MIDDLEBROOK 7H10 WITH HUMAN BLOOD

Cat. no. C42 Middlebrook 7H10 with Human Blood, 10ml Slant 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Middlebrook 7H10 Agar with Human Blood is recommended for use in the isolation and cultivation of Mycobacterium spp., including M. genavense.

SUMMARY

Mycobacterium genavense is a newly described species of mycobacteria which is capable of causing disseminated disease in HIV infected individuals.(2,16) When isolated from clinical specimens, Mycobacterium genavense is most commonly found in blood.

Results of past studies indicated solid media were incapable of supporting growth of M. genavense. However, in 1992 Coyle reported growth of M. genavense on Middlebrook 7H11 supplemented with Mycobactin J.(15) Maier later reported the cultivation of the organism on Middlebrook 7H10 Agar supplemented with 10% human blood.(13) Studies conducted by Desmond found the human blood-supplemented medium to provide more luxuriant growth of M. genavense.(13,14)

Hardy Diagnostics Middlebrook 7H10 with Human Blood is supplemented with OADC Enrichment and 10% human blood. The medium contains a variety of inorganic salts, sodium citrate, vitamins, co-factors, oleic acid, albumin, biotin, catalase, and glycerol. Sodium citrate, when converted to citric acid, holds the inorganic cations in solution. Glycerol is provided as an abundant source of carbon and energy for the 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. The 10% human blood provides necessary components that promote the growth of M. genavense. The human blood components which promote growth of the organism have not yet been identified.(13)

FORMULA

Ingredients per 800ml of deionized water:*

Disodium Phosphate 1.5gm
Monopotassium Phosphate 1.5gm
L-Glutamic Acid (Sodium Salt) 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
OADC Enrichment 100.0ml
Human Blood 100.0ml
Glycerol 5.0ml
Agar 15.0gm

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

Final pH 6.6 +/- 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 (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Fixatives and preservatives should not be used. All specimens, except blood, should be refrigerated if transport to the laboratory is greater than one hour. Once recieved by the laboratory, specimens should be refrigerated until processing. Consult listed references for information on specimen collection. (1-3,6,7,11)

Method of Use:

1. Tissues or body fluids that have been aseptically collected usually do not require digestion and decontamination procedures. (17)

A. Normally sterile tissues may be ground in sterile 0.85% saline or 0.2% bovine serum albumin and then inoculated to the medium. (2)

B. Body fluids should be centrifuged at > 3,000Xg. The sediment can then be inoculated to the medium. (2)

C. Specimens thought to be contaminated should first undergo digestion and decontamination procedures as recommended by the Centers for Disease Control (CDC). Consult listed references for methods. (1-3,6,7,11)

2. Once inoculated, tubes should be incubated in a 5-10% CO 2 atmosphere at 35-37ºC. for up to eight weeks.The caps 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 .

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

4. Observe for typical colonial growth and morphology. Consult listed references for identification procedures for specific organisms recovered. (2,3,6-9,11,13-15)

INTERPRETATION OF RESULTS

It is recommended that biochemical testing and/or chromatographic analysis 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

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, CO 2 incubator, biological safety hoods, and microscopes, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium fortuitum
Group IV
ATCC ® 6841

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

User Quality Control

Physical Appearance

Middlebrook 7H10 with Human Blood should appear opaque, and red in color.

M. fortuitum growing in Middlebrook 7H10 with Human Blood

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Middlebrook 7H10 with Human Blood (Cat. no. C42). Incubated in CO 2 for four days at 35ºC.

Middlebrook 7H10 with Human Blood

Uninoculated tube of Middlebrook 7H10 with Human Blood (Cat. no. C42).

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. 1933. Timely culture for mycobacteria which utilizes a microcolony method. J. Clin. Microbiol.; 31: 2178-2184.

13. Maier, T., et al. 1995. Med. Microbiol. Lett.; 4:173-179.

14. Desmond, E.P. 1994. Clinical Microbiology Newsletter; Vol. 16, No. 7.

15. Coyle, M.B., et al. 1992. Journal of Clin. Micro.; 30:3206-32122.

16. Bottger, E., et al. 1992. Lancet; 340:76-80.

17. Sommers, H.M., et al. 1983. Cumitech 16: Laboratory Diagnosis of the Mycobacterioses, American Society for Microbiology, Washington, D.C.

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

032116gr