Mannitol Salt Agar

Intended Use

Mannitol Salt Agar is used for the selective isolation and enumeration of staphylococci from clinical and nonclinical materials.

Meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)^1-3 performance specifications, where applicable.

Summary and Explanation

Koch, in 1942, reported that only staphylococci grow on agar media containing 7.5% sodium chloride.⁴ Chapman further studied this phenomenon in greater detail and concluded that the addition of 7.5% sodium chloride to phenol red mannitol agar results in an improved medium for the isolation of plasmacoagulating staphylococci.⁵ Mannitol Salt Agar is listed User as one of several media recommended for the enumeration of gram-positive bacteria in cosmetics,⁶ clinical specimens,^7-11 and pharmaceutical products.¹ The USP General Chapter recommends Mannitol Salt Agar as a test medium for isolating Staphylococcus aureus in the Microbiological Examination of Nonsterile Products.¹

Principles of the Procedure

Mannitol Salt Agar is a nutritive medium due to its content of peptones and beef extract, which supply essential growth factors, such as nitrogen, carbon, sulfur and trace nutrients. The 7.5% concentration of sodium chloride results in the partial or complete inhibition of bacterial organisms other than staphylococci. Mannitol fermentation, as indicated by a change in the phenol red indicator, aids in the differentiation of staphylococcal species. Agar is a solidifying agent.

User Quality Control

Identity Specifications
Mannitol Salt Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material
                                    and may contain many light to dark red flecks.
Solution:                       11.1% solution, soluble in purified water upon
                                    boiling. Solution is medium to dark, red to
                                    rose; clear to slightly hazy.
Prepared Appearance:     Light to medium rose red, trace orange; clear
                                    to hazy.
Reaction of 11.1%
Solution at 25°C:            pH 7.4 ± 0.2
Cultural Response

Mannitol Salt Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 42-48 hours. Incubate plates with Staphylococcus aureus ATCC 6538 and E. coli ATCC 8739 at 30-35°C for 18-72 hours.

Formula

Mannitol Salt Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 5.0 g
Peptic Digest of Animal Tissue...................................... 5.0 g
Beef Extract................................................................ 1.0 g
Sodium Chloride........................................................ 75.0 g
D-Mannitol................................................................ 10.0 g
Phenol Red............................................................ 25.0 mg
Agar......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 111 g of the powder in 1 L of purified water. 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.

Sample Collection and Handling

For clinical specimens, refer to laboratory procedures for details on specimen collection and handling.^7-11
For cosmetic and pharmaceutical samples, follow appropriate standard methods for details on sample collection and preparation according to sample type and geographic location.^1,13-15

Procedure

Refer to appropriate standard references for details on test methods to obtain isolated colonies from specimens or samples using Mannitol Salt Agar.^1,6,7,11 Incubate plates at 35 ± 2°C in an aerobic atmosphere for 24-48 hours, or as instructed in the standard reference.^1,6,7,11

Expected Results

After the recommended incubation period, the plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Coagulase-positive staphylococci produce growth of yellow colonies with yellow zones. Coagulase negative staphylococci produce small red colonies with no color change to the medium. Micrococcus produce large, white to orange colonies, with no color change to the medium. Most other bacteria will be inhibited.

*Store at 2-8° C.
†QC testing performed according to USP/EP/JP performance specifications.

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