CRITERION™ Reinforced Clostridial Medium

Cat. no. C8720 CRITERION™ Reinforced Clostridial Medium 76gm
Cat. no. C8721 CRITERION™ Reinforced Clostridial Medium 500gm
Cat. no. C8722 CRITERION™ Reinforced Clostridial Medium 2kg
Cat. no. C8723 CRITERION™ Reinforced Clostridial Medium 10kg
Cat. no. C8724 CRITERION™ Reinforced Clostridial Medium 50kg

INTENDED USE

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

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

CRITERION™ Reinforced Clostridial Medium is a broth medium originally formulated by Hirsch and Grinstead to enhance the growth of clostridia from small inocula.(5) 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 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.

Reinforced Clostridial Medium can be modified for the isolation and differentiation of sulfite-reducing clostridia by adding sodium sulfite and ferric citrate to the medium; the medium can also be made solid by increasing the amount of agar.4 Differential Reinforced Clostridial Medium is recommended for identifying sulfite-reducing clostridia, such as Clostridium perfringens, from food and beverage samples using the most probable number (MPN) method.4

FORMULA*

Gram weight per liter: 38.0gm/L
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

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original beige

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 38gm of the dehydrated culture media in one liter of distilled or deionized water. Stir to mix thoroughly.
Note: The medium can be made differential by adding sodium sulfite and ferric citrate or made into a solid medium by increasing the concentration of agar.4

2. Heat to boiling to dissolve completely. Do not overheat.

3. Dispense into desired containers.

4. Sterilize in the autoclave at 121ºC. for 15 minutes.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. U172.

LIMITATIONS

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

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Clostridium sporogenes
ATCC ® 19404**
J 24-48hr 35°C Aerobic Turbidity
Clostridium perfringens
ATCC ® 13124
A 24-48hr 35°C Aerobic Turbidity
Bacteroides fragilis
ATCC ® 23745**
A 24-48hr 35°C Aerobic Turbidity

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

User Quality Control

Physical Appearance

CRITERION™ Reinforced Clostridial Medium powder should appear homogeneous, free-flowing, and beige in color. The prepared medium should appear slightly opalescent to opalescent, and medium amber in color.

REFERENCES

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. 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. Gibbs, B.M. and B. Freame. 1965. Methods for the Recovery of Clostridia from Foods. J. Appl. Microbiol.; 28:95-111.

5. 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.

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

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

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

9. The Official Compendia of Standards. 2008. USP27-NF22. United States Pharmacopeial Convention, Rockville, MD.


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

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