Lactobacilli MRS Broth

Intended Use

Lactobacilli MRS Agar and Lactobacilli MRS Broth are recommended for use in the isolation, enumeration and cultivation of Lactobacillus species.

Summary and Explanation

Lactobacilli MRS Agar and Lactobacilli MRS Broth are based on the formulations of deMan, Rogosa and Sharpe.¹ These media were shown by the authors to support luxuriant growth of all lactobacilli from oral, fecal, dairy and other sources.

Principles of the Procedure

Lactobacilli MRS Agar and Lactobacilli MRS Broth contain peptone and dextrose. These ingredients supply nitrogen, carbon and other elements necessary for growth. Polysorbate 80, acetate, magnesium and manganese provide growth factors for culturing a variety of lactobacilli. The above ingredients may inhibit the growth of some organisms other than lactobacilli.

User Quality Control

Identity Specifications
Lactobacilli MRS Agar
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:                       7.0% solution, soluble in purified water upon
                                   boiling. Solution is medium amber, clear to
                                   slightly opalescent.
Prepared Appearance:    Medium amber, very slightly to slightly opalescent.
Reaction of 7.0%
Solution at 25°C:           pH 6.5 ± 0.2
Lactobacilli MRS Broth
Dehydrated Appearance: Tan, homogeneous, appears moist.
Solution:                        5.5% solution, soluble in purified water upon
                                    boiling. Solution is medium amber, clear to very
                                    slightly opalescent.
Prepared Appearance:     Medium amber, clear to very slightly opalescent.
Reaction of 5.5%
Solution at 25°C:            pH 6.5 ± 0.2
Cultural Response
Lactobacilli MRS Agar or Lactobacilli MRS Broth
Prepare the medium per label directions. Inoculate Lactobacilli MRS Agar and incubate in a 5% CO2 atmosphere at 35° ± 2°C for 24- 72 hours. Inoculate Lactobacilli MRS Broth and incubate at 35° ± 2°C for 18-24 hours.

Formula

Lactobacilli MRS Agar
Approximate Formula* Per Liter
Proteose Peptone No. 3.............................................. 10.0 g
Beef Extract............................................................... 10.0 g
Yeast Extract............................................................... 5.0 g
Dextrose................................................................... 20.0 g
Polysorbate 80............................................................ 1.0 g
Ammonium Citrate....................................................... 2.0 g
Sodium Acetate........................................................... 5.0 g
Magnesium Sulfate...................................................... 0.1 g
Manganese Sulfate.................................................... 0.05 g
Dipotassium Phosphate................................................ 2.0 g
Agar......................................................................... 15.0 g
Lactobacilli MRS Broth Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water. Lactobacilli MRS Agar – 70 g; Lactobacilli MRS Broth – 55 g.Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Direct Counts
1. To obtain direct counts of lactobacilli, pour 15-20 mL sterile, molten (45-50°C) Lactobacilli MRS Agar into sterile Petri dishes containing 1 mL volumes of diluted test sample.
2. Distribute the inoculum throughout the medium by rotating the plate in one direction and then in the reverse direction.
3. Allow the medium to solidify on a flat surface for 5-10 minutes.
4. Alternatively, plates of Lactobacilli MRS Agar can be used for direct recovery of organisms using the streak inoculation technique.
5. Incubate agar plates at 35°C for 3 days, or at 30°C for 5 days, in an aerobic atmosphere supplemented with carbon dioxide.
Broth Enrichment
1. Samples can be inoculated directly into Lactobacilli MRS Broth.
2. Incubate broth tubes at 35°C for 3 days, or at 30°C for 5 days, in an aerobic atmosphere.
3. Subculture growth in broth tubes to appropriate solid media.

Expected Results

Lactobacilli appear as large, white colonies embedded in or on Lactobacilli MRS Agar or as turbidity in Lactobacilli MRS Broth. Growth may be subcultured onto the appropriate media for use in additional procedures. Refer to appropriate references for recommendations on the culture of Lactobacillus spp.^2,3

Limitations of the Procedure

Organisms other than lactobacilli may grow in these media. Isolates must be confirmed as lactobacilli by appropriate biochemical testing.

*Store at 2-8°C.

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