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CLOSTRIDIUM DIFFICILE SELECTIVE AGAR  (CCFA)

for the isolation and presumptive identification of Clostridium difficile from clinical specimens

*Adjusted as required to meet performance specification 

Description
Clostridium difficile was first isolated in 1935 by Hall and O’Toole¹ who proposed the name `difficile’ because it was very difficult to isolate. In 1940 Snyder² isolated Clostridium difficile from infants aged 10 weeks to 1 year. No further isolations were reported until 1960, when the organism was cultured by McBee³ from the intestinal contents of a seal, and in 1962 Smith and King⁴ reported its presence in human infections.
Toxicogenic isolates of Clostridium. difficile have been demonstrated to be a major cause of antibiotic-associated ileo-caecitis in laboratory animals⁵ and pseudomembranous colitis in man^6,7. Keighley⁸ found Clostridium difficile was associated with colitis and diarrhoea without pseudomembranous changes after antibiotic therapy following gastrointestinal operations.

Hafiz and Oakley⁹ devised a medium for the selective isolation of Clostridium difficile based on the observation that the organism has a high tolerance to cresol, which it produces during its growth, and used Reinforced Clostridial Medium (CM0151) plus 0.2% phenol or p-cresol.
George et al.¹⁰ in a study of selective media for the routine isolation of Clostridium difficile from faecal specimens found this medium was inhibitory compared with growth on blood agar. They recommended the use of a fructose containing nutrient medium plus egg yolk, with D-cycloserine and cefoxitin as selective agents for the isolation of Clostridium difficile.

This medium based on the formulation proposed by George et al.¹⁰. The selective agents D-cycloserine (500µg/ml) and cefoxitin (16µg/ml) inhibit growth of the majority of Enterobacteriaceae, as well as Streptococcus faecalis, staphylococci, Gram-negative non-sporing anaerobic bacilli and Clostridia spp. (except Clostridium difficile) which may be found in large numbers in faecal samples.

Levett¹¹, noting reports^12,13 that some strains of Clostridium difficile had low minimum inhibitory concentrations to both cycloserine and cefoxitin, reduced the antibiotic concentrations to 125µg/ml cycloserine and 4µg/ml cefoxitin and combined this with alcohol shock¹⁴ to compensate for the reduction in selectivity. Clostridium difficile was isolated from all of the 33 faecal specimens plated on to CCFA Medium containing cycloserine and cefoxitin at 250µg/ml and 8µg/ml respectively, but from only 25 of 33 specimens plated on to medium containing 500µg/ml cycloserine and 16µg/ml cefoxitin. The specimen should be treated with alcohol before inoculation (see technique).

It can be expected that medium containing the lower concentration of antibiotics will yield a greater growth of contaminating organisms if antibiotics are used alone, but Levett reported that there was no difference in the growth of contaminating organisms on plates containing either concentration of antibiotics following alcohol shock treatment of the specimen.

Phillips and Rogers¹⁵ have described a simple modification to the medium in which the ability of Clostridium difficile to produce p-cresol from p-hydroxyphenyl acetic acid is used for the rapid presumptive identification by gas chromatographic detection of the p-cresol.

Addition of 7% horse blood to the agar base increases the recovery of Clostridium difficile and produces larger colonies compared with Egg Yolk Emulsion used by George et al.¹⁰.

Technique

1. Lightly inoculate the medium with the faecal sample spreading part of the original inoculum in order to obtain well separated colonies.
2. Incubate plates at 35° C for 18-24 hours in a conventional anaerobic gas jar.
   (The use of the Oxoid Anaerobic Jar (HP0011) with an Anaerobic Gas Generating Kit (BR0038) is strongly recommended. Alternatively use Anaerogen (AN0025 or AN0035). Anaerogen does not require the addition of water or a catalyst.)
3. Colonies of Clostridium difficile after 48 hours incubation are 4-6 mm diameter irregular, raised opaque, grey-white.

Technique for Alcohol Shock Treatment

1. Mix equal parts of industrial methylated spirit or absolute alcohol and the faecal specimen.
2. Homogenise using a vortex mixer.
3. Leave at room temperature for 1 hour.
4. Inoculate on to Clostridium Difficile Selective Agar and incubate anaerobically.

Storage conditions and Shelf life

Appearance
Ruby red agar

Quality control

* This organism is available as a Culti-Loop®

Precautions
Oxoid Clostridium difficile Selective agar (CCFA) is for in vitro diagnostic use only, by experienced microbiologists. It must not be used beyond the stated expiry date, or if the product shows any sign of deterioration.
Typical Gram stain morphology of Clostridium difficile may not be evident in colonies picked from this medium because of the antibiotics present. Subculture to blood agar to obtain characteristic morphology¹⁰.

References
1. Hall I. and O’Toole E. (1935) Am. J. Dis. Child. 49. 390.
2. Snyder M. L. (1940) J. Infect. Dis. 66. 1.
3. McBee R. H. (1960) J. Bact. 79. 311.
4. Smith L. D. S. and King E. O. (1962) J. Bact. 84. 65.
5. Bartlett J. G., Onderdonk A. B., Cisneros R. L. and Kasper D. L. (1977) J. Infect. Dis. 136. 701-705.
6. Bartlett J. G., Chang T. W., Gurwith M., Gorbach S. L. and Onderdonk A. B. (1978) N. Engl. J. Med. 298. 531-534.
7. George W. L., Sutter V. L., Goldstein E. C. J., Ludwig S. L. and Finegold S. M. (1978) Lancet. i. 802-803.
8. Keighley M. R. B., Burdon D. W., Alexander Williams J. et al (1978) Lancet ii. 1165-1167.
9. Hafiz S. and Oakley C. L. (1976) J. Med. Microbiol. 9. 129-136.
10. George W. L., Sutter V. L., Citron D. and Finegold S. M. (1979) J. Clin. Microbiol. 9. 214-219.
11. Levett (1985) J. Clin. Pathol. 38. 233-234.
12. George W. L., Kirby B. D., Sutter V. L. and Finegold S. M. in Schlessinger D. Editor Microbiology 1979 Washington D.C. American Society for Microbiology, 2670271.
13. Dzink J. and Bartlett J. G. (1980) Antimicrob. Ag. Chemother. 17. 695-698.
14. Borriello S. P. and Honour H. (1981) J. Clin. Pathol. 34. 1124-1127.
15. Philips K. D. and Rogers P. A. (1981) J. Clin. Pathol. 34. 643-644.