Brazier's CCEY Agar

LAB 160

Description

Brazier's CCEY agar is the formulation currently used by the Anaerobe Reference Unit for the isolation of C.difficile, resulting from work initiated by Ken Phillips and Paul Levett, and completed by Jon Brazier.

Based upon the market leading anaerobe medium, Fastidious Anaerobe Agar, it incorporates additional ingredients to improve the isolation and differentiation of C.difficile from clinical specimens.

Cholic acid is present to promote spore germination following alcohol shock treatment, and p-hydroxyphenylacetic acid to enhance the production of p-cresol, a distinctive metabolite of C.difficile

Selectivity is achieved by addition of supplement X093 (cefoxitin cycloserine) and egg yolk emulsion X073 is added 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.

1.6

Method for reconstitution

Weigh 48 grams of powder and add to 1 litre of deionised water. Allow to soak for 10 minutes, swirl to mix, and sterilise by autoclaving at 121˚C for 15 minutes. Cool to 47˚C and aseptically add the following: 2 vials of X093, 40ml of Egg Yolk Emulsion X073 and 10ml lysed horse blood. Mix well and pour into Petri dishes.

Appearance: Tan opaque gel.

pH: 7.0 + 0.2

Minimum Q.C. organisms: C. difficile

E. coli (inhibition) NCIMB 50034

Storage of prepared medium: Plates - up to 7 days at 2-8˚C in the dark.

Inoculation: Surface streak untreated or alcohol shocked specimens for single colonies.

Incubation: 37˚C for 24-48hrs under anaerobic conditions

Characteristics of C.difficile: Gray opaque flat colonies, raised elevation, 2-3mm diameter, generally circular but tending to elongate in the direction of spreading, 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.

References

Brazier J.S. (1993) Rôle of the Laboratory in Investigations of Clostridium difficile Diarrhoea. Clinical Infectious Diseases 16 (4) 228-33.