Are you familiar with mannitol salt agar (MSA)? Have you ever used Mannitol salt agar in your laboratory? If you have used it, but don’t remember why and when then you are in the right place. In this post, I am discussing MSA, its composition, uses, and the colony of characteristics of organisms that grow on it.
Mannitol salt agar (MSA) is a selective, differential, and indicator medium that is used to isolate and identify Staphylococcus aureus from the clinical specimen.
Mannitol salt agar (MSA) contains:
- Mannitol (1%): Mannitol is one of the major ingredients. Do you remember what is mannitol? It’s sugar.
- Salt (7.5%): Salt is the common ingredient but see the percentage, which is very high compared with other media.
- Agar: the medium contains agar (a solidifying agent).
Composition of Mannitol Salt Agar
- Enzymatic digest of casein (source of nitrogen, vitamin, and carbon)
- Enzymatic digest of animal tissue (source of nitrogen, vitamin, and carbon)
- Beef extract (source of nitrogen, vitamin, and carbon)
- D-Mannitol: Only carbohydrate source present in the medium
- Sodium Chloride
- Phenol Red (Indicator)
Final pH: 7.4 ± 0.2 at 25 oC
(Remember the ingredients in Bold letters)
You can purchase prepared Mannitol Salt Agar from the commercial suppliers or get the powder and prepare the media in your laboratory.
Preparation of Mannitol salt agar
Mannitol salt agar is best prepared from ready to use dehydrated powder, available from most suppliers of culture media. The medium is usually used at a concentration of 11.1 g in every 100 ml distilled water (concentration may vary depending on the manufacturer).
- Prepare the medium as instructed by the manufacturer. Sterilize by autoclaving at 121oC for 15 minutes.
- When the medium has cooled to 50-55oC, mix well, and dispense it aseptically in sterile petri dishes. Date the medium and give it a batch number.
- Store the plates at 2-8oC preferably in plastic bags to prevent loos of moisture.
Shelf life: Several weeks providing there is no change in the appearance of the medium to suggest contamination, deterioration, or alteration of pH.
pH of medium: This should be within the range of pH 7.3 to 7.7 at room temperature.
So what’s the purpose of using salt in mannitol salt agar?
Incorporation of 7.5% sodium chloride in the medium helps to select only those bacteria which can tolerate high salt concentrations. MSA helps to demonstrate the ability of a bacterium to grow in a 7.5% salt environment (growth indicates tolerance for high salt environment – no growth means intolerance). Species of staphylococci are able to tolerate this salt concentration but other pathogenic bacteria may not. This concentration inhibits the growth of most other gram-positive and gram-negative bacteria.
Thus MSA selectively isolates Staphylococcus spp i.e. selective media for Staphylococcus spp.
Why Mannitol (Sugar) is used in Mannitol Salt Agar?
Whenever there is the use of any sugar in any media used in Microbiology, we try to find does a particular microorganism is able to ferment sugar present in this media or not?
For example, in MacConkey Agar we try to find, does a gram negative rod is lactose fermenter or not? In TCBS, we try to identify the isolate on the basis of sucrose fermentation. Vibrio cholerae is able to ferment Sucrose and gives yellow color in TCBS.
Similarly in MSA, we try to find does a particular organism ferments Mannitol or not?
You must be aware by now; all the staphylococci spp are not pathogenic to human. So, next task of microbiologist is differentiating S. aureus from other Staphylococcus spp.
Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment mannitol (It is coagulase test positive) but others (coagulase-negative Staphylococcus) are not. So, if that particular specimen contains S. aureus, it ferments mannitol (whenever sugar is fermented acid is produced) and changes the pH of the medium to acidic. As MSA contains phenol red as a pH indicator, at pH levels below 6.9, the medium is a yellow color. But if coagulase-negative staphylococcus (CONS) grows, they cant ferment mannitol, so the color of the media around the bacterial colony does not change to yellow, it appears pink.
Staphylococcus saprophyticus (coagulse negative Staphylococcus) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling with S. aureus.
So, MSA is also a differential medium.
Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink.
Expected colony characteristics of organisms in Mannitol Salt Agar
- Escherichia coli: Does not grow
- Staphylococcus epidermidis: Colorless to pink colonies
- Staphylococcus aureus: Yellow colonies; may have a yellow halo around colonies.
- When grown on mannitol salt agar some species of Micrococcus (Micrococcus is a normal flora of human skin, mucosa, and oropharynx), such as M. luteus (yellow) can produce yellow colonies. M. roseus (red) produces pink colonies on MSA. Find out difference between Micrococcus and Staphylococcus here
- Enterococcus faecalis and Enterococcus faecium (most common enterococcal species that has been isolated from human infections) are salt tolerant bacteria. They can ferment mannitol and produce lactic acid, producing yellow colored colonies on MSA. Catalase test can help to differentiate between Enterococcus (-ve) and Staphylococcus (+ve).