Middlebrook 7H9 Agar Base

Intended Use

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

Composition

**Formula adjusted, standardized to suit performance parameters

Final pH ( at 25°C): 6.6±0.2

Directions

Suspend 9.85 grams in 450 ml purified/distilled water. 1ml glycerol may be added if desired. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add 1 vial of Middlebrook OADC Growth Supplement (FD018). Mix well and distribute as desired.

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 7H9 Agar Base developed by Middlebrook and Cohn (3) is used for cultivation of Mycobacteria. This medium can also be used for sensitivity testing of Mycobacteria and for subculturing of stock cultures on addition of Middlebrook OADC Growth Supplement (FD018) and glycerol. 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 (1,4). 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 (5,6). 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. It require a settling period before pH testing of the prepared medium. If the pH is tested immediately after preparation and is out of specification, retest the medium after sometime to obtain final pH result.

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 1.97% w/v aqueous solution at 25°C. pH : 6.6±0.2

pH: 6.40-6.80

Cultural Response: Cultural characteristics observed with added Middlebrook OADC Growth Supplement (FD018) 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 (5,6).

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. and Cohn M. L., 1958, Am. J. Public Health, 48:844.
4. Finegold S. M., and Baron E. J., 1990, Bailey and Scotts Diagnostic Microbiology, 8th Ed., The C.V. Mosby Co., St. Louis.
5. Isenberg, (Ed.), Clinical Microbiology Procedures Handbook 2nd Edition
6. 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.