CRITERION™ Middlebrook 7h9 Broth Base

Cat. no. C6301 CRITERION™ Middlebrook 7H9 Broth Base 500gm

INTENDED USE

Hardy Diagnostics CRITERION™ Middlebrook 7H9 Broth Base, supplemented with ADC Enrichment, is recommended for use in the cultivation of Mycobacterium spp. from sterile fluids. The medium can also be used for preparing dilutions of mycobacteria for antimicrobial testing.

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 growth medium known as 7H9, which contains albumin and oleic acid, to enhance the growth of tubercle bacilli; the medium also protects these microorganisms against a variety of toxic agents.(5) CRITERION™ Middlebrook 7H9 Broth Base is designed to be supplemented with Middlebrook ADC Supplement (Cat. no. X95). Middlebrook ADC Supplement provides nutrients necessary for mycobacterial growth and must be added aseptically to the medium after autoclaving.

Bovine albumin, dextrose, and catalase are components of Middlebrook ADC Enrichment. Albumin, which acts as a protective agent, binds free fatty acids that may be toxic to Mycobacterium spp. Dextrose serves as an energy source. Toxic peroxides that may be present in the medium are destroyed by catalase. Additionally, the basal medium contains glycerol, biotin and sodium citrate. Biotin helps to stimulate the revival of damaged cells as well as provides substrates needed for a variety of carboxylation and decarboxylation reactions. Sodium citrate, when converted to citric acid, holds inorganic cations in solution.

Along with the ADC Enrichment, additional supplements can be added to the medium, as needed: glycerol (2ml/L) to provide an abundant source of carbon and energy for tubercle organisms and polysorbate 80 (0.5ml/L) for improved growth. Prepared Middlebrook 7H9 Broth can also be used as a subculture medium for Mycobacterium species and for the preparation of inocula for drug susceptibility testing.

FORMULA*

Gram weight per 900ml deionized water: 4.7gm
Disodium Phosphate 2.5gm
Monopotassium Phosphate 1.0gm
L-Glutamic Acid 0.5gm
Ammonium Sulfate 0.5gm
Sodium Citrate 0.1gm
Magnesium Sulfate 50.0mg
Ferric Ammonium Citrate 40.0mg
Zinc Sulfate 1.0mg
Copper Sulfate 1.0mg
Pyridoxine 1.0mg
Calcium Chloride 0.5mg
Biotin 0.5mg
ADC Enrichment (Sold separately)** 100.0ml

pH before ADC Enrichment 6.6 +/- 0.1 at 25ºC.

Final pH with ADC Enrichment 6.8 +/- 0.3 at 25ºC.

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

** Middlebrook ADC Enrichment (sold separately - Cat. no. X95):

Bovine Albumin 5.0gm
Dextrose 2.0gm
Beef Catalase 3.0mg

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.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

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

1. Suspend 4.7gm of the dehydrated culture media in 900ml of distilled or deionized water. Stir to mix thoroughly.

2. Heat as necessary 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 ADC (Cat. no. X95). Other enrichments may be added, as desired.

6. Dispense into containers as desired.

7. Protect media from light.

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Consult listed references for information on specimen collection.(1-3,6,7,11)

Method of Use:

1. Middlebrook 7H9 Broth is used primarily for specimens from sterile body sites and for growth of pure cultures of mycobacteria for use in laboratory studies. Using aseptic techniques, inoculate a homogenized or centrifuged specimen directly to the medium. Consult listed references for methods.(1-3,6,7,11)

2. Incubate medium in a 5-10% CO2 atmosphere at 35 +/- 2ºC., for up to eight weeks. Protect from light. Caps of tubes should be loosened for at least one week to allow circulation of CO2. Tighten caps, thereafter, to prevent dehydration. Loosen caps briefly once a week in order to replenish CO2.

3. Examine cultures within five to seven days after inoculation and weekly thereafter for up to eight weeks.

Mycobacterial growth from the broth can be used for additional laboratory test procedures as required. It is recommended that biochemical testing be done for complete identification.

Cultures from skin lesions suspected to be M. marinum or M. ulcerans should be incubated at 25-33ºC for primary incubation. Cultures suspected to contain M. avium or M. xenopi exhibit optimum growth at 40-42ºC and incubate a duplicate culture at 35-37ºC.(15)

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth of Mycobacterium species on this medium.(1-3,6,7,11)

Turbidity at the bottom of or throughout the tube indicates growth.

LIMITATIONS

Middlebrook 7H9 media require 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)

Negative culture results do not rule out active mycobacteria infection.

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 loops, slides, decontamination supplies, applicator sticks, pipets, incinerators, CO2 incubator, biological hoods, and microscopes, etc., as well as serological and biochemical reagents, such as sterile ADC Enrichment (Cat. no. X95), are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium tuberculosis H37Rv
ATCC ® 27294***
G 21 days 35°C CO 2 ** Growth; turbidity at bottom or throughout tube
Mycobacterium kansasii,
Group I
ATCC ® 12478***

G 21 days 35°C CO 2 ** Growth; turbidity at bottom or throughout tube
Mycobacterium scrofulaceum , Group II
ATCC ® 19981***
G 21 days 35°C CO 2 ** Growth; turbidity at bottom or throughout tube
Mycobacterium intracellulare , Group III
ATCC ® 13950***
G 21 days 35°C CO 2 ** Growth; turbidity at bottom or throughout tube
Mycobacterium fortuitum ,
Group IV
ATCC ® 6841***

G 21 days °35°C CO 2 ** Growth; turbidity at bottom or throughout tube

** Atmosphere of incubation is enriched with 5-10% CO 2 .

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

User Quality Control

Physical Appearance

CRITERION™ Middlebrook 7H9 Broth Base should appear homogeneous, free-flowing, and light beige in color. The prepared medium should appear clear, and colorless to 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 of 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.

13. Dubos, R.J. 1947. Experimental analysis of tuberculous infection. Experientia; 3, 45.

14. Dubos, R.J.; et al. 1946. The effect of water soluble lipids on the growth and biological properties of tubercle bacilli. Am. Rev. Tuberc.; 54, 204.

15. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS. Centers for Disease Control, Atlanta, GA.



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