MIDDLEBROOK 7H11 AGAR

Cat. no. C36 Middlebrook 7H11 Agar, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. G357 Middlebrook 7H11 Agar, 86x120mm Omniplate, 40ml 10 plates/box
Cat. no. R49 Middlebrook 7H11 Agar, 13x100mm Tube, 3ml Slant 20 tubes/box
Cat. no. X25 Middlebrook 7H11 Agar, 50ml HardyFlask™, 12ml Slant 20 flasks/box
Cat. no. W35 Middlebrook 7H11 Agar, 15x100mm Plate, 28ml 10 plates/bag
Cat. no. C38 Middlebrook 7H11 Selective Agar, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. X28 Middlebrook 7H11 Selective Agar, 50ml HardyFlask™,
12ml Slant
20 flasks/box
Cat. no. J75 Middlebrook 7H11 / 7H11 Selective Agar, 15x100mm Biplate, 16ml/16ml 10 plates/bag
Cat. no. W40 Middlebrook 7H11 Selective Agar, 15x100mm Plate, 28ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Middlebrook 7H11 Agar is recommended for use in the isolation and cultivation of Mycobacterium species.(1,8)

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) In 1968, Cohn incorporated casein hydrolysate into 7H10 medium to stimulate the growth of mycobacteria that would not otherwise grow on this medium. This formulation was then designated as 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 inorganic cations in solution. Casein hydrolysate is incorporated into 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 target organisms. It is also involved in a variety of carboxylation and decarboxylation reactions. OADC Enrichment contains the following additives required for growth: 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.

Middlebrook 7H11 Selective Agar was formulated in 1972 for use in isolating and cultivating Mycobacterium species from specimens containing mixed flora. Using antimicrobics as selective agents, Mitchison, et al. formulated an agar medium (7H10 Base) designed to isolate mycobacteria.(10) This 7H10 basal medium was later modified by Mitchison, et al. using 7H11 as the base. The antimicrobics in 7H11 Selective Agar are as follows: carbenicillin and polymyxin B, which are active against most of the Enterobacteriaceae; trimethoprim lactate, which inhibits Proteus species; and amphotericin B which is active against yeasts.

FORMULA

Ingredients per 900ml of deionized water:*

Glycerol 5.0ml
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 1.0mg
Biotin 0.5mg
Agar 15.0gm

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

Middlebrook 7H11 Selective Agar contains the following additional selective ingredients per liter:

Carbenicillin 50.0mg
Polymyxin B 25.0mg
Trimethoprim 20.0mg
Amphotericin B 10.0mg

Final pH 6.8 +/- 0.3 at 25°C.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8°C 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

For Cat. nos. C36, C38, J75, R49, W35, W40, X25, and X28.

For Cat. no. G357

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.(1-3,6,7,13)

Method of Use:

1. Inoculate the Middlebrook 7H11 Agar with specimen, after decontamination and neutralization, according to test procedures recommended by the Centers for Disease Control (CDC). Consult listed references for methods.(1-3,6,7,13)

2. Incubate medium in a CO2 atmosphere at 35-37°C. Protect from light. Tubed media should be incubated for one week with loosened caps to allow the circulation of CO2 for the initiation of growth. Caps should be tightened after one week in order to prevent dehydration of media.

3. Examine the media within five to seven days, and weekly thereafter for up to eight weeks.

4. Examine plates under light for the appearance of macroscopic growth. For the rapid microcolony method, refer to the procedure outlined on the Instructions for Use (IFU) for Cat. no. SP57, Middlebrook 7H11 Agar, Thin Pour plate.

5. Examine tubes under light and magnifying mirror for macroscopic growth. Record and describe colony morphology on the first day growth is observed.

6. Consult appropriate references for recording the number of colonies and for aid in the biochemical identification of acid-fast bacilli.(1,2,6,13)

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth of Mycobacterium species on this medium.(1-3,6,7,13) Examine and record each type of colony morphology, pigment, and growth rate. Biochemical testing is required for definitive identification.

LIMITATIONS

Middlebrook 7H11 Agar requires incubation in a 5-10% CO2 atmosphere in order to recover mycobacteria. Mycobacteria, for unknown reasons, are not recovered well from candle extinction jars.(7)

Keep inoculated media away from light or excessive heat. Exposure to such conditions 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, 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 CO2** Growth; colonies visible in 2 weeks, mature in 3 weeks
Mycobacterium kansasii
Group I
ATCC ® 12478***

G 21 days 35°C CO2** Growth; colonies visible in 2 weeks, mature in 3 weeks
Mycobacterium scrofulaceum
Group II
ATCC ® 19981***

G 21 days 35°C CO2** Growth; colonies visible in 2 weeks, mature in 3 weeks
Mycobacterium intracellulare
Group III
ATCC ® 13950***

G 21 days 35°C CO2** Growth; colonies visible in 2 weeks, mature in 3 weeks
Mycobacterium fortuitum
Group IV
ATCC ® 6841***

G 21 days 35°C CO2** Growth; colonies visible in 4 days

The above organisms are used for performance testing of Middlebrook 7H11 Agar. The following organisms are additionally tested on Middlebrook 7H11 Selective Agar:

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC ® 25923
B 24hr 35°C Aerobic Partial to complete inhibition
Candida albicans
ATCC ® 10231
B 24hr 35°C Aerobic Partial to complete inhibition

** 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 7H11 and 7H11 Selective Agars 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 7H11 Agar (Cat. no. C36). Incubated in CO2 for 21 days at 35°C.

M. kansasii growing on Middlebrook 7H11 Agar

Mycobacterium kansasii Group I (ATCC® 12478) colonies growing on Middlebrook 7H11 Agar (Cat. no. C36). Incubated in CO2 for 21 days at 35°C.



M. scrofulaceum growing on Middlebrook 7H11 Agar

Mycobacterium scrofulaceum Group II (ATCC ® 19981) colonies growing on Middlebrook 7H11 Agar (Cat. no. C36). Incubated in CO2 for 21 days at 35°C.

M. intracellulare growing on Middlebrook 7H11 Agar

Mycobacterium intracellulare Group III (ATCC ® 13950) colonies growing on Middlebrook 7H11 Agar (Cat. no. C36). Incubated in CO2 for 21 days at 35°C.



M. fortuitum growing on Middlebrook 7H11 Agar

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Middlebrook 7H11 Agar (Cat. no. C36). Incubated in CO2 for 21 days at 35°C.

M. fortuitum growing on Middlebrook 7H11 Agar

Uninoculated tube of Middlebrook 7H11 Agar (Cat. no. C36).

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. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Tille, P.M., 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. Mitchison, D.A., et al. 1972. J. Med. Microbiol.; 5:165-175.

11. Mitchison, D.A., et al. 1973. J. Clin. Pathol.; 26:250-252.

12. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

13. Vestal, A.L. 1975. Procedures of the isolation and identification of mycobacteria. DHEW (CDC 75-8230). Centers for Diseases Control. Atlanta, GA.


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