REINFORCED CLOSTRIDIAL MEDIUM, USP

Cat. no. U172 Reinforced Clostridial Medium, USP, 4oz Glass Bottle, 100ml 16 bottles/box

INTENDED USE

Hardy Diagnostics Reinforced Clostridial Medium is recommended for the cultivation and enumeration of clostridia, and other anaerobic and facultative bacteria from foods. The medium meets the harmonized USP/EP/JP standards for use as an enrichment medium in performing the microbial examination of nonsterile products.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Reinforced Clostridial Medium Is A Broth Medium Originally Formulated By Hirsch And Grinstead To Enhance The Growth Of Clostridia From Small Inocula.(4) Barnes And Ingram Utilized The Medium To Dilute Vegetative Cells And Barnes Et Al. Later Prepared The Medium As A Solid Version To Enumerate Clostridia From Food.(2,3) The Broth Medium Is Highly Nutritious And Can Also Be Used As An Enrichment To Optimize The Growth Of Clostridia Before Subculturing To Solid Agar.

Reinforced Clostridial Medium contains peptones and beef extract, which provide carbon, nitrogen, vitamins, and minerals to support bacterial growth. Dextrose provides an energy source. Sodium chloride helps maintain osmotic balance. Soluble starch, supplied in low concentrations, helps to detoxify metabolic by-products. Cysteine hydrochloride is added as a reducing agent and sodium acetate acts as a buffer. The small quantity of agar in the medium acts to inhibit the dispersion of carbon dioxide, while diffusing oxygen and other reducing substances.

FORMULA

Ingredients per liter of deionized water:*

Beef Extract 10.0gm
Casein Peptone 10.0gm
Dextrose 5.0gm
Sodium Chloride 5.0gm
Sodium Acetate 3.0gm
Yeast Extract 3.0gm
Soluble Starch 1.0gm
L-Cysteine HCl 0.25gm
Agar 0.5gm

Final pH 6.8 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

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

PRECAUTIONS

PROCEDURE

Consult listed references for more information on the specific methods and procedures needed to isolate clostridia.(2-6,8)

1. Transfer 10ml of the sample into 100ml of Reinforced Clostridial Medium.

2. Incubate containers with tight caps at 35ºC. for 48 hours.

3. After incubation, growth is evident by the appearance of turbidity in the medium. Subculture the medium to a nonselective solid agar, such as Columbia Blood Agar (Cat. no. A16), and incubate specimen under anaerobic conditions at 35ºC. for 48 hours for the detection of clostridia.

INTERPRETATION OF RESULTS

The anaerobic growth of gram-positive rods, with or without endospores, yielding a negative catalase reaction indicates the presence of clostridia.

LIMITATIONS

Due to nutritional variation, some strains may grow poorly or fail to grow at all on this medium.

The media may need to be pre-reduced. This can be achieved by boiling the media in a hot water bath before use to drive oxygen out that may have been introduced from agitation during shipment.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Clostridium sporogenes
ATCC® 19404**
J 48hrs 30-35°C Aerobic*** Turbidity
Clostridium sporogenes
ATCC® 11437**
J 48hrs 30-35°C Aerobic*** Turbidity

** Tested in accordance with USP <62>.(8)

***Bottles are incubated in an aerobic incubator with tight caps to create an atmosphere of low oxygen tension.

USER QUALITY CONTROL

Physical Appearance

Reinforced Clostridial Medium should appear slightly opalescent to opalescent, and medium amber in color.

REFERENCES

1. Anderson, N.L., et al. 2005. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Barnes, E.M., J.E. Despaul, and M. Ingram. 1963. The Behaviour of a Food Poisoning Strain of Clostridium welchii in Beef. J. Appl. Microbio. ; 26:415-427.

3. Barnes, E.M. and M. Ingram. 1956. The Effect of Redox Potential on the Growth of Clostridium welchii Strains Isolated from Horse Muscle. J. Appl. Microbio. ; 19:117-127.

4. Hirsch, A. and E. Grinsted. 1954. Methods for the Growth and Enumeration of Anaerobic Spore-formers from Cheese, with Observations on the Effect of Nisin. J. of Dairy Res. ; 21:101-110.

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

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

7. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. The Official Compendia of Standards. USP-NF . United States Pharmacopeial Convention, Rockville, MD.


ATCC is a registered trademark of the American Type Culture Collection.

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