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Cycloserine Cefoxitin Fructose Agar (CCFA)

Cycloserine Cefoxitin Fructose Agar (CCFA) is an enriched selective and differential medium for the isolation and presumptive identification of Clostridium difficile, a recognized cause of pseudomembraneous (antimicrobial agent-associated) colitis and Clostridium difficile associated diarrhea (CDAD).  The basic nutritive base consists of animal peptones and fructose and is supplemented with cefoxitin and cycloserine at concentrations that inhibit the growth of most normal fecal flora. Cycloserine will inhibit gram-negative bacteria, while cefoxitin will inhibit both gram-positive and gram-negative organisms. Neutral red is added as a pH indicator.  The pH of the media is decreased due to the fermentation of fructose, which will turn the indicator from pink/orange to yellow.  Most strains of Clostridium difficile are not inhibited on the CCFA medium, and, when growing on this medium, will exhibit a characteristic yellow, ground-glass colonial morphology.  This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.

            Anaerobe Systems PRAS Cycloserine
           Cefoxitin Fructose Agar (CCFA) AS-213

Cycloserine Cefoxitin Fructose Agar Products

Item #


CCFA mono plate

1 plate



Proteose Peptone #2, 40.0 g

Sodium Phosphate, 5.0 g

Potassium Phosphate, 1.0 g

Sodium Chloride, 2.0 g

Magnesium Sulfate, 0.1 g

Fructose, 6.0 g

Neutral Red (1% soln), 3.0 ml

Cycloserine, 500.0 mg

Cefoxitin, 15.5 mg

Agar, 15.0 g

Distilled Water, 1000.0 ml


Final pH 7.2 +/- 0.2 at 250C.

Final weight 16.0g +/- 1.6.



For IN VITRO DIAGNOSTIC USE only.  Approved biohazard precautions and aseptic techniques should be observed when using this product.  This product is for use only by properly-trained and qualified personnel.  Sterilize all biohazard waste prior to disposal.


Storage and Shelf Life

Storage:  Upon receipt, store at 2 – 8oC in original container until used.  Avoid overheating or freezing.  Do not use medium if there are signs of deterioration (shrinking, cracking or discoloration due to oxidation of media) or contamination.  The expiration date applies to the product in its original packaging and stored as directed.  Do not use product past the expiration date shown on the container.

Shelf Life:  90 days from date of manufacture.


Specimen Collection:  Specimens for anaerobic culture should be protected from air (oxygen) during collection, transport and processing.  Consult appropriate references for detailed instructions concerning collection and transport of anaerobes.

Methods for Use:  CCFA should be inoculated directly with clinical material or a broth that has been previously inoculated from clinical material.  Inoculated plates should be streaked to obtain isolated colonies and immediately placed in an anaerobic atmosphere and incubated at 35-37oC for 18-48 hours.  Quantitation of C. difficile in a specimen may be clinically useful, which can be achieved by thoroughly mixing a serial 10-fold dilution of the specimen in an anaerobic environment followed by plating the dilutions onto CCFA media.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references.


Materials Required But Not Provided

Standard microbiological supplies and equipment such as loops, saline blanks, slides, staining supplies, microscope, incinerator / autoclave, incubators, anaerobic chamber, other culture media and serological and biochemical reagents.



CCFA will not provide complete information for identification of bacterial isolates.  Rare strains of C. difficile may be inhibited.  Plates should be examined no later than after 48 hours of incubation for optimal selectivity as after 3 – 5 days incubation significant numbers of colonies other than C. difficile may grow.  A test for aerotolerance should be used to confirm that each colony type is an obligate anaerobe.  Consult reference materials for additional information.


Quality Control

CCFA, if used properly, should support good growth of members of C. difficile.  After 24-48 hours, most colonies of C. difficile are large, circular, and yellow, with yellow coloration extending 2 –3 mm beyond the colony into the initially orange medium.  These colonies fluoresce golden yellow/chartreuse under long-wavelength UV light.  Most other bacteria are inhibited on this medium.  At 48 hours, colonies of most other organisms (e.g. Lactobacilli and yeast), which may grow, are very small (pinpoint to 0.5 mm in diameter) and do not fluoresce golden-yellow.

The following organisms are routinely used for quality control performance testing at Anaerobe Systems. 

Organism Tested



Time (Hours)

Special Reaction

Bacteroides fragilis


No Growth



Prevotella melaninogenica


 No Growth



Fusobacterium necrophorum


No Growth



Fusobacterium nucleatum


No Growth



Clostridium perfringens


No Growth



Peptostreptococcus anaerobius


No Growth



Staphylococcus aureus   or    Enterococcus faecalis

25923     29212

No Growth



Escherichia coli


No Growth



Clostridium difficile



24 hours

Yellow coloration


User Quality Control: The final determination to the extent and quantity of user laboratory quality control must be determined by the end user.

If sterility testing is to be performed on this product, the appropriate percentage of the original shipment amount should be incubated anaerobically and aerobically for 48 – 96 hours.

If the nutritive/inhibitory capacity of this medium is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth/inhibition.



Expected Growth

Special Reaction

B. fragilis



E. coli



S. aureus



C. difficile

24 hours

Yellow coloration, chartreuse fluorescence


Physical Appearance: CCFA should appear translucent pink/orange in color.



1.        Dowell, V. R., Jr. and T. M. Hawkins.  1974Laboratory Methods in Anaerobic Bacteriology.  CDC Laboratory Manual.  USDHEW C. D. C. Atlanta, GA 30333.


2.        Dowell, V. R., Jr. and G. L. Lombard.  1977Presumptive Identification of Anaerobic Non-sporeforming Gram-negative BacilliUSDHEW, CDC.  Atlanta, GA 30333.


3.        Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson and A. Y. Armfield.  1977Media for the Isolation, Characterization, and Identification of Obligately Anaerobic BacteriaUSDHEW, CDC, Atlanta, GA 30333.


4.        Holdeman, L. V., F. P. Cato and W. E. C. Moore.  1977.  Anaerobe Laboratory ManualVirginia Polytechnic Institute and State University.  Blacksburg, VA 24061.


5.        Jousimies-Somer, H. R., P. Summanen, D. M. Citron, E. J. Baron, H. M. Wexler and S. M. Finegold.  2002.  Wadsworth – KTL Anaerobic Bacteriology ManualStar Publishing Co., Belmont, CA 94002.


6.        NCCLSQuality Control for Commercially Prepared Microbiological Culture Media; Approved Standard- Third Edition.  (2004).  NCCLS document M22-A3.  NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898.


7.        George, W. L., V.L. Sutter, D. Citron, S. Finegold.  1979.  Selective and Differential Medium for Isolation of Clostridium difficileJournal of Clinical Microbiology9:214-219.