Mannitol salt agar (MSA) is designed to isolate staphylococci, especially Staphylococcus aureus, but the list of organisms that actually grow on it is longer and messier than most textbooks suggest. The medium uses a high salt concentration to kill off most bacteria while letting salt-tolerant species thrive, and it uses the sugar mannitol plus a pH indicator to distinguish which of those survivors ferment mannitol and which do not. In practice, though, the plate does not draw a clean line between S. aureus and everything else. Several other staphylococcal species, enterococci, and even some spore-forming bacteria can grow on MSA, and a recently emerging variant of S. aureus itself breaks the classic color rule the medium depends on.
How the Medium Works
MSA contains roughly 7.5% sodium chloride, which is about ten times saltier than most general-purpose growth media. That salt concentration is lethal or inhibitory to most gram-negative bacteria and many gram-positive ones, effectively narrowing the field to organisms that can handle extreme osmotic stress. S. aureus survives in part by accumulating small molecules called compatible solutes, such as glycine betaine and proline, that help the cell maintain water balance without disrupting its internal chemistry.1PubMed Central. Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline, and taurine Other salt-tolerant organisms use similar tricks, which is why MSA is not as exclusive as its name implies.
The differential side of the plate comes from mannitol and the pH indicator phenol red. When an organism ferments mannitol, it produces acid, which drops the local pH and turns the agar around the colony from its resting red-pink color to bright yellow. Organisms that grow but do not ferment mannitol leave the agar unchanged or slightly pink. The classic teaching is straightforward: yellow colonies equal S. aureus, pink or red colonies equal other staphylococci. The reality is considerably more complicated.
The Primary Target and Why It Usually Works
Staphylococcus aureus is the organism MSA was built to find. In clinical labs, food safety testing, and environmental screening, the expected result is that S. aureus grows as round, golden-yellow colonies surrounded by a yellow halo where mannitol fermentation has acidified the medium.2Natural Science. Detection of Pathogenic Bacteria Staphylococcus aureus and Salmonella sp. from Raw Milk Samples of Different Cities of Pakistan For the vast majority of S. aureus strains, this works reliably. One large analysis of over 1,800 strains found that about 97.8% fermented mannitol as expected.3Nature / Scientific Reports. Non-mannitol-fermenting Staphylococcus aureus strains: a new perspective on diagnostic challenge That high percentage is why the medium has remained a workhorse for decades, especially in labs without access to more advanced identification technology.
The yellow color change is the key diagnostic feature. When you streak a clinical swab or food homogenate onto MSA and see bright yellow colonies after 24 to 48 hours of incubation at around 37°C, the working assumption is that you are looking at S. aureus. From there, most protocols call for a confirmatory test, typically a coagulase test, to verify the identification before reporting the result. MSA on its own is a screening tool, not a definitive answer.
Coagulase-Negative Staphylococci That Mimic the Real Thing
One of the biggest practical problems with MSA is that several coagulase-negative staphylococci (CoNS) also grow on the medium and ferment mannitol, producing yellow colonies that look identical to S. aureus. A study that collected 171 CoNS strains from laboratories across Nigeria found that all of them grew on MSA and fermented mannitol, spanning 13 different species. These strains had been previously misidentified as S. aureus based on their MSA appearance alone.4PubMed. Comparison of growth on mannitol salt agar, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, VITEK 2 with partial sequencing of 16S rRNA gene for identification of coagulase-negative staphylococci The conclusion was blunt: MSA could not differentiate between S. aureus and these CoNS species.
The species involved are not obscure. Research on diabetic foot infections identified mannitol-fermenting CoNS belonging to Staphylococcus haemolyticus, S. xylosus, S. lugdunensis, S. saprophyticus, S. hominis, and others.5International Journal of Research in Pharmaceutical Sciences. Mannitol Fermenting Methicillin-Resistant Coagulase Negative Staphylococci Isolated From Diabetic Foot Infections Some of these, like S. lugdunensis, are clinically significant pathogens in their own right. If a lab relies on MSA color alone without confirmatory testing, it can easily report a CoNS infection as S. aureus, leading to the wrong antibiotic choice or an inflated estimate of S. aureus prevalence in a facility.
This problem is especially acute in resource-limited settings. Labs that cannot afford automated identification systems or molecular testing often depend heavily on culture-based media like MSA. The misidentification rate in those environments can be significant, and it has real consequences for patients and for surveillance data.
Non-Staphylococcal Organisms That Tolerate the Salt
The 7.5% NaCl barrier is effective against most bacteria, but “most” is not “all.” Several genera outside the staphylococci have enough salt tolerance to colonize MSA plates, creating unexpected growth that can confuse results.
Enterococci are the most commonly encountered interlopers. These bacteria are naturally adapted to harsh environments, including the high-bile, high-salt conditions of the intestinal tract. A study examining wild boar samples confirmed that Enterococcus species are salt tolerant and able to grow on MSA.6PubMed Central. A Preliminary Study on Antimicrobial Susceptibility of Staphylococcus spp. and Enterococcus spp. Grown on Mannitol Salt Agar in European Wild Boar (Sus scrofa) Hunted in Campania Region-Italy Enterococcal colonies on MSA can sometimes ferment mannitol, producing color changes that overlap with the expected S. aureus appearance. In environmental or veterinary samples, where enterococci are abundant, this can be a meaningful source of false positives.
Beyond enterococci, a comparison of six selective staphylococcal media found that strains of Bacillus, Corynebacterium, and Micrococcus grew on all six media tested, including MSA.7Journal of Food Protection. A Comparison of Six Selective Media for the Enumeration and Isolation of Staphylococci Bacillus species are spore-formers found widely in soil and food; their spores are notoriously hard to kill, and some vegetative cells handle high salt without difficulty. Micrococcus species are common skin commensals that look morphologically similar to staphylococci under the microscope, making them easy to confuse at the colony level. Corynebacterium species are also normal skin flora.
None of these non-staphylococcal organisms are the “target” of MSA, and experienced microbiologists can often distinguish them by colony morphology, Gram stain, or catalase testing. But in high-throughput screening, or when plates are read by less experienced personnel, they add noise that can lead to miscounts or misidentifications.
When S. aureus Itself Breaks the Color Rule
Perhaps the most surprising recent development is the emergence of S. aureus strains that do not ferment mannitol at all. These produce intensely pink colonies on MSA rather than the expected yellow, making them look like harmless coagulase-negative staphylococci to anyone reading the plate by color.
One investigation found that prior to 2020, all 322 clinical S. aureus isolates tested grew yellow on MSA, exactly as expected. After 2020, roughly 10% of clinical isolates produced intensely pink colonies. These hyper-pink strains were missing the gene for mannitol-1-phosphate dehydrogenase, a key enzyme in the mannitol fermentation pathway.8PubMed Central. Observations on emergence of mannitol-use-deficient Staphylococcus aureus The pink color was not the pale pinkish-red typically seen with CoNS but a distinctly strong pink that stood out visually. The researchers noted that this newly emerging clone could easily be confused with CoNS on MSA, potentially causing labs to dismiss a genuine S. aureus isolate as a contaminant or commensal.
A separate analysis of over 1,800 strains estimated that about 2.2% of S. aureus isolates did not ferment mannitol on MSA.3Nature / Scientific Reports. Non-mannitol-fermenting Staphylococcus aureus strains: a new perspective on diagnostic challenge The discrepancy between 2.2% and 10% likely reflects different study populations and time periods, but both figures point to the same concern: relying on mannitol fermentation as the sole marker for S. aureus on MSA has a real and possibly growing blind spot. If this genetic variant continues to spread, the medium’s differential value erodes further.
This matters most in settings where MSA results are not followed up with coagulase testing or molecular confirmation. A pink colony on MSA gets mentally filed as “not S. aureus,” and the actual pathogen walks out the back door undetected.
Modified MSA for MRSA Screening
One of the most common adaptations of MSA in clinical practice is adding an antibiotic to screen specifically for methicillin-resistant Staphylococcus aureus (MRSA). The logic is simple: MSA already selects for staphylococci, so adding an antibiotic that kills methicillin-susceptible strains should leave only MRSA growing on the plate.
The most studied version adds cefoxitin, a cephalosporin antibiotic, at a concentration of 4 mg per liter. In one evaluation using a well-characterized collection of MRSA strains, including strains with low-level resistance, this MSA-cefoxitin combination supported the growth of about 97% of resistant strains while inhibiting 100% of susceptible ones.9PubMed Central. Mannitol salt agar-cefoxitin combination as a screening medium for methicillin-resistant Staphylococcus aureus This made cefoxitin-based selective media more reliable than the older approach of using oxacillin, which tended to miss certain resistant strains.
A surveillance study comparing different formulations in practice found that MSA with cefoxitin detected about 88% of MRSA isolates, compared to about 75% for MSA with oxacillin.10American Journal of Infection Control. Surveillance Evaluation of oxoid mannitol salt agar with cefoxitin for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from surveillance specimens Neither version catches everything, and some MRSA strains grow slowly on modified MSA, requiring the full 48-hour incubation window before they become visible. But for hospitals running MRSA screening programs on a budget, antibiotic-supplemented MSA remains a practical option that is cheaper than chromogenic agars or molecular PCR-based assays.
An earlier formulation using oxacillin-supplemented MSA was also evaluated for primary MRSA identification and showed a positive predictive value of about 94%, with a false-positive rate around 6% and a false-negative rate near 1%.11PubMed Central. Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus Those false positives mostly came from other staphylococci with borderline resistance profiles, reinforcing the theme that MSA-based approaches always require some level of confirmatory follow-up.
What MSA Cannot Grow
For all its limitations in specificity, MSA does a solid job of excluding certain groups. Most gram-negative bacteria, including Escherichia coli, Pseudomonas, Klebsiella, and Salmonella, simply cannot tolerate the 7.5% NaCl environment and will not produce colonies. This makes MSA useful in mixed samples where gram-negative organisms would otherwise overgrow the staphylococci you are trying to find. Streptococci, despite being gram-positive, are also generally inhibited by the salt concentration, which helps separate staphylococci from streptococcal species in clinical specimens from skin and wound cultures.
It is also worth noting that not even all staphylococci thrive on MSA. The comparison study of six selective media found that none of the media tested, MSA included, supported growth of every staphylococcal species in their panel of 37 strains representing 23 species.12ScienceDirect. A Comparison of Six Selective Media for the Enumeration and Isolation of Staphylococci Some less common staphylococcal species grow poorly or not at all on MSA, which means the medium can miss certain strains even within its target genus. For routine clinical work focused on S. aureus and a handful of common CoNS, this gap rarely matters. For research or environmental studies trying to capture the full diversity of staphylococci in a sample, it is a real limitation.
Practical Mistakes and How to Avoid Them
The most common error with MSA is treating it as a definitive identification tool rather than a screening step. A yellow colony on MSA is not confirmed S. aureus. It is a candidate that needs verification, ideally with a coagulase test or, in settings where CoNS identification matters, a more advanced method like mass spectrometry or molecular sequencing. Labs that skip this step risk misidentifying mannitol-fermenting CoNS as S. aureus.
Reading plates too early is another common pitfall. Some organisms, including certain MRSA strains and slower-growing staphylococci, need the full 48-hour incubation period to produce visible colonies or a clear color change. Reading at 24 hours and discarding plates can lead to false negatives. Conversely, leaving plates too long can cause color changes to diffuse across the agar, making it harder to tell which colonies are fermenting mannitol and which are simply sitting in someone else’s acid zone.
Temperature matters too. MSA is typically incubated at 35 to 37°C. Lower temperatures slow growth and can reduce mannitol fermentation enough to weaken the yellow color change, while higher temperatures can inhibit certain staphylococcal species. When the agar is used for food safety testing, sample types and expected flora differ from clinical settings, and labs sometimes adjust incubation parameters accordingly.
Where MSA Fits in Food Safety and Environmental Testing
Outside the clinical lab, MSA is widely used in food microbiology and environmental monitoring. In food testing, it serves as a first-pass screen for S. aureus in dairy products, meats, and ready-to-eat foods. Raw milk testing protocols, for instance, often call for streaking samples onto MSA and incubating for 48 hours before looking for characteristic yellow colonies.2Natural Science. Detection of Pathogenic Bacteria Staphylococcus aureus and Salmonella sp. from Raw Milk Samples of Different Cities of Pakistan Environmental monitoring in hospitals and food processing facilities uses MSA swabs to check surfaces for staphylococcal contamination.
In these contexts, the same caveats about specificity apply, and a few additional ones emerge. Environmental and food samples often contain a wider diversity of salt-tolerant organisms than clinical swabs do. Soil-dwelling Bacillus spores, skin-associated Micrococcus, and intestinal Enterococcus species all show up on MSA plates from these sources. Food safety labs are generally aware of this and use MSA as one step in a multi-test protocol. But quality control audits occasionally find that facilities are reporting staphylococcal counts based on MSA colony numbers without accounting for the non-target organisms that may be inflating the tally.
Why MSA Persists Despite Its Flaws
Given all of these limitations, it is reasonable to ask why anyone still uses MSA at all. The answer is largely economic and practical. MSA is cheap to prepare, easy to store, requires no special equipment to read, and works well enough for its primary purpose: pulling staphylococci out of mixed bacterial populations and flagging likely S. aureus by color. In well-funded clinical labs, it is often supplemented or replaced by chromogenic agars that give more specific color reactions, or by molecular methods like PCR that detect species-specific genes directly. But in community hospitals, veterinary labs, food testing facilities, and labs in lower-income countries, MSA remains a practical first step that does useful work for a fraction of the cost of its alternatives.
The key is understanding what the medium can and cannot tell you. It can tell you that something salt-tolerant grew. It can tell you whether that something ferments mannitol. It cannot tell you with certainty what species you are looking at, and it will occasionally miss the very organism it was designed to find. Treated as one layer of evidence rather than the final word, MSA continues to earn its place on the bench.