Summary :
Mannitol Salt Agar (MSA) is a commonly used culture media in microbiology laboratories. It has several applications and is particularly useful for the isolation and identification of Staphylococcus species. In this article, we explore its principle, its preparation, its interpretation and its different uses.
◉ Overview
Mannitol Salt Agar, or Chapman Agar, is a selective culture media used for the isolation, enumeration and differentiation of Staphylococcus from clinical, food, antiseptic and cosmetic samples.
This medium is both selective and differential agar :
- MSA's high salt concentration creates a selective environment that inhibits the growth of many bacteria except for salt-tolerant Staphylococcus species.
- It makes it possible to differentiate Staphylococcus species according to their capacity to ferment mannitol.
The interpretation of results on Mannitol Salt Agar plates involves observing colony growth and color changes (from red to yellow) to presumptively identify Staphylococcus aureus. Further confirmatory tests are typically performed to definitively identify the species.
Mannitol Salt Agar (@lablife)
◉ Principle of Mannitol Salt Agar
The principle of Mannitol Salt Agar revolves around its selectivity and differential properties, which allow for the isolation and identification of Staphylococcus species, particularly Staphylococcus aureus.
- The selectivity of this medium is based on the presence of sodium chloride (7.5%) which inhibits most Gram negative and Gram positive bacteria. Staphylococcus species, on the other hand, can tolerate this high salt concentration and grow on MSA.
- The differentiation is based on the ability or not to ferment the mannitol (the only sugar in the medium). If there is fermentation, this induces acidification which leads, at pH levels below 6.9, to a yellow coloration of the medium in the presence of phenol red (pH indicator).
S. coagulase negative (left) and S. aureus (right).(8)
Note :
- In clinical samples, mannitol positive isolates are suggestive of Staphylococcus aureus and should be further tested.
- A non-fermenting bacteria that resists the high salt concentration results in a red to pink area due to the degradation of the peptone
◉ Preparation / Composition
☰ For 1 liter of medium:
Composition Mannitol Salt Agar |
|||
|---|---|---|---|
| Ingredients | gram / liter | ||
| Peptone | 10 g | ||
| Beef meat extract | 1 g | ||
| Sodium chloride | 75g | ||
| Mannitol | 10g | ||
| Phenol red | 0,025g | ||
| Agar | 15g | ||
| pH final | 7,4 ± 0,2 | ||
Dehydrated Mannitol Salt Agar
Dehydrated medium
- Suspend 111 g of the powder in 1 liter of distilled or deionized water.
- Mix well.
- Heat to boil for 1 minute shaking frequently until completely dissolved
- Sterilize in autoclave at 121°C for 15 minutes.
Prepared medium
- Melt the content of the bottle in a water bath at 100°C (loosing the cap partially removed) until completely dissolved.
- Then screw the cap and check the homogeneity of the dissolved medium, if it is the case turning the bottle upside down.
- Cool at 45-50°C, mix well avoiding foam formation and aseptically distribute into Petri dishes.
Mannitol Salt Agar in bottles
◉ Interpretation
Staphylococci aureus form lush, pigmented colonies surrounded by a yellow halo due to the fermentation of mannitol. Non-pathogenic staphylococci usually form small red colonies which do not change the color of the medium.
The change in color of the medium demonstrates the fermentation of mannitol, NOT the color of the colony. This is particularly important because many micrococci are pigmented.
Several species of Staphylococcus other than S. aureus are positive for mannitol and produce yellow colonies surrounded by yellow areas (eg S. capitis, S. xylosus, S. cohnii, S. sciuri, S. simulans ..). Therefore, further biochemical testing is needed to identify S. aureus or other species.
Chapman with Oxacillin can be used to screen nasal specimens for MRSA because the 7.5% salt and 6 g of oxacillin in this medium inhibits most other organisms that normally colonize the nostrils.
The addition of 5% v / v of egg yolk emulsion allows the detection of the lipase activity of staphylococci as well as the fermentation of mannitol. The salt cleans the egg yolk emulsion and the production of lipase is detected as a yellow opaque area around the colonies.
MicrobeLibrery: (A).Staphylococcus aureus: large yellow halo around growth indicates fermentation of mannitol.
(B). Staphylococcus epidermidis: Growth but not color change to the media indicating no fermentation of mannitol.
(C). Staphylococcus saprophyticus: small yellow halo around growth indicates fermentation of mannitol. (10% of S. saprophyticus ferment mannitol)
(D). E. coli: no growth. Inhibited by the 7.5% NaCl.
- Other genera such as Micrococcus elutes and Micrococcus roses produce yellow and pink colonies respectively
- Enterococcus faecalis and Enterococcus faceium are also salt tolerant and can ferment mannitol to produce yellow colonies on agar
- Group D streptococci can grow on this medium and show slight fermentation of mannitol. However the colonies are small and can easily be differentiated from staphylococci by Gram stain or by the catalase test.
- Inoculated dishes that are stored in the refrigerator may show color loss over time.
Sources
- ASM : Mannitol Salt Agar Plates Protocols
- Bio-rad : CHAPMAN - MANNITOL SALT AGAR
- Liofilchem® : Mannitol Salt Agar
- BD Mannitol Salt Agar
- ASM MicrobeLibrery
- mycrobe.org : Mannitol Salt Agar (MSA)
- Color Atlas of Medical Bacteriology
- University Ave- MANNITOL SALT AGAR (MSA)