Cycloserine-Cefoxitin Fructose Agar (CCFA)

Cat. no. AG501 Cycloserine-Cefoxitin Fructose Agar*, 15x100mm Plate, 18ml 1 plate/pouch

* All AnaeroGRO™ plated media is provided in standard 15x100mm monoplates or biplates. Each plate or set of plates is packaged in an oxygen-free gas flushed foil pouch containing a desiccant and an oxygen scavenger sachet.


Hardy Diagnostics AnaeroGRO™ Cycloserine-Cefoxitin Fructose Agar (CCFA) is an enriched selective and differential medium recommended for the isolation and cultivation of Clostridium difficile from fecal specimens. C. difficile is a recognized cause of intestinal infections and pseudomembranous colitis following antibiotic therapy.(5,7)


Clostridium difficile is considered the most common cause of pseudomembranous colitis (PMC) or antibiotic-associated diarrhea (AAD) frequently infecting patients on recent antibiotic therapy. Other risk factors associated with PMC include advancing age and recent major surgery, and this organism is easily transmitted from patient-to-patient in hospitals, health care settings, and long-term care facilities.

Early studies on PMC revealed that traditional media selective for the growth of clostridia were inhibitory to C. difficile; however, George et al. developed a selective and differential growth medium containing cycloserine, cefoxitin, fructose, and egg yolk (CCFA) to facilitate the isolation and differentiation of C. difficile from fecal specimens.(2,4,10)

Ingredients in CCFA, such as animal peptones and fructose, have been optimized to improve recovery of C. difficile and the medium contains neutral red as a pH indicator. As amino acids are utilized by the organism, the pH increases resulting in a color change in the medium from orange to yellow. CCFA is also made selective by the addition of cycloserine and cefoxitin. Cycloserine inhibits Escherichia coli while partially inhibiting other gram-negative bacilli and streptococci. Cefoxitin is a broad spectrum antimicrobial that inhibits gram-positive and gram-negative microorganisms, excluding Enterococcus faecalis and Clostridium difficile.

On CCFA medium, C. difficile colonies appear as yellow, with a ground-glass appearance, and are circular with a slightly filamentous edge, flat to low with a rounded elevation, lipase and lecithinase negative and will exhibit a characteristic golden-yellow fluorescence when examined under long-wave ultraviolet light.(4,5,7,9)

AnaeroGRO™ Cycloserine-Cefoxitin Fructose Agar (CCFA) is packaged in an oxygen-free, reduced state to prevent the formation of toxic oxidized by-products that may damage obligate anaerobes and inhibit the growth of more fastidious species. Culture media that is exposed to environmental oxygen leads to a build-up of reactive oxygen species (ROS) that initiate damaging free radical reactions, which inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to the AnaeroGRO™ media to neutralize the growth inhibiting effects of peroxide and other reactive oxygen species (ROS) that may develop during the medium's brief exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free environment.


Ingredients per liter of deionized water:*

Proteose Peptone 40.0gm
Fructose 6.0gm
Disodium Phosphate 5.0gm
Sodium Chloride 2.0gm
Reducing Agents/Peroxide Inhibitors 1.5gm
Monopotassium Phosphate 1.0gm
Magnesium Sulfate 0.1gm
Neutral Red 0.03gm
Cycloserine 500.0mg
Cefoxitin 16.0mg
Agar 15.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat, cold and oxygen exposure. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate anaerobic transport media (Cat. no. 28050510 or S120D) and refrigerated until inoculation. Consult listed references for information on specimen collection.(1-9)

Method of Use:

1. Open the AnaeroGRO™ pouch just prior to use and apply 2-3 drops of liquid stool, biopsy material, or lumen contents directly onto the medium. Streak the inoculum to obtain isolated colonies. An inoculum can also be obtained from a broth, such as Thioglycollate Broth with H & K (Cat. no. AG22H), which has been previously inoculated with clinical material.

2. Immediately following inoculation, place the medium in an anaerobic atmosphere and incubate at 35-37ºC. for 18-48 hours. Use an indicator of anaerobiosis, such as resazurin (Cat. no. BR55).

3. Observe daily for characteristic colonial morphology.


Clostridium difficile is differentiated by the growth of large colonies (approximately 4mm in diameter) that are circular with a slightly filamentous edge, low undulate to flat in profile, ground-glass in appearance, and yellow with yellow coloration extending 2-3mm beyond the colony into the medium. Colonies of C. difficile typically fluoresce golden-yellow under a long-wave ultraviolet light.(4,5,7,9)

Microorganisms other than C. difficile may grow on CCFA, but do not produce a yellow color change in the medium nor show golden-yellow fluorescence under long-wave UV light. Such organisms are generally smaller than colonies of C. difficile.

Refer to the Wadsworth-KTL Anaerobic Bacteriology Manual or other texts for more information on the identification of anaerobes.(7)


The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into an anaerobic atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic bacteria.

Rare strains of C. difficile may be inhibited on CCFA.

For optimal results, plates should not be examined beyond 48 hours of incubation. Extended incubation may result in significant growth of colonies other than C. difficile.

Organisms other than C. difficile may grow on CCFA, but can be distinguished by their morphological differences.

An aerotolerance test should be performed on colonies suspected to be C. difficile. Aerotolerance testing is used to confirm that each colony type is an obligate anaerobe.

Failure to cultivate and/or isolate obligate anaerobes may be due to the following:


Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, transport media (Cat. no. 28050510 or S120D), incubators, incinerators, anaerobic culture materials, such as gas generators (Cat. no. AN25US), compact systems (Cat. no. AN010C), sealing clips (Cat. no. AN005C), chambers, jars (Cat. no. 16000), and oxygen indicators (Cat. no. BR55), etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Clostridium difficile
ATCC® 9689**
A 24-48hr 35°C Anaerobic Growth; large, yellow colonies that fluoresce golden-yellow under UV light
Clostridium perfringens
ATCC® 13124**
B 24-48hr 35°C Anaerobic Partial to complete inhibition
Bacteroides levii
ATCC® 29147
B 24-48hr 35°C Anaerobic Partial to complete inhibition
Bacteroides fragilis
ATCC® 25285
B 24-48hr 35°C Anaerobic Partial to complete inhibition
Fusobacterium necrophorum
ATCC® 25286
B 24-48hr 35°C Anaerobic Partial to complete inhibition
Peptostreptococcus anaerobius
ATCC® 27337
B 24-48hr 35°C Anaerobic Partial to complete inhibition
Escherichia coli
ATCC® 25922
B 24-48hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC® 25923
B 24-48hr 35°C Aerobic Partial to complete inhibition

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.


Physical Appearance

AnaeroGRO™ Cycloserine-Cefoxitin Fructose Agar (CCFA) should be clear, and pinkish-beige in color.

C. difficile colonies on AnaeroGRO™ CCFA agar under UV light

Clostridium difficile (ATCC® 9689) colonies on AnaeroGRO™ CCFA Agar (Cat. no. AG501) under UV light. Incubated anaerobically for 48 hours at 35ºC.

C. perfringens inhibited on AnaeroGRO™ CCFA agar under ambient light

Clostridium perfringens (ATCC® 13124) growth inhibited on AnaeroGRO™ CCFA Agar (Cat. no. AG501) under ambient light. Incubated anaerobically for 48 hours at 35ºC.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Dowell, V.R., Jr. 1981. Media for the Selective Isolation of Clostridium difficile. Les Anaerobes Microbiologie-Pathologie. Symposium International. New York.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. George, W.L., V.L. Sutter, D. Citron, and S.M. Finegold. 1979. Selective and Differential Medium for Isolation of Clostridium difficile. J. Clin. Microbiol.; 9(2):214-219.

5. Holdeman, L.V., E.P. Cato, and W.E.C. Moore. 1977. Anaerobe Laboratory Manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, VA.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, New York, N.Y.

8. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

9. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

10. Wust, J., N.M. Sullivan, U. Hardegger, and T.D. Wilkins. 1982. Investigation of an Outbreak of Antibiotic-Associated Colitis by Various Typing Methods. J. Clin. Microbiol.; 16:1096-1101.

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