LAB223 MRS Agar (ISO) IMAGE: Lactococcus lactis colonies in LAB223 MRS Agar (ISO) Selective enumeration of mesophilic Lactic Acid Bacteria  Formulated to ISO 15214: 1998 pH 5.7 - no modification required  Complete formulation - no additions or supplements necessary   MRS Agar (ISO) is a medium for the enumeration of mesophilic lactic acid bacteria according to ISO 15214:1998. This medium was originally developed in 1960 by de Man, Rogosa & Sharpe for the cultivation and enumeration of Lactobacillus spp. from various sources and is intended as a substitute for Tomato Juice Agar. This original formulation has been adapted and adjusted to pH 5.7 according to ISO 15214:1998. Nutrition is provided by enzymatic digest of casein, glucose, meat & yeast extracts whilst polyoxyethylenesorbitan monooleate, magnesium and manganese sulphates act as growth stimulants. Selectivity against streptococci & moulds is provided by ammonium citrate and sodium acetate. Used at low pH, ammonium citrate allows growth of lactobacilli whilst inhibiting a number of other organism groups. Although MRS Agar (ISO) is optimised to be selective for Lactobacilli, some growth of Leuconostoc spp. and Pediococci may also occur. Method For Reconstitution Disperse 70 grams of powder in 1 litre of deionised water. Allow to soak for 10 minutes, swirl to mix, then sterilise by autoclaving at 121oC for 15 minutes. Cool to 47°C and mix well before dispensing into sterile Petri dishes. Dry the agar surface before use. Inoculation Using the pour plate method, inoculate 1ml of the test sample (serial dilutions should be used) in to the Petri dish, before pouring over the molten agar. Mix carefully & allow to solidify. Overlays may be used if required. Surface inoculations may also be used. Formulation Enzymatic digest of casein 10.0 Meat extract 10.0 Yeast extract 4.0 Triammonium citrate 2.0 Sodium acetate 5.0 Magnesium sulphate heptahydrate Manganese sulphate tetrahydrate Dipotassium hydrogen phosphate Glucose 0.2 Polyosyethylenesorbitan monooleate Agar Grams per litre Incubation Incubate microaerobically at 30°C for 72 hours + 3 hours. Interpretation Count all colonies. Some Leuconostoc spp. may form large, slimy colonies which may hinder the development of other colonies, thus causing an underestimation of the number of lactic acid bacteria. Due to the possible development of microorganisms other than lactic acid bacteria, it may be necessary in some cases to confirm the colonies obtained using simple techniques such as Gram stain or test for catalase. Amount/ litre 0.05 2.0 20.0 1.08 15.5 70.0 pH 5.7 + 0.1 Code Product Size LAB223-B5 MRS Agar ISO 5kg LAB223-B2.5 MRS Agar ISO 2.5kg LAB223-A MRS Agar ISO 500g LAB223-E MRS Agar ISO 100g Minimum Q.C. organisms (according to ISO/TS 11133) Lactobacillus sakei subsp. sakei ATCC 15521 Lactococcus lactis subsp. lactis ATCC 19435 Escherichia coli ATCC 8739 (inhibited) References de Man, J.C., Rogosa, M and Sharpe, M.E. (1960). A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135. ISO 11133-2:2003/Amd.1:2011(E) Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media – Part 2: Practical guidelines on performance testing of culture media – Amendment 1: test microorganisms for commonly used culture media. ISO 15214:1998 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of mesophilic lactic acid bacteria – Colony count technique at 30oC.