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Cycloserine Cefoxitin Fructose Agar with Horse Blood and Taurocholate (CCFA-HT)

Cycloserine Cefoxitin Fructose Agar with Horse Blood and Tarocholate (CCFA-HT) 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. Horse blood is added as an enriched nutrient source and Taurocholic acid is added as a stimulator of spore germination.  Cycloserine will inhibit gram-negative bacteria, while cefoxitin will inhibit both gram-positive and gram-negative organisms.  The pH of the media is decreased due to the fermentation of fructose. Most strains of Clostridium difficile are not inhibited on the CCFA-HT medium, and, when growing on this medium, will exhibit a characteristic 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/ Horse
Blood & Taurocholate
(CCFA-HT) AS-2136




Cycloserine Cefoxitin Fructose Agar W/ Horse Blood and Taurocholate Products

Item #
Description
Size
Price

AS-2136

CCFA-HT mono plate

1 plate

3.46




Formula

Proteose Peptone #2, 40.0 g

Sodium Phosphate dibasic, 5.0 g

Potassium Phosphate monobasic, 1.0 g

Sodium Chloride, 2.0 g

Magnesium Sulfate, 0.1 g

Fructose, 6.0 g

Horse Blood, 70.0 mL

Sodium Taurocholate, 1.0 g

Cycloserine, 250.0 mg

Cefoxitin, 15.5 mg

Agar, 15.0 g

Distilled Water, 1000.0 ml

 

Final pH 7.35 +/- 0.3 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 – 8oC in original packaging 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 at 2 – 8oC.

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:  CCFA-HT 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-HT 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

CCFA-HT 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 because after 3 – 5 days of incubation significant numbers of colonies other than C. difficile may grow.  At 48 hours, colonies of most other organisms (e.g. Lactobacilli, Clostridia and yeast), which may grow, are very small (pinpoint to 0.5 mm in diameter) and do not fluoresce golden-yellow.  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-HT, if used properly, should support good growth of species of C. difficile.  After 24-48 hours, most colonies of C. difficile are large, grey, slightly filamentous and low umbonate to flat.  These colonies fluoresce golden yellow/chartreuse under long-wavelength UV light.

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

B. fragilis

Inhibited

E. coli

Inhibited

S. aureus

Inhibited

C. difficile

24 hours

 

Physical Appearance: CCFA-HT should appear opaque burgundy red 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.  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.  1977Anaerobe 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.  2002Wadsworth – 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.

 

8.        Wilson KH, Kennedy MJ, Fekety FR:  Use of Sodium Taurocholate to Enhance Spore Recovery on a Medium Selective for Clostridium difficile.  Journal of Clinical Microbiology 1982, 15(3): 443-446.

 

9.        Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD:  Comparison of Five Cultural Procedures for Isolation of Clostridium difficile from Stools.  Journal of Clinical Microbiology 1992, 30(2): 514-516.

 

10.     Edelstein, Martha. “Isolation and Identification of Clostridium difficile; Tissue Culture and Cytotoxicity Assay.”  Clostridium difficile: Its Role in Intestinal Disease.  Eds. Rolfe RD, Finegold SM.  San Diego:  Academic Press Inc, 1988.  287-307.