Overview

CCFA (Cycloserine-Cefoxitin Fructose Agar, George et al. 1979 JCM 9:214) is the reference selective medium for the isolation of Clostridioides difficile (formerly Clostridium difficile; reclassified Lawson et al. 2016 Anaerobe 40:95). It is the foundational tool of C. difficile infection (CDI) laboratory diagnosis, outbreak surveillance, ribotyping, and FMT donor screening.

The selective and differential mechanism combines four components: D-cycloserine (250–500 mg/L; broad-spectrum cell-wall inhibitor, D-alanine analogue inhibiting D-Ala-D-Ala ligase — C. difficile is intrinsically resistant); cefoxitin (8–16 mg/L; cephamycin β-lactam — C. difficile is resistant via inducible β-lactamase and altered PBPs); D-fructose (6 g/L; C. difficile is a strong fructose fermenter, acidifying near the colony); and neutral red (pH indicator producing characteristic yellow-on-red colonies as fructose fermentation drops local pH below 6). On UV (365 nm), C. difficile colonies show distinctive chartreuse-green fluorescence. The horse-stable / faecal odour (p-cresol from p-hydroxyphenylacetate decarboxylation) is a tentative identification clue.

This product also supports the optional CCFA-TA variant (with sodium taurocholate spore-germination supplement, Wilson et al. 1982 JCM 15:443; ~20–40 % higher C. difficile recovery from stool) and is fully compatible with proprietary CHROMagar™ C. difficile licensed supplements (sold separately by CHROMagar Microbiology, Paris) — the chromogenic substrate produces black colonies via β-glucosidase cleavage and gives faster reading (24–48 h vs 48–72 h for CCFA) with reported sensitivity and specificity > 95 % per Eckert et al. 2013 JCM 51:1002.

We also have

YCFA Modified Medium · YCFA Full Recipe · YCFA Base · Modified Chopped Meat Broth (ATCC 1490) · Chopped Meat with Carbohydrates · Beef Granules

Package Contents

Each GMExpression CCFA kit contains:

  • 10 × CCFA Agar plates (90 mm) — pre-poured, pre-reduced, individually sealed in oxygen-impermeable foil pouches with oxygen indicator. C. difficile ATCC 9689 QC release-tested.
  • Vial CYC — D-Cycloserine stock (50 mg/mL in distilled water, filter-sterilised) for top-up reinforcement of selectivity (optional; standard plates are already supplemented).
  • Vial CFX — Cefoxitin stock (8 mg/mL in distilled water, filter-sterilised); single-use aliquots (cefoxitin half-life ~7 days at 4 °C in solution).
  • Vial TA (optional) — Sodium taurocholate stock (100 mg/mL filter-sterilised) for the CCFA-TA spore-germination variant.
  • Instruction manual (A5 booklet, v1.0) with full George 1979 CCFA protocol, Wilson 1982 CCFA-TA modification, CHROMagar compatibility note, QC organism panel, and a CDI laboratory-workflow annex with cross-references to ESCMID / IDSA / CDC C. difficile guidelines.

Customisation options on request: CCFA dehydrated base (without supplements; longer shelf life); CCFA-EY variant with sterile egg yolk emulsion (lecithinase / lipase differential — see § C4 EYA); CCFA + 5 % horse blood variant (haemolysis differentiation); milder antibiotic concentrations (250 mg/L cycloserine + 8 mg/L cefoxitin, modern Wadsworth recommendation for clinical use) or original George 1979 strength (500 + 16 mg/L) for surveillance / environmental work.

Composition — per 1 L equivalent unless stated otherwise

CCFA Agar (George et al. 1979 JCM 9:214; per 1 L)

ComponentConcentrationFunction
Proteose Peptone No. 2 (Difco)40.0 gHigh-grade peptone; George 1979 specifies this exact peptone
Disodium hydrogen phosphate (Na2HPO4)5.0 gBuffer (alkaline)
Potassium dihydrogen phosphate (KH2PO4)1.0 gBuffer (acidic)
Sodium chloride (NaCl)2.0 gOsmotic balance
Magnesium sulphate heptahydrate (MgSO4·7H2O)0.1 gTrace cofactor
D-Fructose6.0 gDifferential substrate — fermented by C. difficile, acidifies near colony
Agar15.0 gSolidifying agent
Neutral red (1 % aqueous, pre-autoclave)30 mg (= 3 mL of 1 % stock)pH indicator — yellow < pH 6 (C. difficile colonies); pink-red pH 7–8 (background)
D-Cycloserine (post-autoclave, filter-sterilised)250 mg/L (clinical) — 500 mg/L (George 1979 original / surveillance)Selective agent 1
Cefoxitin (post-autoclave, filter-sterilised)8 mg/L (clinical) — 16 mg/L (George 1979 original / surveillance)Selective agent 2

Pre-autoclaving pH: 7.4 ± 0.2 at 25 °C.

Optional variants

VariantAdditional componentEffect
CCFA-TA (Wilson 1982)Sodium taurocholate 1.0 g/L (post-autoclave)Stimulates C. difficile spore germination; ~20–40 % higher recovery from stool
CCFA-EY (Wadsworth)Sterile egg yolk emulsion 40–50 mL/L (4–5 % v/v)Lecithinase / lipase differential — C. difficile is negative for both (confirms species)
CCFA-BloodSterile defibrinated horse or sheep blood 50 mL/L (5 % v/v)Enhanced colony morphology; haemolysis reading
CHROMagar™ C. difficile (proprietary; supplement sold separately)β-glucosidase chromogenic substrate + proprietary selective antibiotics + sodium taurocholate (per CHROMagar Microbiology CDIFF kit)Black colonies; faster reading (24–48 h); sensitivity / specificity > 95 % (Eckert 2013)

→Culturomics and Media Navigator

Need help matching this medium to your microorganism — or starting from a target organism and finding the medium that fits? Use the GMExpression Culturomics and Media Navigator: a curated, cross-referenced tool that lets you (i) start from a medium and view the bacterial taxa it is recommended for, or (ii) start from a target organism (genus, species, or DSMZ/ATCC strain ID) and view the panel of GMExpression media that will support it. The Navigator integrates CCFA + CHROMagar Compatible C. difficile Media with the wider YCFA, Chopped Meat Broth, BHI-S, Wilkins-Chalgren, Modified GAM, Anaerobic Columbia Blood Agar, GMM, M2GSC, BBE, KVLB, TPY, and Egg Yolk Agar catalogue.

Use and Applications

  • CDI laboratory diagnosis — culture of C. difficile from stool for confirmation following positive toxin EIA or NAAT. Required for ribotyping, AST, and outbreak investigation. CCFA / CHROMagar are isolation media, not diagnostic media in themselves; positive culture must be confirmed for toxigenicity by orthogonal testing per current ESCMID / IDSA / CDC guidelines.
  • CDI surveillance and outbreak investigation — environmental sampling of hospital wards (high-touch surfaces, commodes, mattresses) for C. difficile contamination. The most-frequently-reordered selective anaerobic medium in clinical microbiology.
  • CDI strain typing — culture-based isolation is the precursor to PCR ribotyping (027 hypervirulent NAP1; 078 community-acquired; 017 toxin A-negative B-positive; 014/020; 002; 106), MLST, and whole-genome sequencing.
  • Antimicrobial-susceptibility testing of C. difficile — requires pure culture as starting material (typically transferred from CCFA primary isolation to non-selective Wilkins-Chalgren or BHI-S for MIC determination).
  • FMT donor screening — many faecal microbiota transplant programmes culture donor stool on CCFA (often CCFA-TA for maximum sensitivity) to exclude asymptomatic C. difficile carriage.
  • Research on C. difficile — sporulation studies, virulence-factor characterisation, host-pathogen interactions; C. difficile must first be isolated on CCFA then sub-cultured to non-selective media for downstream work.
  • Veterinary C. difficile isolation — porcine, bovine, equine, canine; ribotypes overlap with human community-acquired strains (especially RT 078).

Compatible Microorganisms

CCFA — designed target (positive growth)

  • Clostridioides difficile (ATCC 9689, type strain; ATCC 700057, hypervirulent typing reference; ATCC BAA-1382 toxin-positive). Colony morphology: small (1–4 mm), ground-glass / fried-egg appearance, yellow on pink-red background. Odour: horse-stable / faecal (p-cresol from p-hydroxyphenylacetate decarboxylation via hpdBCA). UV fluorescence: chartreuse-green at 365 nm (useful for tentative identification before sub-culture). All toxigenic and non-toxigenic ribotypes recovered: 027 (NAP1; hypervirulent), 078 (community-acquired), 017 (toxin A-negative, B-positive), 014/020, 002, 106, and others.

CCFA — partially suppressed (non-target)

  • Clostridium sordellii, C. bifermentans, C. perfringens — partially cycloserine-resistant; may grow as smaller colonies; differentiated by colony morphology + UV fluorescence (negative).
  • Bacteroides fragilis group — cefoxitin-resistant; can grow but is bile-and-esculin-negative on CCFA (no bile/esculin in formulation); differentiated by colony appearance and lack of yellow-on-red phenotype.
  • Enterococcus spp. — partially cefoxitin-susceptible; usually suppressed.

CCFA — suppressed (selective check)

  • Facultative Gram-negatives — E. coli, Klebsiella, Enterobacter, Pseudomonas (cycloserine + cefoxitin combination).
  • Gram-positive cocci — Staphylococcus, Streptococcus (cycloserine).
  • Most Bifidobacterium, Lactobacillus, Eubacterium, Fusobacterium.

CHROMagar C. difficile — designed target

  • Clostridioides difficile — black colonies via β-glucosidase chromogen cleavage. Sensitivity and specificity > 95 % (Eckert 2013).

Preparation

1Use pre-poured plates (recommended). Pre-poured pre-reduced GMExpression CCFA plates are released from QC ready to inoculate. Remove from foil pouch inside the AAE or just before inoculation; equilibrate at room temperature for 10 min. Verify the oxygen indicator inside the pouch shows reduced state.
2If preparing from dehydrated base. Weigh all base ingredients: 40 g Proteose Peptone No. 2, 5 g Na2HPO4, 1 g KH2PO4, 2 g NaCl, 0.1 g MgSO4·7H2O, 6 g D-fructose, 15 g agar, 3 mL of 1 % neutral red stock solution.
3Suspend and soak. Combine with 1000 mL distilled water. Soak 5 min for agar hydration.
4Heat-dissolve. 95–100 °C with stirring; medium should be clear and pink-red (from neutral red). Adjust pH to 7.4 ± 0.2 at 25 °C.
5Autoclave. 121 °C × 15 min. After autoclaving, slight darkening (Maillard with fructose) is expected; excessive browning indicates over-autoclaving or fructose containing trace glucose / sucrose — use food-grade D-fructose.
6Cool to 50 °C. In a water bath, with magnetic stirring to ensure uniform temperature.
7Add cycloserine and cefoxitin (filter-sterilised stocks). From Vial CYC (50 mg/mL): add 5–10 mL = 250–500 mg/L final. From Vial CFX (8 mg/mL): add 1–2 mL = 8–16 mg/L final. Clinical recommendation (modern Wadsworth): 250 mg/L cycloserine + 8 mg/L cefoxitin. Surveillance / environmental: 500 + 16 mg/L (George 1979 original).
8Optional variants — add at this step. Taurocholate (CCFA-TA): 10 mL of Vial TA (100 mg/mL) = 1 g/L; for spore-germination enhancement and ~30 % higher recovery from stool. Egg yolk (CCFA-EY): 40–50 mL of sterile 50 % egg yolk emulsion (4–5 % v/v); for lecithinase / lipase differentiation. Blood (CCFA-Blood): 50 mL sterile defibrinated horse / sheep blood (5 % v/v).
9Mix and pour. Mix gently with magnetic stirring (avoid antibiotic-concentration gradients on the plate). Pour 25 mL per 90 mm plate within 15 min of antibiotic addition. Allow to set 30 min at room temperature on a level surface.
10Bag and store. 4 °C protected from light in sealed bags. Shelf life: 4 weeks for selective-grade work (cefoxitin loses ~15–20 % activity per month at 4 °C; cycloserine ~10 % per month).
11CHROMagar variant. For CHROMagar™ C. difficile, use the proprietary CHROMagar Microbiology CDIFF kit per manufacturer's published instructions. The GMExpression CCFA base agar is not a substitute for the CHROMagar proprietary formulation but provides a compatible workflow option.

Critical control points

  • Antibiotic concentration choice. Modern Wadsworth (250 mg/L cycloserine + 8 mg/L cefoxitin) gives ~20 % higher C. difficile recovery from stool than George 1979 original (500 + 16 mg/L) but allows more facultative-anaerobe breakthrough. Choose by application: clinical diagnosis → modern milder; surveillance / environmental → George original.
  • Cefoxitin stability. Cefoxitin in aqueous solution has a half-life of ~7 days at 4 °C; prepare a fresh stock for each batch of medium. Aliquot Vial CFX into single-use portions on first opening; freeze unused aliquots at −20 °C.
  • Antibiotic mixing. If cycloserine or cefoxitin is added to medium below 50 °C, localised undissolved precipitates produce regions of higher antibiotic concentration — these appear as colony-free zones on the plate. Mitigation: add antibiotics to 50 °C medium with magnetic stirring; pour plates within 15 min.
  • Cefoxitin pH sensitivity. Cefoxitin loses ~20 % activity per 0.5 pH unit decrease. C. difficile fermenting fructose produces lactate and acetate, locally acidifying around the colony — this is the desired differential phenomenon but if bulk medium pH drifts below 7.0 during storage, cefoxitin selectivity is compromised. Mitigation: tighter post-autoclaving pH verification; store plates upright in sealed bags to minimise CO2 uptake.
  • Cycloserine procurement (Australia). D-cycloserine is a TGA Schedule 4 prescription medicine in Australia (MDR-TB regimen). Bulk powder import requires a TGA permit. Use Sigma-Aldrich Australia research-grade supply or a TGA-licensed pharmacy compounder for the cycloserine stock.

Cautions

CCFA is isolation, not diagnosis. C. difficile isolated on CCFA gives the species but not toxigenicity. Hospital reporting requires orthogonal confirmation (toxin EIA, NAAT, or 16S sequencing). Direct customers to current ESCMID / IDSA / CDC C. difficile diagnostic algorithms — culture is the typing / surveillance step, not the clinical decision-trigger step.
Yellow-on-red phenotype failure. If C. difficile colonies appear pale or the background is yellow rather than red: (i) bulk medium pH has drifted below 6.5 (the indicator turns yellow throughout); (ii) overlong incubation (> 72 h) — fermentation acidifies the whole plate; (iii) neutral-red concentration is too low — verify 30 mg/L target. Read at 48 h, not 24 h or 96 h.
Horse-stable odour. C. difficile growth produces p-cresol from p-hydroxyphenylacetate via the hpdBCA operon decarboxylation pathway; p-cresol gives the characteristic horse-stable, faecal odour, apparent after 48 h. This is a tentative identification clue — not contamination or medium spoilage. Vent cultures in a fume hood or BSC.
CCFA-TA recovery rate gain. Multiple studies (Wilson 1982; Walters 1983; Bouza 2005) show CCFA-TA recovers ~20–40 % more C. difficile from clinical stool than CCFA alone, via the taurocholate spore-germination stimulant. For surveillance-stringency work, CCFA-TA is recommended over CCFA.
CHROMagar IP / licensing. CHROMagar Microbiology (Paris) holds proprietary patents on the chromogenic-substrate composition of CHROMagar™ C. difficile. A GMExpression-branded chromogenic equivalent requires (a) licensing from CHROMagar Microbiology, (b) using a different non-patented chromogenic substrate, or (c) supplying CCFA only. GMExpression position: CCFA is the public-domain (George 1979) reference medium; the proprietary CHROMagar supplement is sold separately by the IP holder. This product is fully compatible with the CHROMagar workflow but is not a CHROMagar substitute.
Shelf life. Prepared CCFA plates: 4 weeks at 4 °C for selective-grade work (cefoxitin-limited). Stored beyond 4 weeks, selectivity drops below 80 % of nominal; for research-grade work where slight selectivity loss is acceptable, plates remain functional up to 8 weeks. Vials CYC, CFX, TA: see storage section.

Storage and Expiry · Safety

  • Pre-poured plates (sealed in foil pouches with O2 indicator): 4 °C, light-protected. Shelf life 4 weeks for selective-grade work.
  • Dehydrated CCFA base (powder): 15–30 °C, sealed in original packaging. Shelf life 30 months.
  • Vial CYC (Cycloserine 50 mg/mL): 4 °C for 4 weeks; −20 °C for 6 months. Avoid freeze-thaw cycling beyond 5 cycles.
  • Vial CFX (Cefoxitin 8 mg/mL): 4 °C for 7 days only (short half-life in aqueous solution); −20 °C for 30 days. Aliquot to single-use portions on opening.
  • Vial TA (Taurocholate 100 mg/mL): 4 °C for 6 months; chemically stable.
  • Prepared plates, opened pouch: use within 24 h once oxygen-exposed.

Safety notes. D-cycloserine is a TGA Schedule 4 prescription medicine — handle as a controlled antimicrobial; avoid skin contact and inhalation. Cefoxitin is a β-lactam antibiotic — sensitiser; use face mask when weighing dry powder. Vials should be opened only inside a BSC. C. difficile isolates handled subsequently are Risk Group 2 organisms — comply with applicable biosafety regulations (Australia OGTR / state-level requirements). SDS available on request.

References

  1. George WL, Sutter VL, Citron D, Finegold SM. (1979). Selective and differential medium for isolation of Clostridium difficile. Journal of Clinical Microbiology 9(2): 214–219. [CCFA primary reference]
  2. Wilson KH, Kennedy MJ, Fekety FR. (1982). Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. Journal of Clinical Microbiology 15(3): 443–446. (CCFA-TA modification.)
  3. Eckert C, Lalande V, Barbut F. (2013). Evaluation of a chromogenic medium for the isolation of Clostridium difficile. Journal of Clinical Microbiology 51(3): 1002–1003. (CHROMagar evaluation.)
  4. Lawson PA, Citron DM, Tyrrell KL, Finegold SM. (2016). Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O'Toole 1935) Prévot 1938. Anaerobe 40: 95–99. (Taxonomic update.)
  5. Jousimies-Somer HR et al. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed., ch. 6 and ch. 9.
  6. CLSI M11 (current ed.) and M22. (CDI workflow and prepared media QC.)
  7. BD BBL™ Cycloserine Cefoxitin Fructose Agar product information 221731; Oxoid Manual 9th ed., CM0601 + SR0096; CHROMagar Microbiology product literature CHROMagar™ C. difficile (CDIFF).
  8. Carroll KC et al. (eds). (2019). Manual of Clinical Microbiology, 12th ed., chapter on Clostridium / Clostridioides. ASM Press.

Frequently Asked Questions

Q1. Should I use CCFA or CHROMagar C. difficile?
CCFA is the natural starting point: (a) the George 1979 formulation is in the public domain (no IP encumbrance); (b) it is the reference medium cited in every CDI lab textbook; (c) it is cheaper to manufacture; (d) it serves both diagnostic and research markets. CHROMagar is a premium product with higher specificity and faster reading (24–48 h vs 48–72 h for CCFA) but requires the proprietary CHROMagar Microbiology supplement (sold separately). For high-volume clinical surveillance, choose by cost and read-time trade-off; for research and reference work, CCFA is preferred.
Q2. What is the difference between Clostridium difficile and Clostridioides difficile?
Same organism. Lawson et al. 2016 Anaerobe 40:95 reclassified the species from Clostridium difficile to Clostridioides difficile on phylogenetic grounds (the species clusters with Peptostreptococcaceae rather than Clostridiaceae). Both names appear in literature; current ASM-MCM, CLSI, ESCMID, IDSA, and CDC use Clostridioides. Product literature uses Clostridioides as preferred name with "(formerly Clostridium difficile)" parenthetical for customer clarity.
Q3. Why are some C. difficile colonies on CCFA not the expected yellow-on-red?
The yellow-on-red phenotype requires (a) fructose fermentation (intrinsic to C. difficile); (b) neutral red present and uniformly distributed; (c) anaerobic atmosphere with sufficient incubation (48 h minimum). Pale colonies or yellow background causes: (i) bulk-medium pH drift below 6.5 (re-check medium pH; indicator turns yellow throughout below 6.5); (ii) overlong incubation (> 72 h) — fermentation acidifies the whole plate; (iii) neutral-red concentration too low — verify 30 mg/L target.
Q4. Can I use 5 % blood-supplemented CCFA instead of CCFA-EY?
Yes. Blood-supplemented CCFA gives richer growth and enables haemolysis reading (C. difficile is non-haemolytic; C. sordellii and C. bifermentans are β-haemolytic — useful differential). The egg-yolk version (CCFA-EY) gives lecithinase / lipase phenotyping at the colony level; C. difficile is lecithinase-negative and lipase-negative, while C. sordellii and C. perfringens are lecithinase-positive. Both supplements have value; many laboratories use CCFA-blood for primary screening and CCFA-EY when species differentiation is needed.
Q5. How long does autoclaved CCFA agar keep?
At 4 °C protected from light in sealed bags: 4 weeks for selective-grade work. Cefoxitin loses ~20 % activity per month; cycloserine is more stable (~10 % per month). For research-grade work where slight loss of selectivity is acceptable, plates remain functional for 8 weeks. Pre-poured GMExpression plates are released with a 4-week dating from manufacture, vacuum-sealed in O2-impermeable pouches to extend useful life.
Q6. Can I autoclave the cycloserine and cefoxitin?
No. Both antibiotics are heat-labile and lose > 50 % activity in a 121 °C × 15 min autoclave cycle. Always add post-autoclave as filter-sterilised stocks (0.22 µm membrane). Cefoxitin is the more heat-labile of the two — even prolonged exposure to medium temperature above 55 °C should be avoided.
Q7. What does the horse-stable / faecal odour from CCFA cultures mean?
C. difficile growth produces p-cresol from p-hydroxyphenylacetate via the hpdBCA operon decarboxylation pathway; p-cresol gives the characteristic horse-stable, faecal odour. It becomes apparent after 48 h incubation and is a tentative identification clue alongside the yellow colonies, ground-glass morphology, and UV chartreuse-green fluorescence. It is not a sign of contamination or spoilage of the medium itself.
Q8. Do I need both CCFA and CHROMagar in a CDI surveillance laboratory?
No — they serve the same purpose and are alternatives, not complements. The choice is operational: CCFA is cheaper and is the reference for published comparison studies; CHROMagar is faster-reading (24 h vs 48 h for visible colonies) and slightly more sensitive. Most clinical laboratories choose one. Surveillance and outbreak-investigation laboratories sometimes use CCFA-TA (with taurocholate) for maximum sensitivity, and switch to CHROMagar for high-throughput operations where the faster turnaround time justifies the supplement cost.