MIDDLEBROOK 7H9 BROTH

Cat. no. C32 Middlebrook 7H9 Broth, 16x125mm Tube, 10ml 20 tubes/box
Cat. no. U32 Middlebrook 7H9 Broth, 500ml Polycarbonate Bottle, 500ml 1 each
Cat. no. C62 Middlebrook 7H9 Broth with Tween® 80, 16x125mm Tube, 5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Middlebrook 7H9 Broth and Middlebrook 7H9 Broth with Tween® 80 are recommended for use in the cultivation of Mycobacterium spp. The media are also used for preparing dilutions of mycobacteria for antimicrobial testing.

Cat. no. U32 is not intended to be used for the diagnosis of human disease.

SUMMARY

In 1947, Dubos and Middlebrook formulated a tubercle bacilli growth enhancing medium to protect organisms against a variety of toxic agents.(5) This medium is named Middlebrook 7H9 Broth Base. The basal medium of 7H9 broth is supplemented with Middlebrook ADC Enrichment. The supplementation provides nutrients necessary for mycobacterial growth.

Bovine albumin, dextrose, and catalase are components of Middlebrook ADC Enrichment. Albumin acts as a protective agent and 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. Glycerol provides an abundant source of carbon and energy for the tubercle organisms. Biotin helps stimulate the revival of damaged cells, and is involved in a variety of carboxylation and decarboxylation reactions. Sodium citrate, when converted to citric acid, holds inorganic cations in solution.

Middlebrook 7H9 Broth with Tween® 80 does not contain glycerol, but contains Tween® 80. Tween® 80 allows for the replication of microorganisms, and encourages the diffusion of new cells by wetting the surface of the tubercle bacilli. The compound is absorbed by the lipid portion of the bacterial surface and renders the tubercle bacilli dispersible in water.(13,14)

Middlebrook 7H9 Broth supplemented with glycerol is used for the maintenance of stock strains of pure cultures of mycobacteria for use in routine laboratory analyses. Middlebrook 7H9 Broth with Tween® 80 is used for the growth of M. bovis and in the preparation of inocula for the tellurite reduction test, useful in differentiating the M. avium complex (positive) from most other nonphotochromogenic mycobacteria (negative).

FORMULA

Ingredients per 900ml of deionized water:*

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
Glycerol 2.0ml

ADC Enrichment:

Bovine Albumin 5.0gm
Dextrose 2.0gm
Beef Catalase 3.0mg

In addition, Middlebrook 7H9 with Tween® 80 (Cat. no. C62) does not contain glycerol, but contains:

Dextrose 2.0gm
Tween® 80 0.5gm

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 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

For Cat. nos. C32 and C62.

For Cat. no. U32.

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 is primarily used for specimens usually 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, such as the tellurite reduction test. It is recommended that biochemical testing be done for complete identification.

Tellurite Reduction Test:(15)

1. Inoculate tubes using a heavy inoculum from an actively growing solid culture.

2. Incubate tubes at 35ºC. for 7 days. Hand shake the tubes daily to encourage heavy growth. Tubes must be heavily turbid for testing. Note: If not heavily turbid by day 7, discard the tube and begin with a fresh culture.

3. Add two drops of a sterile 0.2% potassium tellurite solution to each tube and shake well to mix contents. Use only fresh potassium tellurite solution.

4. Reincubate the cultures at 35ºC. for 3 days. Do not shake the tubes during the second incubation period.

5. On day three, examine sedimented cells in each tube for formation of a black precipitate. Do not shake tubes during examination.

INTERPRETATION OF RESULTS

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

Turbidity in the bottom layer of the medium or throughout the tube indicates growth.

Tellurite ReductionTest Results:(15)

Positive - formation of a black precipitate of metallic tellurium in and around the sedimented cells.

Negative - growth of cells without a black precipitate. A light brown precipitate or gray precipitate is recorded as a negative result.

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)

M. bovis is inhibited in the presence of glycerol.

Tellurite Reduction Test: If tubes do not show a heavy turbidity after 7 days incubation, repeat the test using a fresh, heavily turbid culture to minimize potentially questionable or false negative results.(15)

Most other rapidly growing mycobacteria also reduce tellurite to metallic tellurium within 3 days.(15)

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 0.2% potassium tellurite solution, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium tuberculosis
H37Ra
ATCC® 25177
G 21 days 35°C CO2** Growth; turbidity at bottom or throughout tube
Mycobacterium kansasii
Group I
ATCC® 12478
G 21 days 35°C CO2** Growth; turbidity at bottom or throughout tube
Mycobacterium scrofulaceum
Group II
ATCC® 19981
G 21 days 35°C CO2** Growth; turbidity at bottom or throughout tube
Mycobacterium intracellulare
Group III
ATCC® 13950
G 21 days 35°C CO2** Growth; turbidity at bottom or throughout tube
Mycobacterium fortuitum
Group IV
ATCC® 6841***
G 21 days 35°C CO2** Growth; turbidity at bottom or throughout tube

** Atmosphere of incubation is enriched with 5-10% CO2.

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

User Quality Control

PHYSICAL APPEARANCE

Middlebrook 7H9 Broth and Middlebrook 7H9 Broth with Tween® 80 should appear clear and colorless.

M. kansasii Group I growing in Middlebrook 7H9 Broth

Mycobacterium kansasii Group I (ATCC® 12478) growing in Middlebrook 7H9 Broth (Cat. no. C32). Incubated in CO2 for 21 days at 35ºC.

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. Kilburn, J.O. V.A. Silcox, and G.P. Kubica. 1969. Differential identification of mycobacteria. V. The tellurite reduction test. Am. Rev. Respir. Dis.; 99(1):94-100.

ATCC is a registered trademark of the American Type CultureCollection.
Tween is a registered trademark of ICI Americas, Inc.

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