MRS Selective Agar Base w/ Clindamycin-Ciprofloxacin

Intended use

Recommended for the selective cultivation of Lactic acid bacteria from food. The composition of this medium are as per the specifications laid down in ISO 20128:2006.

Composition**

Final pH ( at 25°C) 6.5±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Beef extract

Directions

Suspend 65.15 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified / distilled water. 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. Aseptically add rehydrated contents of one vial of Ciprofloxacin Clindamycin Selective Supplement (FD346). Mix well and pour into sterile Petri plates.

Principle And Interpretation

MRS Selective Agar Base has been developed as per specification laid down in ISO 20128 for enumeration of presumptive Lactobacillus acidophilus from milk products (7). Lactobacilli MRS medium is based on the formulation of deMan, Rogosa and Sharpe (2) with slight modification. It supports luxuriant growth of all Lactobacilli from oral cavity (2), dairy products (6), foods (9), and other sources (5).

Proteose peptone and HM peptone B supply nitrogenous and carbonaceous compounds. Yeast extract provides vitamin B complex and dextrose is the fermentable carbohydrate and energy source. Polysorbate 80 supplies fatty acids required for the metabolism of Lactobacilli. Sodium acetate and ammonium citrate inhibit Streptococci, moulds and many other microorganisms. Magnesium sulphate and manganese sulphate provide essential ions for multiplication of lactobacilli. Phosphates provide good buffering action in the media.

Lactobacilli are microaerophilic and generally require layer plates for aerobic cultivation on solid media. When the medium is set, another layer of un-inoculated MRS Agar is poured over the surface to produce a layer plate (5). Lactobacilli isolated on MRS Agar should be further confirmed biochemically. Clindamycin and ciprofloxacin helps in the elimination of contaminating flora.

Type of specimen

Food and Milk products

Specimen Collection and Handling

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (1,7,9,10). 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

1. Due to selective properties, some strains of Lactobacillus may show poor growth.
2. Further biochemical identification must be carried out for confirmation.

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 Cream to light yellow homogeneous free flowing powder

Gelling Firm, comparable with 1.2% Agar gel.

Colour and Clarity of prepared medium Medium to dark amber coloured, clear to slightly opalescent gel forms in Petri plates

Reaction Reaction of 6.51% w/v aqueous solution at 25°C. pH : 6.5±0.2

pH 6.30-6.70

Cultural Response Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours or longer.(with 5% CO2)

Key: * Corresponding WDCM numbers.

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 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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (3,4).

Reference

1. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
2. deMan J., Rogosa M. and Sharpe M., 1960, J. Appl. Bacteriol., 23:130.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
4. 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.
5. MacFaddin J.,1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol.1, Williams and Wilkins, Baltimore.
6. Marshall R.T. (Ed.), 1992, Standard Methods for the Examination of Dairy Products, 16th ed., APHA, Washington, D.C.
7. Milk products -- Enumeration of presumptive Lactobacillus acidophilus on a selective medium -- Colony-count technique at 37 degrees C . ISO 20128:2006 (IDF 192:2006)
8. Sabine and Vaselekos, 1965, Nature, 206:960.
9. Salfinger Y., and Tortorello M.L. , 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
10. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.