Mannitol Salt Agar for the isolation of Staphylococcus aureus

 

Mannitol Salt Agar (MSA) is a selective and differential agar medium commonly used in microbiology for the isolation and identification of Staphylococcus aureus, particularly from clinical specimens. It is named after its main components: mannitol (a sugar alcohol) and sodium chloride (salt). Here's how MSA works for the isolation of Staphylococcus aureus.

                       Golden Yellow colonies of Staphylococcus aureus on Mannitol salt agar.

Principle of Mannitol Salt Agar

Selectivity: MSA is selective because it contains a high concentration of salt (7.5-10% NaCl). This high salt concentration creates an environment that is inhibitory to many bacteria but allows for the growth of staphylococci, particularly Staphylococcus aureus. Staphylococci are salt-tolerant, and this characteristic helps in their isolation.

Differential: MSA is also differential because it contains mannitol, a carbohydrate source, and the pH indicator phenol red. Staphylococcus aureus can ferment mannitol, producing acidic by products in the process. This acid production causes the pH of the medium to drop, turning the phenol red indicator from red (alkaline) to yellow (acidic).

Composition of Mannitol Salt Agar (MSA)

Ingredients	Gms/ Litre
Peptone	10
Beef Extract	1
D-Mannitol	10
Sodium Chloride (Salt)	75
Agar	15
Phenol Red	0.025
pH	7.4 ±2

Preparation of Mannitol Salt Agar

Equipment:

1.     Balance for weighing ingredients.

2.      Autoclave for sterilization.

3.      Flasks or containers for mixing and sterilization.

4.      pH meter or pH indicator paper.

5.      Graduated cylinders and beakers for measuring and mixing.

Procedure:

1.      Weigh the ingredients accurately using a balance.

2.      Add the weighed peptone and beef extract to a flask or container.

3.      Dissolve the peptone and beef extract in distilled water while stirring to create a peptone-beef extract mixture. The exact volume of water will depend on the specific recipe or manufacturer's instructions but is typically around 1 liter. Ensure complete dissolution.

4.      Add the measured mannitol to the peptone-beef extract mixture and stir until it is dissolved.

5.      Add the measured sodium chloride (NaCl) to the mixture. The high salt concentration is essential for the selective property of MSA. Stir until the salt is completely dissolved.

6.      Add agar-agar to the mixture to solidify the medium. Agar serves as the solidifying agent. Stir well to ensure even distribution of agar throughout the medium.

7.      Adjust the pH of the medium to around 7.4, which is the typical pH for MSA. You can use a pH meter or pH indicator paper to monitor and adjust the pH if necessary.

8.      Heat the mixture to dissolve the agar and sterilize the medium. This can be done by placing the container with the mixture in an autoclave and sterilizing it at 121°C (250°F) for 15 minutes. Make sure to cap or cover the container loosely to allow for steam release during autoclaving.

9.      After sterilization, cool the medium to approximately 45-50°C (113-122°F).

10. If using phenol red as the pH indicator, add a small amount (typically 0.025 grams) of phenol red powder dissolved in distilled water to the medium. Stir to mix thoroughly.

11. Pour the sterile and molten MSA medium into sterile Petri dishes or containers, as needed, and allow it to solidify.

12. Once solidified, store the prepared MSA plates in a cool, dry place or in the refrigerator until you are ready to use them for bacterial culture.

Result Interpretation on Mannitol Salt Agar

Yellow Colonies: Yellow colonies on MSA are indicative of Staphylococcus aureus. These colonies are yellow because Staphylococcus aureus can ferment mannitol, producing acidic byproducts that lower the pH of the agar. As a result, the pH indicator (typically phenol red) changes from red (alkaline) to yellow (acidic).

Red Colonies: Red colonies on MSA are indicative of staphylococci other than Staphylococcus aureus. These staphylococci do not ferment mannitol, so the medium remains red (alkaline) around their colonies.

Organisms	Results
Staphylococcus aureus	Yellow colonies with yellow zones.
Staphylococci other than S. aureus (e.g. Staphylococcus epidermidis )	Colorless or Red colonies with red zones.
Streptococci	No growth to trace growth.
Micrococci	Large white to orange.
Gram-negative bacteria	No growth to trace growth.

Quality Control on Mannitol Salt Agar

Positive Control: Staphylococcus aureus ATCC 6538, Medium-sized yellow colonies

Negative Control: Escherichia coli ATCC 25922, Partial to Complete Inhibition.

Uses of Mannitol Salt Agar

1.      Mannitol Salt Agar is primarily used for the selective isolation of staphylococci, including Staphylococcus aureus, from various clinical and non-clinical samples. The high salt concentration (7.5-10% NaCl) inhibits the growth of most other bacteria, making it a selective medium for staphylococci.

2.      Mannitol Salt Agar is a differential medium that allows for the identification of Staphylococcus aureus based on its ability to ferment mannitol. Staphylococcus aureus ferments mannitol and produces acidic by products, which cause the pH indicator (phenol red) to change from red (alkaline) to yellow (acidic). Other staphylococci that do not ferment mannitol will keep the medium red.

3.      Mannitol Salt Agar is frequently used in clinical microbiology laboratories to screen for pathogenic staphylococci, especially Staphylococcus aureus, in various patient samples (e.g., wound swabs, nasal swabs, blood cultures). The ability to quickly identify Staphylococcus aureus colonies based on their yellow coloration aids in the early detection and management of staphylococcal infections.

4.      Mannitol Salt Agar can be used to detect and enumerate coagulase-positive staphylococci in food and dairy products. High counts of these bacteria can be an indicator of poor hygiene and potential food contamination.

Limitations of Mannitol Salt Agar

1.      Mannitol Salt Agar is selective for staphylococci and inhibits the growth of many other bacteria due to its high salt concentration. However, it is not specific to Staphylococcus aureus. Other staphylococci that can grow on MSA may produce false-positive yellow colonies if they ferment mannitol.

2.      Different strains of Staphylococcus aureus may vary in their ability to ferment mannitol. Some strains may ferment mannitol more slowly or weakly, leading to delayed or weak acid production. This variability can affect the interpretation of MSA results.

3.      Some non-staphylococcal bacteria, particularly some species of Micrococcus, can also tolerate high salt concentrations and may grow on MSA plates. This can lead to confusion in interpretation if not all salt-tolerant bacteria are staphylococci.

4.      In some environmental or clinical samples, the presence of salt-tolerant contaminants other than staphylococci can lead to overgrowth and make it difficult to isolate the target bacteria.

5.      Mannitol Salt Agar relies solely on the ability of bacteria to ferment mannitol for differentiation. Other important biochemical characteristics of bacteria, such as the production of specific enzymes or the utilization of other substrates, are not assessed with this medium.

6.      In certain cases, Staphylococcus aureus strains may not ferment mannitol or may do so weakly, resulting in false-negative results on MSA plates.

7.      Mannitol Salt Agar requires the use of specialized ingredients like mannitol and high salt concentrations, which can make it more expensive to prepare compared to other media. Additionally, it requires sterilization and may take additional time and resources to prepare.

8.      Mannitol Salt Agar is primarily designed for the isolation and preliminary identification of staphylococci, particularly Staphylococcus aureus. It is not suitable for the isolation and differentiation of other types of bacteria.

References

1.      BD Mannitol Salt Agar. Becton Dickinson GmbH.

2.      Mannitol Salt Agar. HiMedia.

3.      Mannitol Salt Agar (MSA) (Chapman Medium) European Pharmacopoeia, Usp.

4.      Mannitol Salt Agar. Accumix.