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BRAZIER’S CLOSTRIDIUM DIFFICILE SELECTIVE AGAR

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

Products available

*Adjusted as required to meet performance standards

Desciption
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 C. 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 C. difficile was associated with colitis and diarrhoea without pseudomembranous changes after antibiotic therapy following gastrointestinal operations.

George et al⁹ in a study of selective media for the routine isolation of C. difficile from faecal specimens, recommended the use of a fructose containing nutrient medium plus egg yolk, with D-cycloserine and cefoxitin as selective agents for the isolation of C. difficile.

The selective agents D-cycloserine (500µg/ml) and cefoxitin (16µg/ml) were found to inhibit growth of the majority of Enterobacteriaceae, as well as Streptococcus faecalis, staphylococci, Gram-negative non-sporing anaerobic bacilli and clostridia species (except C. difficile) which may be found in large numbers in faecal samples. Willey and Bartlett reduced the concentrations of these selective agents to 250µg/ml and 10µg/ml, respectively. Later, Levett¹⁰ reported improved recovery of C. difficile using 250µg/ml of D-cycloserine and 8µg/ml cefoxitin. Alcohol shock can be used to increase inhibition of competing flora if required, which has been reported to greatly diminish competing flora and enhance both the isolation and recognition of C.difficile¹¹.

Brazier’s Clostridium difficile Selective agar resulted from work begun by Ken Phillips and Paul Levett, and completed by Jon Brazier. Cholic acid is present to promote spore germination following alcohol shock treatment, and p-hydroxyphenylacetic acid is included to enhance the production of p-cresol, a metabolite of C.difficile that gives off a distinct odour. Cholic acid has been incorporated, due to its ability to stimulating spore germination of C. difficile¹², along with egg yolk emulsion, to help differentiate C. difficile from lecithinase positive clostridia. Finally, the addition of lysed horse blood optimises the recognition of colony fluorescence when cultures are examined using UV light.

Technique
Technique for alcohol shock treatment (if required)

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 and incubate anaerobically.

Technique for inoculation

1. Lightly inoculate the medium with the faecal sample (or alcohol shock preparation) streaking from the original inoculum in order to obtain well separated colonies.
2. Incubate plates at 37°C for 24-48 hours in anaerobic conditions.

Interpretation
Characteristics of C. difficile: Gray/white, opaque, flat colonies, 1.5-3mm diameter, circular but may be elongated in the direction of spread, ground glass appearance and a rough, fimbriate edge. Lecithinase negative. Incubation longer than 48hrs may result in a lighter gray or white centre to the colony. Phenolic odour due to the production of p-cresol. Colonies fluoresce yellow/green under UV light.

Confirm by latex agglutination.

Quality Control

Storage conditions and Shelf life
Oxoid Braizier’s Clostridium difficile Selective Agar plates should be stored in the original packaging, at the temperature stated on the pack or product specification, and protected from light.

Appearance
Prepared medium: amber/brown gel

Precautions
This medium 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.

Sterilise specimens, equipment and media properly after use.

Limitations
Organisms with atypical enzyme patterns may give anomalous results.

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. George W. L., Sutter V. L., Citron D. and Finegold S. M. (1979) J. Clin. Microbiol. 9. 214-219.
10. Levett (1985) J. Clin. Pathol. 38. 233-234.
11. Brazier J.S, (1998) J. Antimicrob. Chemother. 41, Suppl. C, 29–40
12 Brazier J. S. (1993). Clin. Infect. Dis. 16, Suppl.4, S228–33.

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