Middlebrook 7H11 Agar Base w/o Malachite Green

Intended Use

Recommended for isolation, cultivation and determination of antimicrobial susceptibility of Mycobacteria.

Composition

Final pH (at 25°C): 6.6±0.2

Formula adjusted, standardized to suit performance parameters

Directions

Suspend 10.25 grams in 450 ml purified/distilled water containing 2.5 ml glycerol. 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 rehydrated contents of one vial of Middlebrook OADC Growth Supplement (FD018). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Middlebrook 7H11 Agar Base w/o Malachite Green is based on Middlebrook 7H11 Agar which is a modification of Middlebrook 7H10 Agar (1) used for the isolation, cultivation and sensitivity testing of Mycobacterium tuberculosis. It was shown by Cohn et al (2) that the addition of tryptone enhanced the growth of more fastidious Mycobacterium tuberculosis strains which in turn was helpful in drug susceptibility testing (3).

The media consists of many inorganic salts which help for growing Mycobacteria. Citric acid formed from sodium citrate helps in retaining inorganic cations in solution. Glycerol supplies carbon and energy. OADC Supplement contains oleic acid, bovine albumin, sodium chloride, dextrose and catalase. Oleic acid and other long chain fatty acids are metabolized by Mycobacteria. Dextrose is an energy source. Catalase neutralizes toxic peroxides, while albumin protects tubercle bacilli from toxic agents. Malachite green partially inhibits other bacteria.

Type of specimen

Clinical samples : Sputum

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (4,5).

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

• 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 coloured 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

Reaction: Reaction of 2.05% 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 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 in order 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 (4,5).

Reference

1. Middlebrook and Cohn, 1958, Am. J. Public Health, 48:844.
2. Cohn et.al, 1968, Am.Rev.Resp.Dis., 98:295.
3. MacFaddin J.F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore.
4. Isenberg, (Ed.), Clinical Microbiology Procedures Handbook 2nd Edition.
5. 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.