MIDDLEBROOK 7H10 AGAR

Cat. no. W30 Middlebrook 7H10 Agar, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. C34 Middlebrook 7H10 Agar, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. X26 Middlebrook 7H10 Agar, 50ml HardyFlask™, 12ml 20 flasks/box

INTENDED USE

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

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) In 1958, Middlebrook and Cohn improved this formulation and developed a media (7H10) which allowed for more luxuriant and rapid growth of Mycobacterium species. (9)

Middlebrook 7H10 is a non-selective medium supplemented with OADC Enrichment. The medium contains a variety of inorganic salts, sodium citrate, vitamins, co-factors, oleic acid, albumin, biotin, catalase, glycerol, and malachite green. Inorganic salts provide substances essential for the growth of mycobacteria. Sodium citrate, when converted to citric acid, holds inorganic cations in solution. Glycerol is provided as an abundant source of carbon and energy for tubercle organisms. 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. OADC Enrichment 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

Ingredients per 995ml of deionized water:*

Disodium Phosphate 1.5gm
Monopotassium Phosphate 1.5gm
L-Glutamic Acid 0.5gm
Ammonium Sulfate 0.5gm
Sodium Citrate 0.4gm
Ferric Ammonium Citrate 40.0mg
Magnesium Sulfate 25.0mg
Zinc Sulfate 1.0mg
Copper Sulfate 1.0mg
Pyridoxine 1.0mg
Calcium Chloride 0.5mg
Biotin 0.5mg
Malachite Green 0.25mg
Glycerol 5.0ml
Agar 15.0gm


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

Final pH 6.8 +/- 0.2 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 (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: 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. According to test procedures recommended by the Centers for Disease Control, inoculate Middlebrook 7H10 Agar with specimen after decontamination and neutralization. Consult listed references for methods. (1-3,6,7,11)

2. If inoculating plates, fit the plate with a MycoSeal™ (Cat. no. SS9225). The MycoSeal™ allows for penetration of CO 2 to the plate, yet prevents excess moisture loss and dehydration of the agar media.

3. The caps of tubes and bottles should be kept loose for at least one week to allow circulation of carbon dioxide. Tighten caps, thereafter, to prevent dehydration. Loosen caps briefly once a week in order to replenish CO 2 .

4. Incubate medium in a 5-10% CO 2 atmosphere at 35-37ºC.; protect from light. Examine plates within five to seven days after inoculation and weekly, thereafter, for up to eight weeks.

5. To read plates and bottles, invert and place on the stage of a microscope, as focusing is performed through the bottom of the plate and agar. The correct plane of focus is easily determined by focusing on the streak lines still evident on the agar surface. Using transmitted light, scan the plate at 10-20X through the first two streaked quadrants. When detected, colonies are examined at 30X-60X to determine colony morphology. Microcolonies are characterized in descriptive terms relating to their margin and consistency. (12)

INTERPRETATION OF RESULTS

Observations are recorded as follows: (11)

1. Number of days required for colonies to become macroscopically visible.

2. Number of colonies (plates and bottles):

No colonies = Negative
Fewer than 50 colonies = Actual count
50 to 100 colonies = 1+
100 to 200 colonies = 2+
200 to 500 colonies (almost confluent growth) = 3+
More than 500 colonies (confluent growth) = 4+

3. Pigmentation

White, cream or buff = Non-chromogenic
Lemon, yellow, orange, red = Chromogenic

It is recommended that biochemical testing be performed for definitive identification of microorganisms. Consult appropriate references for aid in the biochemical identification of acid-fast bacilli. (1,2,6,11)

LIMITATIONS

Middlebrook 7H10 Agar requires incubation in a 5-10% CO 2 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 loops, slides, decontamination supplies, MycoSeals™, applicator sticks, pipets, incinerators, CO 2 incubator, biological safety hood, microscopes, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium tuberculosis
H37Ra
ATCC ® 25177

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

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

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

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

G 21 days 35°C CO 2 ** Growth; colonies visible in 4 days

User Quality Control

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

PHYSICAL APPEARANCE

Middlebrook 7H10 Agar should appear clear, slightly opalescent, and light amber with a green hue in color.

M. tuberculosis growing on Middlebrook 7H11 Agar

Mycobacterium tuberculosis H37Ra (ATCC ® 25177) colonies growing on Middlebrook 7H10 Agar (Cat. no. C34). Incubated in CO 2 for 21 days at 35ºC.

M. kansasii growing on Middlebrook 7H11 Agar

Mycobacterium kansasii Group I (ATCC ® 12478) colonies growing on Middlebrook 7H10 Agar (Cat. no. C34). Incubated in CO 2 for 21 days at 35ºC.

M. scrofulaceum growing on Middlebrook 7H11 Agar

Mycobacterium scrofulaceum Group II (ATCC ® 19981) colonies growing on Middlebrook 7H10 Agar (Cat. no. C34). Incubated in CO 2 for 21 days at 35ºC.

M. intracellulare growing on Middlebrook 7H11 Agar

Mycobacterium intracellulare Group III (ATCC ® 13950) colonies growing on Middlebrook 7H10 Agar (Cat. no. C34). Incubated in CO 2 for 21 days at 35ºC.

M. fortuitum growing on Middlebrook 7H11 Agar

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Middlebrook 7H10 Agar (Cat. no. C34). Incubated in CO 2 for 21 days at 35ºC.

M. fortuitum growing on Middlebrook 7H11 Agar

Uninoculated tube of Middlebrook 7H10 Agar (Cat. no. C34).

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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032116gr