CRITERION™ MIDDLEBROOK 7H10 AGAR BASE

Cat. no. C6280 CRITERION™ Middlebrook 7H10 Agar Base 38gm
Cat. no. C6281 CRITERION™ Middlebrook 7H10 Agar Base 500gm
Cat. no. C6282 CRITERION™ Middlebrook 7H10 Agar Base 2kg
Cat. no. C6283 CRITERION™ Middlebrook 7H10 Agar Base 10kg
Cat. no. C6284 CRITERION™ Middlebrook 7H10 Agar Base 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ Middlebrook 7H10 Agar Base is recommended for use in qualitative procedures for the isolation and cultivation of Mycobacterium species.

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

In 1947 Dubos and Middlebrook formulated a media (7H9) containing albumin and oleic acid which enhanced the growth of tubercle bacilli, and protected the organisms against a variety of toxic agents.(5) Later, in 1958, Middlebrook and Cohn improved this first formulation and developed a media (7H10) which allowed more luxuriant, faster growth of Mycobacterium species.(9)

CRITERION™ Middlebrook 7H10 Agar Base is a non-selective medium containing a variety of inorganic salts, sodium citrate, vitamins, co-factors, oleic acid, albumin, biotin, catalase and malachite green. The inorganic salts provide substances essential for the growth of mycobacteria. Sodium citrate, when converted to citric acid, holds the inorganic cations in solution. Malachite green is added as a selective agent, which partially inhibits the growth of other bacteria. Biotin and catalase help stimulate the revival of damaged organism. Glycerol may be added to the base medium as a source of carbon and energy for the tubercle organisms. OADC Enrichment which must be added aseptically to the medium after autoclaving contains the following required additives: albumin to protect the tubercle bacilli against toxic agents; oleic acid, a fatty acid utilized in the metabolism of the organism; sodium chloride to maintain osmotic equilibrium; catalase to destroy any toxic peroxides in the medium; and dextrose as an energy source.

FORMULA

Gram weight per 900ml deionized water: 19.0gm
Monopotassium Phosphate 1.5gm
Disodium Phosphate 1.5gm
Ammonium Sulfate 0.5gm
L-Glutamic Acid 0.5gm
Sodium Citrate 0.4gm
Ferric Ammonium Citrate 40.0mg
Magnesium Sulfate 25.0mg
Copper Sulfate 1.0mg
Zinc Sulfate 1.0mg
Pyridoxine 1.0mg
Calcium Chloride 0.5mg
Biotin 0.5mg
Malachite Green 0.25mg
Agar 15.0gm
OADC Enrichment (sold separately) ** 100.0ml

Final pH 6.6 +/- 0.2 at 25ºC.

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

** OADC Enrichment (sold separately - Cat. no. U98):

Bovine Albumin 5.0gm
Dextrose 2.0gm
Sodium Chloride 0.85gm
Beef Catalase 4.0mg
Oleic Acid 50.0mg

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 light beige with a green tint.

Store the prepared culture media at 2-8ºC. Protect from light.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

Note: Hydrate culture media using 900ml of distilled or deionized water per liter of media. The OADC Enrichment added after sterilization will bring the volume to a full liter.

1. Suspend 19.0gm of the dehydrated culture media in 900ml of distilled or deionized water containing 5ml of glycerol.

2. Heat to boiling and mix to dissolve completely.

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

4. Cool to 45-50ºC.

5. Aseptically add 100ml per liter of sterile OADC. Other enrichments may be added as desired.

6. Dispense into containers as desired.

7. Protect media from light.

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. W30.

LIMITATIONS

Prepared Middlebrook 7H10 Agar requires incubation in a 5-10% CO2 atmosphere in order to recover mycobacteria. For unknown reasons, mycobacteria are not recovered well from candle extinction jars.(7)

Keep inoculated media away from light or excessive heat, as exposure results in the release of formaldehyde in the media which may inhibit or kill mycobacteria.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, as well as OADC Enrichment (Cat. no. U98), etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium tuberculosis
H37RV
ATCC ® 25177

G 21 days 35°C CO2** Growth; colonies visible in 2 weeks, mature in 3 weeks
Mycobacterium kansasii
Group I
ATCC ® 12478

G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium scrofulaceum
Group II
ATCC ® 19981

G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium intracellulare
Group III
ATCC ® 13950

G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium fortuitum
Group IV
ATCC ® 6841

G 21 days 35°C CO2** Growth; colonies visible in 4 days

User Quality Control

PHYSICAL APPEARANCE

CRITERION™ Middlebrook 7H10 Agar Base powder should appear homogeneous, free-flowing, and light beige with a greenish tint in color. The prepared media should appear slightly opalescent, and light 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. 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. Cohn, M.L., et al. 1968. Am. Rev. Respir. Dis.; 98:295.

5. Dubos, R.J. and G. Middlebrook. 1947. Am. Rev. Tuberc.; 56:334-345.

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

7. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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

9. Middlebrook, G. and M.L. Cohn. 1958. Am. J. Public Health; 48:844-853.

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

11. Vestal, A.L. 1975. Procedures for the isolation and identification of mycobacteria. DHEW (CDC 75-8230). Centers for Diseases Control. Atlanta, GA.

12. Welch, D.F., et al. 1993. Timely culture for mycobacteria which utilizes a microcolony method. J. Clin. Microbiol.; 31: 2178-2184.


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