Mannitol Salt Agar (MSA): Principle, Composition, Preparation, Uses and Colony Characteristics

By Prof Walter Jaoko

Introduction

What Is Mannitol Salt Agar?

Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. It encourages the growth of a group of certain bacteria while inhibiting the growth of others. This medium is important in medical laboratories as one method of distinguishing pathogenic microbes in a short period of time. It contains a high concentration (about 7.5–10%) of salt (NaCl) which is inhibitory to most bacteria – making MSA selective against most Gram-negative and selective for some Gram-positive bacteria (Staphylococcus, Enterococcus and Micrococcaceae) that tolerate high salt concentrations.

Principle

Mannitol Salt Agar is used for the isolation of staphylococci in a laboratory setting. Mannitol Salt Agar is not intended for use in the diagnosis of disease or other conditions in humans. Mannitol Salt Agar was formulated to isolate staphylococci by inhibiting growth of most other bacteria with a high salt concentration. Chapman (the inventor) added 7.5% Sodium Chloride to Phenol Red Mannitol Agar and noted pathogenic strains of staphylococci (coagulase-positive staphylococci) grew luxuriantly and produced yellow colonies with yellow zones. Non-pathogenic staphylococci produced small red colonies with no color change to the surrounding medium.

Mannitol Salt Agar is highly selective and samples from heavily contaminated sources may be streaked onto this medium without danger of overgrowth. Mannitol Salt Agar is recommended for isolating pathogenic staphylococci from samples, cosmetics, and microbial limit tests. Bacteria that grow in the presence of a high salt concentration and ferment mannitol produce acid products, turning the phenol red pH indicator from red to yellow. Typical pathogenic staphylococci ferment mannitol and form yellow colonies with yellow zones. Typical non-pathogenic staphylococci do not ferment mannitol and form red colonies. Enzymatic digest of casein, enzymatic digest of animal tissue, and beef extract provide the nitrogen, vitamins, and carbon in Mannitol Salt Agar. D-Mannitol is the carbohydrate source. In high concentrations, sodium chloride inhibits most bacteria other than staphylococci. Phenol red is the pH indicator. Agar is the solidifying agent.

Composition of Mannitol salt agar (MSA)

• 5.0 g/L enzymatic digest of casein
• 5.0 g/L enzymatic digest of animal tissue
• 1.0 g/L beef extract (all of above these three ingredients are source of nitrogen, vitamin and carbon)
• 10.0 g/LD-mannitol (1%) – Only Carbohydrate source present in the medium
• 75.0 g/L Sodium chloride (NaCl)
• 0.025 g/L phenol red
• 15.0 g/L agar – a solidifying agent
• pH should be 7.4 ± 0.2 at 25°C

Storage

Dehydrated medium should be stored between 10 to 25°C. Once opened, place the container in a dark, dry place. The dehydrated medium should not be used if there is any lump or the colour has changed from the original.

Preparation of Mannitol salt agar

Mannitol salt agar is available in dehydrated powder form by various manufacturers like Mast, Oxoid, Difco, Himedia and so on.Follow the instructions of the manufacturer to prepare the medium i.e.

• 111.0g dehydrated medium in1000mL distilled water, mix properly and finally Sterilize by autoclaving at 121°C for 15 minutes.
• After cooling to 50-55°C, mix well, and dispense it aseptically in sterile petri dishes. Date the medium and give it a batch number.
• Store the plates at 2-8oC preferably in plastic bags to prevent loose of moisture.
• Shelf life:  It can be used for Several weeks but should be free from of any change in the appearance of the medium showing contamination, deterioration, or alteration of pH.

Result Interpretation on Mannitol Salt Agar

Limitations of Mannitol Salt Agar

• Several Staphylococcus species other than aureus are mannitol positive and produce yellow colonies surrounded by yellow zones on this medium (e.g. S. capitis, S. xylosus, S. cohnii, S. sciuri, S. simulans, and other species). Therefore, further biochemical tests are necessary for the identification of S. aureus or other species.
• Most organisms other than staphylococci are inhibited by the high salt concentration found in Mannitol Salt Agar except for some halophillic marine organisms.
• A few strains of Staphylococcus aureus may exhibit a delayed fermentation of mannitol. Negative plates should be re-incubated overnight before discarding.
• Presumptive Staphylococcus aureus must be confirmed with a coagulase test.

Further References

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
2. Tille, P. Bailey and Scott’s Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
3. Chapman, G.H. 1945. J. Bacteriol.; 50:201.
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
5. Koch, F.E. 1942. Zentr. Bakt. Labt. Orig.; 149:122.
6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.
7. Jorgensen, J.H., et al. 2015. Manual of Clinical Microbiology, 11th ed. American Society for Microbiology, Washington, D.C.
8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
9. American Public Health Association. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. APHA, Washington, D.C.
10. APHA Technical Committee on Microbiological Methods for Foods. 2001. Compendium of Methods for the Microbiological Examination of Foods, 4th ed. APHA, Washington, D.C.