MRS Agar

Description

The quality of the peptones, in addition to the meat and yeast extracts, combines all the necessary growth factors that make MRS medium one of the best media for the cultivation of lactobacilli.

As the selectivity of this medium is low and contaminants tend to grow, subculturing in a (double layer) solid medium, and then in broth, is recommended to increase selectivity. In many cases, growth is encouraged by incubation in a CO₂-enriched atmosphere.

MRS medium is particularly recommended for the enumeration and maintenance of lactobacilli either by the MPN technique in broth, or by inoculation on a plate, overlaying it with a second layer of molten medium. This technique overcomes the need for a CO₂-enriched atmosphere.

Technique

Collect, dilute and prepare samples and volumes as required according to specifications, directives, official standard regulations and/or expected results.

Spread the plate by streaking methodology or by spiral method. Incubate the plates right side up in a CO₂ atmosphere at 30 ± 1 °C for 72 ± 3 h.

(Incubation times longer than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications, etc. This medium can be inoculated directly or after enrichment broth like MRS broth, incubated under microaerophilic conditions to promote lactobacilli enrichment.)

After incubation, enumerate all the colonies that have appeared onto the surface of the agar.

Each laboratory must evaluate the results according to their specifications.

Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor if a diluted sample was streaked. Report results as colony forming units (CFU) per mL or g along with incubation time and temperature.

LC-4109 and LC-4691, tubes and bottles

Melt the medium contained in the tubes (LC-4109) or in the bottles (LC-4691) in a water bath or in a microwave oven, avoiding overheating, before pouring into Petri dishes when cooled to room temperature.

Once solidified on a flat surface, spread the plate by streaking methodology or by spiral method. Incubate the plates right side up aerobically at 30 ± 1 °C for 72 ± 3 h.

(Incubation times greater than those mentioned above or different incubation temperatures may be required depending on the sample, on the specifications, etc.; adequate humidity and presence of carbon dioxide will stimulate the cultures. This medium can be inoculated directly or after enrichment broth like MRS broth, incubated under microaerophilic conditions to promote lactobacilli enrichment.)

Note (LC-4691): the solid media can be melted in different ways: autoclave, bath and, if the customer considers it appropriate, also the microwave. Whenever the microwave option is chosen, it is necessary to take certain safety measures to avoid breaking of the containers, such as loosening the screw cap and putting the bottle or tube in a water bath in the microwave. The fusion temperature and time will depend on the shape of the container, the volume of medium and the heat source. Avoid overheating as well as prolonged heating periods.

LC-4691, bottles

PROTECT FROM LIGHT AT ALL TIMES. AVOID PROLONGED EXPOSURE TO LIGHT.

Physical/chemical control

Microbiological control

Inoculate: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10³-10⁴ (qualitative selectivity).

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Anaerobiosis. Incubation at 30 ± 1 °C for 72 ± 3 h.

Microbiological control according to ISO 11133:2014/Amd 1:2018.

LC-4109 and LC-4691: microbiological control

Melting - pour plates - inoculation: practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10³-10⁴ CFU (qualitative selectivity).

Microaerophilic incubation at 30 ± 1 °C for 72 ± 3 h.

Sterility control

Incubation 48 h at 30-35 °C and 48 h at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Culture Media. CRC Press. Boca Raton, Fla. USA
• CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD, Eds. (2003) Handbook of Culture Media for Food Microbiology. Elsevier Science B.V. Amsterdam
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington DC., USA
• LAWRENCE, D.R. & P.A. LEEDHAM (1979). The detection of acid lactic bacteria. J. Int. Brew. 85:119-121
• ISO Standard 11133:2014 Microbiology of food, animal feed and water. Preparation, production, storage, and performance testing of culture media.
• McFADDIN, J. (1985) Media for the isolation-cultivation-identification-maintenance of medical bacteria. Vol. I. William & Wilkins. Baltimore. USA
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• SMITH, C.E., G.P. CASEY & W.M. INGLEDEW (1987). The use and understanding of media used in Brewing Microbiology. - Update 1987 - Brewer’s Digest 62(10)12-16, 43.
• VAN KEER, C., L. van MELKEBEKE, W VERTRIEST, G. HOOZEE & E. Van SCHOONENBERGHE (1983) Growth of Lactobacillus species on different media. J. Inst. Brew. 89:361-363.