anaeroGRO™

Chopped Meat Media

Cat. no. AG19H Chopped Meat Glucose Broth, 16x125mm Tube with
Hungate Septum Cap, 9ml
20 tubes/box
Cat. no. AG20H Chopped Meat Carbohydrate Broth, 16x125mm Tube with Hungate Septum Cap, 9ml 20 tubes/box
Cat. no. AG21H Chopped Meat Broth, 16x125mm Tube with Hungate
Septum Cap, 9ml
20 tubes/box

INTENDED USE

Hardy Diagnostics AnaeroGRO™ Chopped Meat Media is recommended for the cultivation of microaerophilic, facultative and obligate 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) Growth of spore-forming and non-spore-forming obligate anaerobes is supported by this medium. Chopped 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 Chopped Meat Medium preserves viability of organisms over a long period of time and is useful in maintaining anaerobic stock organisms.(16) The Food and Drug Administration recommends its use in the enumeration and identification of Clostridium perfringens from food.(14)

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 obligate 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. Various formulations are available containing different carbohydrates, Cat. no. AG19H contains glucose while Cat. no. AG20H contains glucose, cellobiose, maltose, and starch. These two media with additional components are better suited to enhance the productions of toxin by anaerobes such as: Clostridium spp. compared to the unsupplemented medium (Cat. no. AG21H). Chopped meat carbohydrate medium is recommended for the use with gas-liquid chromatography for analysis of anaerobic metabolic products.(16)

FORMULA

Ingredients per liter of deionized water:*

Chopped Meat Broth (Cat. no. AG21H):
Peptic Digest of Animal Tissue 17.5gm
Sodium Chloride 5.0gm
Yeast Extract 5.0gm
Cooked Meat 250.0gm
Iron Filings 10.0gm
Hemin 10.0ml
Vitamin K 10.0ml

In addition, AnaeroGRO™ Chopped Meat Glucose Broth (Cat. no. AG19H) contains:

Glucose 3.0gm

In addition, AnaeroGRO™ Chopped Meat Carbohydrate Broth (Cat. no. AG20H) contains:

Glucose 4.0gm
Maltose 1.0gm
Cellobiose 1.0gm
Starch 1.0gm

Final pH 7.1 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1-3,6,11,15,16) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat, cold, and oxygen exposure. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium (Cat. no. S120D) and refrigerated until inoculation.

Method of Use: Consult the listed references for the appropriate cultivation techniques using this medium.(1-3,6,11,15,16)

1. The medium can be inoculated with a pure culture of an isolated colony, macerated tissue or liquid from a clinical specimen.To avoid oxygen exposure liquid specimens may be injected directly through the rubber septum of the hungate screw cap with a needle and syringe.

2. Heavily inoculate in the area of meat particles.

3. Incubate the tubes with caps tightened at 35ºC. for up to seven days in an ambient air incubator.

4. Growth or turbidity should be confirmed by Gram stain and subcultured onto an appropriate plated growth medium, such as Brucella Agar with H and K (Cat. no. AG301).

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth and other identification tests to identify growth of organism in this medium.(1,2,3,6,11,15,16)

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, transports (Cat. no. S120D) 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

** Tubes are incubated in an aerobic incubator with the caps screwed down tightly to create an atmosphere of low oxygen tension within the tube.

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

USER QUALITY CONTROL

Physical Appearance

AnaeroGRO™ Chopped Meat Media 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 Chopped Meat Glucose Broth

Bacteroides fragilis (ATCC® 25285) growing in Chopped Meat Glucose Broth (Cat. no. AG19H). Incubated aerobically (with cap screwed down tightly) for 24 hours at 35ºC.

Chopped Meat Glucose Broth

Uninoculated tube of Chopped Meat Glucose Broth (Cat. no. AG19H).

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. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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.

14. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

15. Summanen, P., and E.J. Baron. 2002. Wadsworth Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, Belmont, CA.

16. Engelkirk, P.G., J. Duben-Engelkirk, and V.R. Dowell, Jr. 1992. Principles and Practice of Clinical Anaerobic Bacteriology. Star Publishing Company, Belmont, CA.

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

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