Cycloserine Cefoxitin Fructose Agar with Lysozyme (CCFAL) 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. CCFAL is an enriched selective and differential medium for the isolation of C. difficile. 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 and lysozyme is added to aid in the germination of spores.  The pH of the media is decreased due to the fermentation of fructose, which will turn the indicator from pink/orange to yellow. Clostridium difficile is not inhibited on the CCFAL 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 w/ Lysozyme (CCFAL) AS-2133

Formula

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

Lysozyme, 5.0 mg

Agar, 15.0 g

Distilled Water, 1000.0 ml

 

Final pH 7.2 +/- 0.2 at 25⁰C.

Final weight 17.0g +/- 1.7.

 

Precautions

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 – 8^oC 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.

 

Procedure

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:  CCFAL 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 35-37^oC 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 CCFAL 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.

 

Limitations

CCFAL 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

CCFAL, 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 pink/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	ATCC #	Results	Time (Hours)	Special Reaction
Bacteroides fragilis	25285	No Growth
Enterococcus faecalis	29212	No Growth
Clostridium sporogenes	3584	No Growth
Clostridium beijerinckii	8260	No Growth
Proteus mirabilis	12453	No Growth
Clostridium perfringens	13124	No Growth
Clostridium innocuum	14501	No Growth
Clostridium sordellii	9714	No Growth
Clostridium difficile	9689	Growth	24	Yellow
Clostridium difficile	700057	Growth	24	Yellow

 

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.

  

Organism	Expected Growth	Special Reaction
B. fragilis	Inhibited
E. coli	Inhibited
S. aureus	Inhibited
C. difficile	24 hours	Yellow coloration, chartreuse fluorescence

 

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

 

References

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

 

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

 

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

 

4.        Holdeman, L. V., F. P. Cato and W. E. C. Moore.  1977.  Anaerobe Laboratory Manual.  Virginia 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 Manual.  Star Publishing Co., Belmont, CA 94002.

 

6.        NCCLS.  Quality 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.        Wilcox MH, Fawley WN, Parnell P:  Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery of Clostridium difficile.  Journal of Hospital Infection 2000, 45: 65-69.

 

8.        George, W. L., V.L. Sutter, D. Citron, S. Finegold.  1979.  Selective and Differential Medium for Isolation of Clostridium difficile.  Journal of Clinical Microbiology.  9:214-219.