Overview:
Clostridium Difficile Agar (CCFA), or Cycloserine-Cefoxitin Fructose Agar, is a selective, differential, and enriched solid medium developed by George et al. in 1979 for the primary isolation and presumptive identification of Clostridium difficile, the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in clinical settings. The base provides proteose peptone and fructose as key nutritional sources for amino acids, peptides, and carbohydrates, supporting the growth of C. difficile while the antibiotics cycloserine (inhibiting Gram-negative bacilli) and cefoxitin (suppressing Gram-positive and Gram-negative flora) ensure selectivity against normal fecal microbiota. Neutral red serves as a pH indicator, turning colonies yellow due to alkaline byproducts from amino acid metabolism. Under anaerobic conditions at 35-37�C for 48 hours, C. difficile forms characteristic 2-4 mm flat, yellow, ground-glass colonies that fluoresce chartreuse under long-wave UV light (360 nm). This medium complies with CDC, CLSI, and ISO 15189 guidelines for C. difficile detection from stool specimens.
Applications:
?? Selective isolation of Clostridium difficile from fecal samples in clinical diagnostics for Clostridioides difficile infection (CDI)
?? Enumeration and presumptive identification in outbreak investigations and surveillance per FDA-BAM and APHA protocols
?? Recovery from rectal swabs or environmental samples in hospital hygiene monitoring
?? Subculture from enrichment broths (e.g., cycloserine-cefoxitin broth) for toxin confirmation via ELISA or PCR
Key Features:
?? High selectivity with cycloserine (500 mg/L) and cefoxitin (16 mg/L) inhibiting >95% of competing enteric flora (e.g., E. coli, Bacteroides) while allowing robust C. difficile growth
?? Fructose enhancement improves recovery of stressed or low-viability cells; neutral red differentiation yields yellow colonies for easy presumptive ID
?? UV fluorescence (chartreuse) under 360 nm confirms C. difficile; inhibits most other anaerobes, with rare exceptions like Lactobacillus (non-fluorescent pinpoint colonies)
?? Validated for >90% recovery of ATCC 9689 (C. difficile) in CLSI and EU standards; anaerobic incubation essential for optimal performance
?? Opaque, pinkish-amber medium post-preparation; examine plates within 48 hours to avoid overgrowth
Composition (per liter):
Proteose Peptone � 40.0 g
Fructose � 6.0 g
Disodium Phosphate (Na?HPO?) � 5.0 g
Sodium Chloride (NaCl) � 2.0 g
Monopotassium Phosphate (KH?PO?) � 1.0 g
Magnesium Sulfate (MgSO?�7H?O) � 0.1 g
Neutral Red � 0.03 g
Agar � 15.0 g
Cycloserine � 0.500 g
Cefoxitin � 0.016 g
Final pH: 7.2 � 0.2 at 25�C
Preparation:
Suspend 69.23 g of the dehydrated base in 995 mL of purified/distilled water. Heat with gentle swirling to boiling until completely dissolved (do not overheat). Autoclave at 121�C (15 psi) for 15 minutes. Cool to 45-50�C in a water bath. Aseptically add 5 mL of rehydrated Cycloserine-Cefoxitin Supplement (500 mg cycloserine and 16 mg cefoxitin, reconstituted in sterile water). Mix thoroughly and pour into sterile Petri plates (20-25 mL per plate). Allow to solidify at room temperature and dry the surface briefly under aseptic conditions. Inoculate fecal emulsions (0.1-1 g equivalent) using a swab or loop in a streaking pattern for isolation. Incubate anaerobically (e.g., in jars with GasPak) at 35-37�C for 48 hours. Prepared plates should appear light amber to pinkish, slightly opalescent, and homogeneous.
Storage:
Dehydrated medium: Store in a tightly closed container at 10�30�C, protected from moisture, light, and humidity; stable until expiry date (typically 3-4 years).
Prepared plates: Store at 2�8�C in sealed plastic bags under anaerobic conditions if possible; use within 2 weeks. Discard if dehydrated, contaminated, discolored, or overgrown.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle under biosafety level 2; use Universal Precautions for fecal specimens�wear gloves, lab coat, and eye protection. Autoclave all waste before disposal. Antibiotics may vary by lot; perform QC with ATCC 9689 prior to use. Prolonged incubation (>48 hours) reduces selectivity; confirm positives with toxin assays. Not suitable for non-spore-forming anaerobes.