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

An infection control nurse in a hospital ward is conducting an MRSA screening programme. Nasal swabs are collected from all newly admitted patients in the ICU. In the laboratory, these swabs are plated onto a specialised medium and incubated overnight. The next morning, yellow colonies on a red-pink background immediately flag a sample as potentially positive for Staphylococcus aureus — before any further testing is done.

That immediate visual signal — yellow colonies on a pink medium — is the differential principle of Mannitol Salt Agar, and it is the reason MSA became the standard screening medium for S. aureus isolation from specimens with mixed flora.

Mannitol salt agar (MSA) is a selective, differential, and indicator medium used to isolate and identify Staphylococcus aureus from the clinical specimen. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. Members of the genus Staphylococcus can tolerate high salt concentration (7.5%) and grow on mannitol salt agar.

Principle

Mannitol salt agar (MSA) is a selective and differential medium.

Figure: Yellow colonies of S. aureus in Mannitol Salt Agar (MSA).Image source: ASM

Selective medium

Incorporating 7.5% sodium chloride in the medium helps select only those bacteria that can tolerate high salt concentrations. Staphylococci species can tolerate this salt concentration, but other pathogenic bacteria may not. This concentration inhibits the growth of most other gram-positive and gram-negative bacteria. Thus MSA selectively isolates Staphylococcus spp i.e. selective media for Staphylococcus spp.

Differential Medium

Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment mannitol, but coagulase-negative staphylococci (CONS) are not.  So, if that particular specimen contains S. aureus, it ferments mannitol and changes the pH of the medium to acidic. As MSA contains phenol red as a pH indicator, at pH levels below 6.9, the medium is a yellow color. But if CONS grow, they cant ferment mannitol, so the color of the media around the bacterial colony does not change to yellow; it appears pink. So, MSA is also a differential medium.

Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink. Other commonly used media that contain Phenol red as pH indicator are; TSI Agar, urea base agar, and XLD agar.

Uses of Mannitol Salt Agar

1. Primary isolation of Staphylococcus aureus from clinical specimens MSA is used as a selective plating medium for specimens where S. aureus is suspected but where mixed flora is expected to obscure its growth on non-selective media. Common specimens include:

• Skin and wound swabs (particularly from skin infections, abscesses, and burn wounds)
• Nasal and throat swabs
• Food samples in public health investigations

2. MRSA screening MSA supplemented with oxacillin (6 µg/mL) or cefoxitin is used in some laboratories as a screen plate for methicillin-resistant S. aureus (MRSA) from nasal, axillary, groin, and perianal surveillance swabs in hospital settings. Chromogenic MRSA agars have largely replaced this in high-resource laboratories, but oxacillin-MSA remains in use in resource-limited settings.

3. Environmental and food microbiology MSA is used to enumerate staphylococci in food samples (particularly dairy products and processed meats) and environmental surfaces during hygiene audits, where the high salt tolerance of staphylococci is the key selection principle.

4. Teaching and demonstration MSA is one of the clearest visual teaching media in microbiology — the yellow vs. pink colony distinction gives immediate, unambiguous results that demonstrate selective and differential media principles simultaneously.

Composition of Mannitol Salt Agar

Final pH: 7.4 ± 0.2 at 25°C

Note on NaCl concentration: MSA contains 7.5% NaCl — approximately 15 times the salt concentration of normal saline (0.9%). This extreme osmotic environment is lethal or severely inhibitory to most bacteria, including Gram-negative rods and most streptococci. Staphylococci survive because they possess active osmoregulation mechanisms, including accumulation of compatible solutes (such as glycine betaine) that counterbalance external osmotic pressure.

Preparation of Mannitol Salt Agar

You can purchase prepared mannitol salt agar from commercial suppliers, get the powder, and prepare the media in your laboratory. Mannitol salt agar is best prepared from ready-to-use dehydrated powder, available from most suppliers of culture media. The medium is usually used at 11.1 g in every 100 ml of distilled water (concentration may vary depending on the manufacturer).

1. Prepare the medium as instructed by the manufacturer. Sterilize by autoclaving at 121oC for 15 minutes.
2. When the medium has cooled to 50-55oC, mix well, and dispense it aseptically in sterile Petri dishes. Date the medium and give it a batch number.
3. Store the plates at 2-8oC in plastic bags to prevent moisture loss.

Figure: Growth of S. aureus and CONS on MSA (Image source: Pearson Education)

Shelf life: Several weeks, providing there is no change in the appearance of the medium to suggest contamination, deterioration, or alteration of pH.

The medium’s pH should be within the range of pH 7.3 to 7.7 at room temperature.

Staphylococcus saprophyticus (coagulse-negative Staphylococci) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus.

Colony Characteristics in Mannitol Salt Agar

Critical note on coagulase testing from MSA: Do not perform the coagulase test directly from colonies isolated on mannitol salt agar. The high salt concentration of the medium can cause false-positive results in the tube coagulase test. Subculture the suspect colony onto blood agar or TSA and perform the coagulase test from fresh growth (18–24 hours) on the subcultured plate.

Troubleshooting

1. When grown on mannitol salt agar, some Micrococcus (Micrococcus is a normal flora of human skin, mucosa, and oropharynx), such as M. luteus (yellow) can produce yellow colonies. M. roseus (red) produces pink colonies on MSA. Find out the difference between Micrococcus and Staphylococcus here
2. Enterococcus faecalis and Enterococcus faecium(the most common enterococcal species that have been isolated from human infections) are salt-tolerant bacteria. They can ferment mannitol and produce lactic acid, producing yellow-colored colonies on MSA. Catalase testcan help to differentiate between Enterococcus (-ve) and Staphylococcus (+ve).

How to Remember

The name is the mechanism:

• Mannitol — the only sugar in the medium; S. aureus ferments it, most CONS do not
• Salt — 7.5% NaCl selects for salt-tolerant staphylococci; almost everything else is inhibited
• Agar — solidifying agent

The colour logic — two questions, two answers:

The false-positive trap — three organisms to remember:

1. S. saprophyticus — a CONS that can ferment mannitol; yellow colonies; UTI pathogen in young women
2. Enterococcus faecalis — salt-tolerant; ferments mannitol; yellow colonies; ruled out by catalase test
3. Micrococcus — skin commensal; some species produce yellow colonies; ruled out by microdase (modified oxidase) test

A clinical memory anchor: In MRSA screening programmes, the yield from MSA plates is not just yellow colonies — it is the absence of yellow colonies that matters most. A fully pink or colourless plate in a surveillance swab is a negative screen. The value of MSA is that one plate answers two questions (is Staphylococcus present? is it a mannitol fermenter?) without further testing for the majority of samples.

References

1. Kampf, G., Lecke, C., Cimbal, A. K., Weist, K., & Rüden, H. (1998). Evaluation of mannitol salt agar for detection of oxacillin resistance in Staphylococcus aureus by disk diffusion and agar screening. Journal of clinical microbiology, 36(8), 2254–2257. https://doi.org/10.1128/JCM.36.8.2254-2257.1998
2. Han, Z., Lautenbach, E., Fishman, N., & Nachamkin, I. (2007). Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens. Journal of medical microbiology, 56(Pt 1), 43–46. https://doi.org/10.1099/jmm.0.46777-0
3. Tille, P. M. (2017). Bailey and Scott's Diagnostic Microbiology (14th ed.). Elsevier.
4. Clinical and Laboratory Standards Institute (CLSI). (2023). M22: Quality Control for Commercially Prepared Microbiological Culture Media (5th ed.). CLSI.