Middlebrook 7H10 Agar Base, Special

Intended Use

Recommended for isolation, cultivation and sensitivity testing of Mycobacterium tuberculosis.

Composition**

Final pH ( at 25°C) 6.6±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 9.75 grams in 450 ml purified/distilled water containing 2.5 ml glycerol. Heat to boiling to dissolve the medium completely. Distribute in 180 ml amounts in flasks and sterilize at 15 lbs pressure (121°C) for 10 minutes. Cool to 45-50°C and aseptically add 50 ml Middlebrook OADC Growth Supplement (FD018). Mix well and pour into sterile screw capped tubes or containers.

Note : Keep prepared medium in the dark before and after inoculation.

Principle And Interpretation

Solid media for Mycobacterial cultivation may be egg-based (Lowenstein Jensen Media) or agar-based (Middlebrook Media) (1). Dubos and Middlebrook (2) developed various formulations containing oleic acid and albumin, which protect Mycobacterium from toxic agents, helping for the growth of tubercle bacilli.

Middlebrook 7H10 Agar Base was formulated as per Middlebrook, Cohn et al (3) reformed the original oleic acid-albumin agar and observed rapid and luxuriant growth of Mycobacterium species, which they called as 7H10. Kubica and Dye (4) reported less contamination on 7H10 Agar than egg-based media commonly used for the cultivation of Mycobacteria.

Middlebrook 7H10 Agar Base, Special was formulated by Middlebrook and Cohn (5). On enrichment with OADC Growth Supplement (FD018) and glycerol, it is recommended for cultivation and sensitivity testing of M. tuberculosis. Middlebrook media consists of many inorganic salts, which help, in growth of Mycobacteria. Citric acid formed from sodium citrate helps in retaining inorganic cations in solution. Glycerol supplies carbon and energy. Middlebrook OADC Growth Supplement (FD018) contains oleic acid, bovine albumin, sodium chloride, dextrose and catalase. Oleic acid and other long chain fatty acids are essential for metabolism of Mycobacteria. Some free fatty acids are toxic to Mycobacteria but albumin binds to those fatty acids and prevents toxic action on Mycobacteria. Dextrose serves as an energy source. Catalase neutralizes toxic peroxides. Malachite green partially inhibits other bacteria (1,6). Mycobacteria are strict aerobes and therefore increased CO2 tension and aerobic conditions must be provided during incubation. Care should be taken while decontamination of the specimen. Also proper specimen collection (sputum and not saliva) should be ensured. Samples should be carefully handled to avoid contamination.

Type of specimen

Clinical samples : Sputum

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (7,8). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Due to nutritional variations, some strains may show poor growth.
2. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
3. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Light yellow to light green homogeneous free flowing powder

Gelling Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium Light amber coloured clear to slightly opalescent gel with greenish tinge forms in Petri plates

Reaction Reaction of 1.95% w/v aqueous solution containing 0.5% glycerol at 25°C. pH : 6.6±0.2

pH 6.40-6.80

Cultural Response Cultural characteristics observed with added Middlebrook OADC GrowthSupplement (FD018) and glycerol after an incubation at 35 - 37°C for 2 - 4 weeks.

Storage and Shelf Life

Store below 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle inorder to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. Dubos R. J. and Middlebrook G., 1947, Am. Rev. Tuberc., 56:334.
3. Middlebrook G., Cohn M. L., Dye W. E., Russel W. F. and Levy D., 1960, Acta. Tuberc. Scand., 38:66.
4. Kubica G. P. and Dye W. E., 1967, Laboratory Methods for Clinical and Public Health Mycobacteriology, PHS Publication No. 1547, U.S. Govt. Printing Office, Washington, D.C.
5. Middlebrook G. and Cohn M. L., 1958, Am. J. Public Health, 48:844.
6. Finegold S. M., and Baron E. J., 1990, Bailey and Scotts Diagnostic Microbiology, 8th Ed., The C.V. Mosby Co., St. Louis.
7. Isenberg, (Ed.), Clinical Microbiology Procedures Handbook 2nd Edition
8. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.