Mannitol Salt Agar (MSA): Introduction, Composition, Principle, Test Requirement, Procedure, Result -Interpretation, Limitation, and Keynotes

Introduction of Mannitol salt agar (MSA)

Mannitol Salt Agar (MSA) is a selective, differential, and indicator medium which is used to isolate and identify S. aureus from clinical and non-clinical specimens. Other Staphylococcus species and Micrococcus also grow on this medium. Selective is due to very high salt (7.5%) compared with other media. Gram-Positive Staphylococcus: not fermenting mannitol, the medium does not change color (e.g. epidermis whereas Staphylococcus aureus yellow color colonies). Gram Positive Streptococcus: No growth and also Gram-Negative: No growth

Composition of Mannitol salt agar (MSA)

Mannitol Salt Agar (MSA) typically contains:

Ingredients	Gms/Litre
Proteose peptone	10.0
Beef extract	1.0
D- mannitol	10.0
Sodium chloride	75.0
Phenol red	0.025
Agar	15.0
Distilled Water	1000 ml
Final pH at 25°C	7.4 ± 0.2

Principle of MSA

Proteose peptone and beef extract are sources of nitrogen, vitamin, and carbon while D-mannitol is the only carbohydrate source present in the medium. A high concentration of salt (75.0 gm/L NaCl) acts as a selective agent. Phenol red is an indicator and agar is a solidifying agent.

Differential due to mannitol (sugar)fermentation (whenever sugar is fermented acid is produced) and changes the pH of the medium to acidic. Similarly, indicator due to having phenol red- at pH levels below 6.8, the medium is yellow color whereas in the neutral pH (6.8 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink. Therefore Coagulase-negative staphylococcus (CoNS) grows, and they can’t ferment Mannitol, so the color of the medium around the bacterial colony does not change to yellow, it appears pink color.

Preparation of Mannitol salt agar

Mannitol salt agar is available in dehydrated powder form by various manufacturers like Mast, Oxoid, Difco, Himedia, and so on.

Follow the instructions of the manufacturer to prepare the medium i.e. 111.0 gm dehydrated medium in 1000 mL distilled water, mix properly and finally Sterilize by autoclaving at 121°C for 15 minutes.

After cooling to 50-55°C, mix well, and dispense it aseptically in sterile Petri dishes. Date the medium and give it a batch number.

Store the plates at 2-8°C preferably in plastic bags to prevent loss of moisture.

Shelf life:  It can be used for several weeks but should be free from any change in the appearance of the medium showing contamination, deterioration, or alteration of pH.

Test Requirements for MSA

• Type of specimen  -As specimen feces, food samples, and water may be used.
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922)

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
6. Examine colonial characteristics.

Colony characteristics in Mannitol salt agar (MSA)

Escherichia coli: Inhibited growth

Staphylococcus epidermidis: Colorless to pink colonies

Staphylococcus aureus: Yellow; may have a yellow halo around colonies.

Micrococci: Red colonies

Mannitol Salt Agar ( MSA) with the growth of Staphylococcus aureus, CoNS and but lacking growth of E. coli

Inhibited growth of Pseudomonas aeruginosa, E. coli but the growth of S. aureus on MSA

Yellow colony of S. aureus on MSA

No growth of Rothia om MSA agar

Keynotes on MSA

1. Some species of Micrococcus (Micrococcus is a normal flora of human skin, mucosa, and oropharynx), such as M. luteus can produce yellow colonies. M. roseus produces pink colonies on MSA. In this condition Micrococcus and Staphylococcus can be further differentiated on the basis of Gram stain reaction, oxidase test(modified), bacitracin (0.04U) susceptibility test, and so on.
2. Enterococcus faecalis and E. faecium (the most common enterococcal species that has been isolated from clinical specimens) are salt-tolerant bacteria. They can ferment mannitol and produce lactic acid that changes indicator producing yellow-colored colonies on MSA. Catalase test can be a key feature to differentiate between Enterococcus and Staphylococcus which are negative and positive respectively.
3. Staphylococcus saprophyticus (coagulase-negative Staphylococcus) may ferment mannitol, producing a yellow halo around colonies in MSA thus resembling S. aureus.

Bibliography

1. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MannitolSaltAgar.htm
2. http://himedialabs.com/TD/M118.pdf
3. https://en.wikipedia.org/wiki/Mannitol_salt_agar
4. https://www.bd.com/resource.aspx?IDX=8979
5. https://www.interchim.fr/ft/J/JH992A.pdf
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC105027/