M-FC Agar Base, Modified

Intended Use

Recommended for detection and enumeration of Klebsiella in water samples by using membrane filtration technique.

Composition

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 49.6 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. Add 10 ml of 1% Rosolic Acid (FD058).Cool to 45-50°C and add 50 mg Carbenicillin. Mix well and pour into sterile Petri plates.

Principle And Interpretation

M-FC Agar Base, Modified is used for the enumeration of Klebsiella using membrane filter technique. Klebsiella are widely distributed in nature, occurring in soil, water, grains, vegetation etc. Wood pulp, paper mills, textile finishing plants and sugarcane processing operations contain large numbers of Klebsiella in their effluents and are often in the predominant coliform in such effluents. M-FC Agar, Modified is formulated as per APHA (1) for enumeration of Klebsiella. M-FC Agar is modified by replacing lactose by inositol and adding Carbenicillin.

Proteose peptone, tryptose and yeast extract in the medium provide necessary nutrients for the growth of faecal coliforms. Inositol is the fermentable carbohydrate and the carbon source in the medium. Bile salts mixture inhibits the growth of contaminating gram-positive microorganisms. Aniline blue is a triphenyl methane dye, which suppresses the growth of many gram-positive microorganisms. Also, along with rosolic acid it forms the indicator system in the medium. Carbenicillin inhibits accompanying coliforms and other bacteria and helps in selective isolation of Klebsiella species. Sample volume is selected to yield 20 to 60 Klebsiella colonies per membrane. This membrane filter is aseptically placed on agar surface. Occasional false positive results may be occurred due to Enterobacter species. Klebsiella colonies appear deep blue to blue grey due to aniline blue present in the medium. Klebsiella colonies will form blue or bluish gray coloured. Presumptive colonies should be further confirmed by performing the biochemical tests.

Type of specimen

Water samples

Specimen Collection and Handling

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (1). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

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 specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

• Occasional false positive results may be occurred due to Enterobacter species.
• Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.

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 greyish yellow homogeneous free flowing powder

Gelling Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium After Addition of 1% Rosolic Acid : Red coloured clear to slightly opalescent gel forms in Petri plates

Reaction Reaction of 4.96% w/v aqueous solution at 25°C. pH: 7.4±0.2

pH 7.20-7.60

Cultural Response

Cultural characteristics observed with added 1% Rosolic Acid (FD058) and 50 mg/l Carbenicillin after an incubation at 35-37°C for 18-24 hours.

Key: *Corresponding WDCM numbers, #Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (2,3).

Reference

1. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
2. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
3. 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.