Clostridium Difficile Supplement

Description

Clostridium difficile was first isolated from meconium and infant feces in 1935 by Hall and O'Toole, who proposed the name "difficile" because it was very difficult to isolate. Keighley associated Clostridium difficile with colitis and diarrhea after antibiotic therapy following gastrointestinal operations. In 1979, George et al. developed a medium called CCFA (cycloserine-cefoxitin-fructose agar), which is a bloodless medium, based on the egg yolk agar of McClung and Toabe with fructose as a glucose replacement. The combination of Clostridium Difficile Agar with the supplement is based on this medium.

The selective agents D-cycloserine and cefoxitin inhibit the growth of most Enterobacteriaceae, as well as Enterococcus faecalis, staphylococci, Gram-negative non-sporing anaerobic bacilli and Clostridia species (except Clostridium difficile), which may be found in large quantities in fecal samples.

Technique

Aseptically reconstitute 1 vial with 5 mL of sterile distilled water. Mix gently until complete dissolution and add aseptically to 500 mL of Clostridium Difficile Agar Base (LC-1447), autoclaved and cooled to 50 °C, together with 7% of defibrinated horse blood. Mix well and distribute into Petri dishes.

Instructions for use

• In a Petri dish, add 12-15 mL of molten agar and let it solidify.
• Lightly inoculate the medium with the faecal sample, spreading 10 µL of the original inoculum in order to obtain well separated colonies.
• Incubate plates at 35-37 °C for 18-48 hours in a conventional anaerobic jar.
• Colonies of Clostridium difficile after 48 hours of incubation are 4-6 mm in diameter, irregular, raised, opaque, grey-white.

Reconstitute the original freeze-dried vial by adding 5 mL of sterile distilled water.

Note: each vial is sufficient to supplement 500 mL of Clostridium Difficile Agar Base.

Physical/chemical control

Microbiological control

Reconstitute 1 vial as indicated in Composition; shake and dissolve completely.

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Inoculate: practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity).

5% CO₂ atmosphere. Incubation at 35-37 °C for 24-48 h.

Sterility control

Add 5 mL of the sample to 100 mL of TSB and to 100 mL of Thioglycollate.

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• George W.L., Sutter V.L., Goldstein E.C.J., Ludwig S.L. and Finegold S.M. (1978) Lancet. i. 802-803.
• Hall I. and O'Toole E. (1935) Am. J. Dis. Child. 49. 390.
• Keighley M.R.B., Burdon D.W., Alexander Williams J. et al (1978) Lancet ii. 1165-1167.