MRS Broth Test Principle and Procedure

In this MRS broth test principle and procedure post we have briefly explained MRS broth test principle, objectives, requirements, procedure, uses and limitations.

MRS Broth Test Principle and Procedure

In the 1950s, tomato juice agar was used to isolate Lactobacillus from food products. Rogosa recommended a medium for isolation of lactobacilli from oral and fecal specimens, but it was found to be inadequate for recovery of Lactobacillus spp. from dairy products.

A modification of this formulation was developed by de Man, Rogosa, and Sharpe by eliminating tomato juice. This medium became known as MRS broth test medium. It was found to be superior to previous formulations because it supported the growth of slower-growing lactobacilli. Recently, MRS broth test has been used in clinical laboratories to differentiate certain genera of gram-positive cocci on the basis of gas production.

Principle

Gelatin peptone and beef extract provide essential nutrients and amino acids necessary for the growth of bacteria. Yeast extract is a source of B-complex vitamins and enhances bacterial growth. Dextrose provides a ready source of energy, and fermentation of dextrose is detected by gas production in the Durham tube.

Dipotassium phosphate aids in the maintenance of osmotic equilibrium. Polysorbate 80 supplies fatty acids required for bacterial metabolism. Ammonium citrate and sodium acetate are selective agents which inhibit the growth of certain organisms, including gram-negative bacteria and molds.

Composition

Media Composition: Dextrose – 20.0   g , Ammonium Citrate – 2.0   g, Gelatin Peptone – 10.0 g, Dipotassium Phosphate – 2.0   g, Beef Extract – 8.0 g, Polysorbate 80 – 1.0   g, Sodium Acetate – 5.0   g, Magnesium Sulfate – 0.2   g, Yeast Extract – 4.0   g, Manganese Sulfate – 0.05   g, Demineralized Water – 1000.0 ml, pH 6.2 ± 0.2 at 25°C.

Plate Preparation

In one litre of distilled water, dissolve 52.25 gms of the medium. By heating with regular agitation, thoroughly combine the ingredients and dissolve them.

Boil for one minute, or until the mixture is completely dissolved. Fill appropriate containers with the mixture and sanitise in an autoclave at 121°C for 12 minutes. The prepared medium should be kept between 2 and 8°C.

The colour is bright amber. The dehydrated medium should be beige in colour, uniform, and free-flowing. If there are any physical changes, the medium should be discarded.

1. Use an isolated colony from an 18-24 hour culture to inoculate MRS broth test medium. Alternatively, a 0.1 ml aliquot from a pure culture in Todd Hewitt Broth for 18-24 hours can be employed.

2. Inoculate MRS broth test with DT to determine gas generation. Incubate aerobically for up to 7 days at 33-37°C.

3. Keep an eye out for signs of growth and/or gas production. For definite identification of Lactobacillus and some gram-positive cocci.

Interpretation

MRS Broth Test

Left: Lactobacillus fermentum ATCC 9338, Centre: Escherichia coli ATCC 25922, Right: Uninoculated tube (Control)

Gas Production

Gas production shown by a bubble in the Durham tube, growth with considerable turbidity of the broth (Leuconostoc spp.) Turbidity as a result of growth, no gas production (Lactobacillus spp).

Growth

Positive: Turbidity in medium, growth on subculture, Negative: No turbidity and no growth on subculture.

Applications

1. MRS broth test utilised to identify some Lactobacillus and Leuconostoc species that create gas. MRS broth test is part of a series of confirmatory tests on MRS Agar-isolated organisms.

2. Lactobacilli are commonly found in the oral cavity, dairy products, meals, faeces, and other places.

Limitations

1. Because organisms other than lactobacilli can thrive in this medium, biochemical tests to validate lactobacilli isolates should be undertaken.

2. Because of the different nutrients in the media, some strains may develop badly or not at all.

3. For comprehensive identification, biochemical, immunological, molecular, or mass spectrometric testing should be done on colonies from pure culture.

Further Readings

Reference