COOKED MEAT MEDIUM

Cat. no. K19 Cooked Meat Medium, 16x125mm Tube, 10ml 20 tubes/box
Cat. no. K219 Cooked Meat Medium, 20x125mm Tube, 15ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Cooked Meat Medium is recommended for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms, especially Clostridium species.

SUMMARY

The use of animal tissue for culturing anaerobic organisms was first employed by Theobald Smith in 1890. (4) Von Hibler later used brain tissue for cultivating and classifying anaerobic bacilli. (5) Robertson replaced brain tissue with beef heart and used this medium to differentiate putrefactive and saccharolytic species. (8)

The formulation presently used is a modified version of Robertson's formulation. This medium is also referred to as Chopped Meat Medium. (2)

Nutritional requirements needed by most bacteria are provided by beef heart, peptone and dextrose. Dextrose, yeast extract, hemin and vitamin K are added to enhance the growth of anaerobic microorganisms. Amino acids and other nutrients are supplied by the muscle protein in the heart tissue granules. Reducing substances, which permit the growth of strict anaerobes, are supplied by the muscle tissue and the iron filings. (9) It is thought that the meat particles act as a reducing and detoxifying substance, thereby disabling harmful by products that may be produced by the replicating organism. (11) Because reducing substances are more available in denatured protein, the meat particles are cooked before use in the medium.

Growth of spore-forming and non-spore-forming obligate anaerobes is supported by this medium. Cooked Meat Medium is also useful as an enrichment broth for cultivating organisms from a very small inoculum. (2,3,7,9,10) Additionally, researchers have found that Cooked Meat Medium preserves viability of organisms over a long period of time and is useful in maintaining anaerobic stock organisms. (13) The Food and Drug Administration recommends its use in the enumeration and identification of Clostridium perfringens from food. (14)

FORMULA

Ingredients per liter of deionized water:*

Peptic Digest of Animal Tissue 17.5gm
Dextrose 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 5.0gm
Cooked Meat Medium 250.0gm
Iron Filings 10.0gm
Hemin 10.0ml
Vitamin K 10.0ml

Final pH 6.8 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or 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

Specimen Collection: Consult listed references for information on specimen collection. (1-3,6) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Method of Use: Consult the listed references for the appropriate cultivation techniques using this medium. (1-3,6) It is recommended that liquid media for anaerobic incubation should be reduced prior to inoculation by placing tubes (with loosened caps) under anaerobic conditions for 18-24 hours. Alternatively, the media may be reduced by bringing the media up to 100ºC. in a boiling waterbath. Loosen screw caps slightly before heating, and tighten during cooling to room temperature. The boiling serves to reduce media intended for the culture of anaerobic organisms.

1. The medium can be inoculated with a pure culture of an isolated colony, macerated tissue or liquid from a clinical specimen.

2. Heavily inoculate in the area of meat particles.

3. Incubate the tubes with caps tightened at 35 +/- 2.0ºC. for up to 7 days.

4. Growth or turbidity should be confirmed by gram stain and subcultured onto an appropriate plated growth medium.

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth and other identification tests to identify growth of organism in this medium. (1-3,6)

LIMITATIONS

Meat particles in the medium may cause turbidity, which could be misinterpreted as positive growth.

Meat particles blacken only in the presence of alkali, which is a result of ammonia production by proteolytic enzymes.

The reactions observed in the medium are useful for characterization, not speciation, of the organism.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, 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
Bacteroides fragilis
ATCC ® 25285
A 24-48hr 35°C Aerobic Growth
Streptococcus pyogenes
ATCC ® 19615
A 24-48hr 35°C Aerobic Growth
Clostridium perfringens
ATCC ® 13124
A 24-48hr 35°C Aerobic Growth

User Quality Control

PHYSICAL APPEARANCE

Cooked Meat Medium should appear amber in color, with approximately one inch of chopped meat on the bottom. Black iron filings should also be present on the bottom of the medium.

B. fragilis growing in Cooked Meat Medium

Bacteroides fragilis (ATCC ® 25285) growing in Cooked Meat Medium (Cat. no. K19). Incubated aerobically (with cap screwed down tightly) for 24 hours at 35ºC.

Cooked Meat Medium

Uninoculated tube of Cooked Meat Medium (Cat. no. K19).

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

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Smith, T. 1890. Centr. Bakteriol.; 7:509.

5. Von Hibler, E. 1899. Centr. Bakteriol.; 25:513, 594, 631.

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

7. MMacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

8. Robertson, M. 1916. J. Pathol. Bacteriol.; 20:327.

9. Willis. 1977. Anaerobic Bacteriology: Clinical and Laboratory Practice, 3rd ed. Butterworths, London.

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

11. Dowell, Lombard, Thompson and Armfield. 1979. Media for Isolation, Characterization and Identification of Obligately Anaerobic Bacteria, CDC Laboratory Manual, DHEW Publications No. (CDC) 79-8272. CDC, Atlanta.

12. Holman, W.L. 1919. J. Bacteriol.; 4:149.

13. Claros, M.C., et al. 1995. J. Clin. Micro., 33; 9:2505-2507, American Society for Microbiology.

U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

022316gr