Introduction of Mannitol salt agar (MSA)
Mannitol Salt Agar (MSA) is a selective, differential, and indicator medium which is used to isolate and identify Staphylococcus aureus from the clinical specimens.
Other Staphylococcus species and Micrococcus also grow on this medium.
Selective due to is very high salt (7.5%) compared with other media
Gram-Positive Staphylococcus: not fermenting mannitol, the medium does not change color (e.g. epidermidis whereas Staphylococcus aureus yellow color colonies)
Gram-Positive Streptococcus: No growth and also Gram-Negative: No growth
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) grow, they can’t ferment Mannitol, so the color of the medium around the bacterial colony does not change to yellow, it appears pink or color.
Composition of Mannitol salt agar (MSA)
Mannitol Salt Agar (MSA) typically contains:
5.0 gm/L enzymatic digest of casein
5.0 gm/L enzymatic digest of animal tissue
1.0 gm/L beef extract (all of above these three ingredients are sources of nitrogen, vitamin, and carbon)
10.0 gm/LD-mannitol (1%) – Only Carbohydrate source present in the medium
75.0 gm/L Sodium chloride (NaCl)
0.025 gm/L phenol red
15.0 gm/L agar – a solidifying agent
7.4 ± 0.2 at 25°C
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.
- Inoculating loop/ wire
- Bunsen burner
- Test organisms
- Control strains
Testing Procedure/ Using MSA
- Allow the plates/ tubes to warm at 37°C or to room temperature, and the agar surface to dry before inoculating ( in case of agar plate).
- Inoculate and streak the specimen as soon as possible after collection.
- If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface ( use only agar plate).
- Streak for isolation with a sterile loop.
- Incubate plates aerobically at 35-37ºC. for 18-24 hours.
- 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.
As shown above figure.
Keynotes on MSA
- 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.
- 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 those are negative and positive respectively.
- Staphylococcus saprophyticus (coagulase-negative Staphylococcus) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus.