MIDDLEBROOK 7H11 AGAR, THIN POUR

Cat. no. SP57 Middlebrook 7H11 Agar, Thin Pour,
10x100mm SpaceSaver™ Plate, 18ml
15 plates/bag

INTENDED USE

Hardy Diagnostics Middlebrook 7H11 Agar, Thin Pour, is recommended for use in the isolation and cultivation of Mycobacterium species, using the microcolony method.

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) Cohn, in 1968, incorporated casein hydrolysate into the 7H10 medium, and obtained a media that stimulated the growth of mycobacteria that would not otherwise grow on the 7H10 medium. This formulation was then designated 7H11 Agar, and is recommended over 7H10 Agar. (4,7)

Middlebrook 7H11 Agar contains inorganic compounds that supply essential growth stimulating inorganic salts as well as vitamins and necessary co-factors. Glycerol is provided as a source of carbon and energy for the tubercle organisms. Sodium citrate is converted to citric acid, which holds the inorganic cations in solution. Casein hydrolysate is incorporated into the 7H11 Agar as a growth stimulant for strains of drug resistant Mycobacterium tuberculosis . (2,8) Malachite green is added as a selective agent, which partially inhibits the growth of other bacteria. Biotin helps stimulate the revival of damaged organism as well as being involved in a variety of carboxylation and decarboxylation reactions. 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 microcolony method, first described by Welch, et al., in 1993, utilizes Middlebrook 7H11 Agar in a plate with a smaller fill amount than the standard plate. (12) In this procedure, plates are inoculated with a clinical specimen, sealed, incubated, and examined microscopically at regular intervals for the presence of Mycobacterium colonies. Welch, et al., also discovered that the conventional macroscopic method required an average of 23 days until detection of colonies was possible. However, the microcolony method on thinly poured 7H11 Agar plates detected the presence of colonies in an average of 11 days. (12) In addition to reducing the interval before colonies are detected, the microcolony method allows for the easy detection of mixed clinical specimens from mycological infections, and may yield a presumptive identification. (12)

FORMULA

Ingredients per 900ml of deionized water:*

Disodium Phosphate 1.5gm
Monopotassium Phosphate 1.5gm
Pancreatic Digest of Casein 1.0gm
L-Glutamic Acid 0.5gm
Ammonium Sulfate 0.5gm
Sodium Citrate 0.4gm
Magnesium Sulfate 50.0mg
Ferric Ammonium Citrate 40.0mg
Malachite Green 1.0mg
Pyridoxine 10.mg
Biotin 0.5mg
Glycerol 5.0ml
Agar 15.0gm

OADC Enrichment:
Bovine Albumin 5.0gm
Beef Catalase 4.0gm
Dextrose 2.0gm
Sodium Chloride 0.85gm
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 (CDC), inoculate the Middlebrook 7H11 Agar with specimen, after decontamination and neutralization. Consult listed references for methods.(1-3,6,7,11)

2. After inoculation, fit the plate with a MycoSeal™ (Cat. no. SS9225). The MycoSeal™ allows for penetration of CO2 to the plate, yet prevents excess moisture loss and dehydration of the plated media.

3. Incubate medium in a 5-8% CO2 atmosphere at 35-37ºC., for up to four weeks. Protect from light.

4. Examine the sealed plates microscopically twice a week, for up to four weeks. To read plates, 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 40X through the first two streaked quadrants. When detected, colonies are examined at 100X-400X to determine colony morphology. Microcolonies are characterized in descriptive terms relating to their margin and consistency.(12)

5. Consult appropriate references for recording the number of colonies, colony morphology, and for aid in the biochemical identification of acid-fast bacilli.(1,2,6,11)

INTERPRETATION OF RESULTS

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

LIMITATIONS

Middlebrook 7H11 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 loops, slides, decontamination supplies, MycoSeals™ (Cat. no. SS9225), applicator sticks, pipets, incinerators, CO 2 incubator, 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 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 7H11 Agar should appear clear, slightly opalescent, and light amber with a green hue in color.

M. kansasii growing on Middlebrook 7H11 Agar

Mycobacterium kansasii Group I (ATCC ® 12478) colonies growing on Middlebrook 7H11 Agar (Cat. no. SP57). Incubated in CO 2 for 12 days at 35ºC.

M. scrofulaceum growing on Middlebrook 7H11 Agar

Mycobacterium scrofulaceum Group II (ATCC ® 19981) colonies growing on Middlebrook 7H11 Agar (Cat. no. SP57). Incubated in CO 2 for 12 days at 35ºC.


M. intracellulare growing on Middlebrook 7H11 Agar

Mycobacterium intracellulare Group III (ATCC ® 13950) colonies growing on Middlebrook 7H11 Agar (Cat. no. SP57). Incubated in CO 2 for 12 days at 35ºC.

M. fortuitum growing on Middlebrook 7H11 Agar

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Middlebrook 7H11 Agar (Cat. no. SP57). Incubated in CO 2 for 12 days at 35ºC.



Middlebrook 7H11 Agar

Uninoculated plate of Middlebrook 7H11 Agar (Cat. no. SP57).




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.


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032116gr