LACTOBACILLI MRS AGAR Principle, Composition, Preparation and Colonies Characteristics

lactobacillus casei on Lactobacilli agar MLT GEEKS

get link De Man, Rogosa & Sharpe, originally developed M.R.S in 1960; It is an alternative non-selective suitable media for most fastidious lactic acid bacteria and is a substitute for Tomato Juice Agar. Lactobacillus MRS is a growth medium recommended for the growth of all Lactobacillus species. This media shows a low degree of selectivity; therefore, secondary accompanying microflora may grow well and compete for nutrients. However, most of these accompanying microorganisms can be inhibited by the addition of various concentrations of selective agents, such as source site cycloheximide, polymyxin, thallium acetate, sorbic acid, acetic acid, or sodium nitrite to the medium.

Importance of Lactobacilli

Lactobacilli species are long, slender, non-sporeforming, gram-positive rods that are generally facultatively anaerobic, most of these grow well with reduced oxygen tension and increased CO2.

Lactobacilli are important microorganisms for the dairy, food, and beverage industry.

  • The microbial spoilage of fruit juice is most commonly due to aciduric organisms such as lactic acid bacteria and yeast.
  • Lactic acid organisms are important to the dairy industry for determining the cause of acid defects in dairy products as well as in evaluating the lactic starter cultures in cured cheese and cultured milks.
  • Species such as Lactobacillus brevis are contaminating organisms in the production of beer that, if present, can be responsible for its spoilage. These lactobacilli damage beer by causing turbidity and a poor flavor due to diacetyl, a strongly flavored by-product of their metabolism.
  • Finally, lactobacilli are used by the vegetable food industry for the fermentation of cabbage to sauerkraut.

Principle of Lactobacillus MRS Agar

MRS medium is based on the formulation of deMan, Rogosa and Sharpe with slight modification. It supports luxuriant growth of all Lactobacilli from oral cavity, dairy products, foods, faeces and other sources.

  • Proteose peptone and beef extract 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.
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Lactobacilli are microaerophillic 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.

Note that Lactobacilli isolated on MRS Agar should be further confirmed by biochemically testing.

Composition of Lactobacillus MRS Agar

Composition Grams/Litres
Proteose peptone 10.000
Beef extract 10.000
Yeast extract 5.000
Dextrose 20.000
Polysorbate 80 1.000
Ammonium citrate 20.000
Sodium acetate 5.000
Magnesium sulphate 0.1000
Manganese sulphate 0.050
Dipotassium phosphate 2.000
Agar 12.000
Final pH ( at 25°C) 6.5±0.2

How to Prepare Lactobacillus MRS Agar

The preparation of Lactobacillus MRS Agar depends on each laboratory workload or the numbers of culture plates available. Anyway, note that a standard Petri dish is equivalent to 20ml when poured completely.

  • Suspend 67.15 grams in 1000 ml distilled water.
  • Heat to boiling to dissolve the medium completely
  • Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  • Mix well and pour into sterile Petri plates.

NB: It is also advisable to incubate a petri dish for 24hour for quality control in order to avoid wastage of resources and ease early detection of contamination after the preparation

Appearance of  Lactobacillus MRS Agar after preparation

Uninoculated MRS agar
Uninoculated MRS agar

Medium to dark amber colored, clear to slightly opalescent gel forms in Petri plates

Type of Inoculums

  • Faeces
  • Foods
  • water samples
  • Vaginal discharge
  • High vaginal smear
  • Milk
  • Brewing (Beer)

Culture Procedure

  • Pick isolated colonies with an inoculating needle and streak on a plate of Lactobacillus MRS Agar
  • incubated at 35ºC. for 24-72 hours in a CO2incubator or under microaerophilic conditions (5% carbon dioxide, 5-10% oxygen). Lower temperatures of 22-25ºC. may be used for psychrotrophic counts or 42ºC. for thermophilic counts.

NB: It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

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Plate reading, Colonies morphology and interpretation

Organisms Growth Characterisics
Lactobacillus delbrueckii Growth; clear colonies
Lactobacillus fermentum Growth; clear colonies
Lactobacillus acidophilus Growth; clear colonies

Reference.

  • deMan J., Rogosa M. and Sharpe M., 1960, J. Appl. Bacteriol., 23:130.
  • Marshall R.T. (Ed.), 1992, Standard Methods for the Examination of Dairy Products, 16th ed., APHA, Washington,D.C
  • Downes F. P. and Ito K., (Eds.), 2001, Compendium of Methods For the Microbiological Examination of Foods, 4th Ed., APHA, Washington, D.C
  • Sabine and Vaselekos, 1965, Nature, 206:960. 5.MacFaddin J.,1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol.1, Williams and Wilkins, Baltimore.

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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