Mannitol Salt Agar (MSA) is utilized as a differential and selective medium to isolate and detect Staphylococcus aureus in clinical and non-clinical samples. It is recommended for identification and enumeration of coagulase positive Staphylococci that are present in milk, food as well as other specimens. MSA also promotes an increase in the number of particular group of specific bacteria, while limiting the growth of other bacteria.
Principle of Mannitol Salt Agar (MSA)
Mannitol Salt Agar contains beef extract as well as proteose peptone which is very nutritious, as they are growth factors as well as trace nutrients like minerals, vitamins, nitrogen and amino acids that are essential to growth.
The medium has 7.5 percent sodium chloride that results in the complete or partial inhibiting of bacteria that are not staphylococci. Mannitol is the carbohydrate that can be fermented into a source. Its fermentation results in acid production. Staphylococcus aureus is a bacterium that thrives in this medium and ferments mannitol in order to create yellow colonies.
The majority of the coagulase non-positive varieties that comprise Staphylococci and Micrococci don’t ferment mannitol but form tiny colonies in red. The colour of the colonies as well as the medium is because of the reactivity of the phenol red (indicator) with the medium’s pH. Phenol red appears bright red when pH is 8.4 while phenol red turns bright yellow when pH is 6.8.
Agar is the agent that solidifies. Additionally the 5% v/v Egg Yolk Emulsion could be added in a media that allows the detection of the lipase activity of staphylococci and mannitol ferment. The salt is able to clear the egg yolk-emulsion as well as lipase production is seen as a transparent yellow zones within the colonies.
Composition of Mannitol Salt Agar (MSA)
| Ingredients | Gms/Litre |
| Proteose peptone | 10.0 |
| Sodium chloride | 75.0 |
| D- mannitol | 10.0 |
| Beef extract | 1.0 |
| Phenol red | 0.025 |
| Agar | 15.0 |
Final pH 7.4 +/- 0.2 at 25ºC.
Selective medium
The inclusion of 7.5 percent sodium chloride in the medium allows for selection of only the bacteria that are able to endure high salt concentrations. MSA can demonstrate the capacity for a bacterium’s growth in an 7.5 percent salt-based environment (growth signifies tolerance to the high salt conditions but no growth does not mean intolerance). Staphylococci species can take this salt level, but other pathogenic bacteria might not. This salt concentration hinders the growth of a majority of other gram positive and gram negative bacteria. Therefore, MSA specifically isolates Staphylococcus species i.e. specific media specifically for Staphylococcus spp.
Differential Medium
Staphylococci that are pathogenic, i.e. Staphylococcus aureus can ferment mannitol, but other (coagulase-negative staphylococci) are not. Therefore, if a particular specimen is contaminated with S. aureus that ferments mannitol, it alters in the pH. This can be acidic. Because MSA includes the color red phenol as an indicator of pH for pH levels that are less than 6.9 it is colored yellow. However, if coagulase-negative staphylococci (CONS) develops and they are unable to ferment mannitol. As a result, it’s color for the medium surrounding the colony of bacteria is not changed to yellow and appears to be pink. Thus, MSA is also a different medium.
Take note that in the pH range of neutral (6.9 and 8.4) that the hue of the phenol red is red when it is above pH 8.4 the hue of phenol red is pink. Other media commonly used to contain Phenol Red as a pH indicator include TSI Agar Urea base agar, and XLD Agar.
Preparation of Mannitol Salt Agar (MSA)
- Suspend 111 grams of Mannitol Agar in 1000ml distillate water.
- Boil until the medium dissolves completely.
- Sterilize with autoclaving at 15lbs. Pressure (121degC) during 15 minutes.
- If desired, sterile egg Yolk Emulsion (E7899) is able to be added to the final concentration of 5% V/V after autoclaving.
- Put the Mannitol Salt Agar that has been cooled into sterile petri dishes , and allow it to cool to ambient temperature.
Shelf life of Mannitol Salt Agar (MSA):
A few weeks provided there isn’t any changes in the appearance or color of the medium which could suggest the presence of contamination, degradation or change in the pH.
pH of Mannitol Salt Agar (MSA):
It should fall within the pH range of 7.3 up to 7.7 at temperatures at room temperature.
Physical Properties of Mannitol Salt Agar (MSA)
- Appearance: Light yellow to pink homogeneous free flowing powder
- Gelling: Firm,comparable with 1.5% Agar gel
- Colour and Clarity of prepared medium: Red coloured clear to slightly opalescent gel forms in Petri plates
- Reaction: Reaction of 11.1% w/v aqueous solution at 25°C. pH : 7.4±0.2
- pH: 7.20-7.60
Cultural Response: The cultural aspects that are observed following an incubation period of 35-37°C for 18-72 hours. The rate of recovery is considered to be 100% for the growth of bacteria upon Soybean casein digest agar.
Storage of Mannitol Salt Agar (MSA)
The powder is extremely hygroscopic, keep the powder at 10-30 degrees C in a dry area and keep it in the container that was tightly sealed. Keep the prepared plates and bottles in a temperature of 10-25 degrees, far from light. Don’t use the product after the expiry date as indicated on the label, or if the product has any indication of contamination or signs of deterioration.
Test Procedure on Mannitol Salt Agar (MSA)
- Inoculated plates through straight streaking substance to be examined on the surface of the agar.
- Incubate at an aerobic temperature of 35 degrees Celsius +/- 24 to 48 hours.
- The Harmonized USP/EP/JP microbiological method for testing of non-sterile items recommends inoculating the product into Tryptic Soy Broth.
- Subculture on a dish of Mannitol Salt Agar , and then keep incubating at 30-35degC for 18 to 72 hours.
Results and Colony Characteristics in Mannitol Salt Agar (MSA)
| Organisms | Results |
| Staphylococcus aureus | Yellow colonies surrounded by yellow zone |
| Staphylococcus epidermidis | Pink or Red colonies |
| Micrococci | Red colonies |
| Escherichia coli | No growth |
Quality Control on Mannitol Salt Agar
- Inoculum to increase productivity: 10-100 CFU
- Inoculum to be selected For selectivity: CFU 104-106
- Conditions for incubation include aerobically at 35 +2 degC for 24 to 48 hours.
- *30-35degC for 18-72 h (USP/EP/JP Growth Promotion Testing).
Positive Control: Staphylococcus aureus ATCC 6538, Medium-sized yellow colonies
Negative Control: Escherichia coli ATCC 25922, Partial to Complete Inhibition.
Troubleshooting
- If grown on mannitol in agar, certain kinds that belong to Micrococcus (Micrococcus is a common human skin flora that can be found in the mucosa and oropharynx) including M. Lutus (yellow) can form colonies of yellow. The M. roseus (red) produces pink colonies on MSA. Find out the difference among Micrococcus and Staphylococcus here.
- Enterococcus Faecalis and Enterococcus Faecium (the most prevalent enterococcal species which has been identified from human infection) are salt-tolerant bacteria that can tolerate salt. They are able to ferment mannitol and produce lactic acid, resulting in white-colored colonies that are visible on MSA. Catalase tests can help identify the difference between Enterococcus (-ve) as well Staphylococcus (+ve).
Uses of Mannitol Salt Agar (MSA)
- It is used for specific isolation and differentiation of Staphylococcus aureus in clinical samples.
- It also serves to count staphylococci in dairy and food products.
- This medium is also listed within the Bacteriological Analytical Manual for Cosmetics Testing.
- It can also be used for the bacteriological testing of water from swimming pools, spas and drinking waters with membrane filters.
Limitations of Mannitol Salt Agar (MSA)
- There are a variety of Staphylococcus species, other than aureus are mannitol-positive and create yellow colonies, that are surrounded by yellow zones on the medium (e.g. S. capitis, S. the xylosus S. Cohnii S. sciuri, S. simulans and many other species). So, further tests of biochemistry are required to determine S. aureus and other species.
- All organisms, except staphylococci are unable to be inhibited by the large salt concentration within Mannitol Salt Agar except for some marine organisms with halophilic properties.
- Some species of Staphylococcus aureus could exhibit delayed fermentation of the mannitol. Negative plates must be incubated for a few hours before getting rid of.
- Presumptive Staphylococcus aureus has to be confirmed by the blood test for coagulase.
