar was presumptive Clostridium and since
the inoculum was C. sporo genes PA 3679, blackening was associated with
this organism in inoculated samples. In non-inoculated samples, blackening
was associated with Clostridium-like organisms and biochemical tests using
RapID ANA II kit (Innovative Diagnostic Systems) were performed to
identify black colonies observed in FDT prepared from non-inoculated
samples.

 

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