Clostridioides difficile (Prevot) Lawson et al.

1. Thaw the frozen vial under anaerobic conditions. 
2. Under anaerobic conditions, transfer the entire contents of the thawed vial into a single tube (5-6 mL) of #2107 broth.  Mix well. 
3. Additional tubes of broth may be inoculated with 0.5 mL each from the suspension and 0.1 mL may be inoculated onto a slant.  Streak several blood plates for colony morphology and purity.
4. Incubate the tubes and plate under anaerobic conditions at 37°C for 24 to 48 hours. Incubate the purity plate aerobically at 37°C.

ANAEROBIC CONDITIONS:

Anaerobic conditions for transfer may be obtained by either of the following:

• Use of an anaerobic gas chamber, or
• Placement of test tubes under a gassing cannula system connected to anaerobic gas.

Anaerobic conditions for incubation may be obtained by any of the following:

• Loose screw caps on test tubes in anaerobic chamber,
• Loose screw caps on test tubes in an activated anaerobic gas pack jar, or
• Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.

Anaerobe Systems Brucella Blood Agar Plates (AS-111) can be used to analyze colony morphology and purity.

No growth should occur on the aerobic blood plate. Turbid growth is evident is broth at 24 hours. Subsequent transfers may reach peak viability in 6 to 12 hours.

The presence of tcdA and tcdB genes is confirmed by PCR. The binary toxin gene cdt is not amplified by PCR.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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