Mannitol Salt Agar (MSA)- Composition, Principle, Preparation, Results, Uses

Staphylococcus aureus is a gram-positive, spherical bacterium that belongs to the family Staphylococcaceae. It is one of the most common causes of skin and soft tissue infections, such as boils, abscesses, impetigo, and cellulitis. It can also cause more serious infections, such as pneumonia, endocarditis, osteomyelitis, septicemia, and toxic shock syndrome. Some strains of S. aureus produce toxins that can cause food poisoning, scalded skin syndrome, and enterotoxin-mediated diarrhea.

S. aureus is a facultative anaerobe that can grow in the presence or absence of oxygen. It is catalase-positive and coagulase-positive, meaning that it can produce enzymes that break down hydrogen peroxide and clot plasma, respectively. These characteristics help to distinguish S. aureus from other staphylococci and other gram-positive cocci.

S. aureus is widely distributed in nature and can be found on the skin and mucous membranes of humans and animals. It can also survive on inanimate objects, such as clothing, bedding, furniture, and medical equipment. S. aureus can be transmitted by direct contact with infected or colonized individuals or by indirect contact with contaminated objects or surfaces.

S. aureus has a remarkable ability to adapt to different environments and resist various antimicrobial agents. It can acquire resistance genes through horizontal gene transfer or mutation. Some strains of S. aureus are resistant to multiple antibiotics, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). These strains pose a serious threat to public health and require special infection control measures.

S. aureus can be identified by various laboratory methods, such as culture, microscopy, biochemical tests, serological tests, and molecular techniques. One of the commonly used media for the isolation and differentiation of S. aureus is Mannitol Salt Agar (MSA), which will be discussed in detail in the following sections.

Mannitol Salt Agar (MSA) is a selective and differential medium that allows the growth and differentiation of staphylococci from other bacteria. The principle of MSA is based on the following characteristics of staphylococci:

• Staphylococci are able to tolerate high concentrations of salt (7.5%) that inhibit the growth of most other bacteria. Salt acts as a selective agent that creates a hypertonic environment for the bacterial cells, causing them to lose water and shrink. Only staphylococci and some halophilic marine bacteria can survive in such conditions.

• Staphylococci can ferment mannitol, sugar alcohol to produce acid. Mannitol is the sole source of carbon and energy in MSA. When mannitol is fermented, the pH of the medium drops, and the color of the indicator phenol red changes from red to yellow. This allows the differentiation of mannitol-fermenting staphylococci (such as S. aureus) from non-mannitol-fermenting staphylococci (such as S. epidermidis). Phenol red is red at pH above 8.4 and yellow at pH below 6.8.

• Staphylococci can produce lipase, an enzyme that hydrolyzes fats and oils. Lipase activity can be detected by adding egg yolk emulsion to MSA. Egg yolk contains lecithin and cholesterol, which are substrates for lipase. When lipase breaks down these substrates, it produces glycerol and fatty acids that clear the egg yolk emulsion and form a yellow opaque zone around the colonies.

Therefore, MSA can be used to isolate and identify staphylococci based on their salt tolerance, mannitol fermentation, and lipase production.

Sodium chloride, also known as table salt, is a key ingredient in Mannitol Salt Agar. It serves two main purposes: to create a hypertonic environment and to enhance the lipase activity of staphylococci.

A hypertonic environment is one where the concentration of solutes (such as salt) is higher outside the cell than inside. This causes water to move out of the cell by osmosis, resulting in cell shrinkage and dehydration. Most bacteria cannot tolerate such high salt concentrations and are inhibited from growing on MSA. However, staphylococci are halotolerant, meaning they can survive and grow in salty conditions. This makes MSA a selective medium that allows the isolation of staphylococci from other bacteria.

Sodium chloride also enhances the lipase activity of staphylococci. Lipase is an enzyme that breaks down fats into fatty acids and glycerol. Staphylococci produce lipase to digest the fats in the egg yolk emulsion that can be added to MSA. The salt clears the egg yolk emulsion and allows the detection of lipase production as a yellow opaque zone around the colonies. This is an additional test that can help differentiate between different species of staphylococci based on their ability to produce lipase.

Therefore, sodium chloride plays an important role in MSA by creating a selective and differential medium for staphylococci. It inhibits most other bacteria and enhances the lipase activity of staphylococci, making MSA a useful tool for microbiological testing.

Mannitol is a sugar alcohol that can be fermented by some bacteria to produce acid. In Mannitol Salt Agar (MSA), mannitol serves as a differential factor that distinguishes between different species of staphylococci based on their ability to ferment it. The fermentation of mannitol lowers the pH of the medium, which is detected by the pH indicator phenol red. Phenol red changes color from red to yellow in acidic conditions. Therefore, mannitol-fermenting bacteria produce yellow colonies with yellow zones around them on MSA, while non-fermenting bacteria produce red or pink colonies with no color change in the medium.

One of the most important mannitol-fermenting bacteria is Staphylococcus aureus, which is a pathogenic species that causes various infections in humans and animals. Staphylococcus aureus can be differentiated from other coagulase-negative staphylococci (CNS) that do not ferment mannitol on MSA. However, some CNS species, such as S. capitis, S. xylosus, S. cohnii, S. sciuri, S. simulans, and others, can also ferment mannitol and produce yellow colonies on MSA. Therefore, additional tests are required to confirm the identity of S. aureus or other species.

Mannitol fermentation is influenced by several factors, such as temperature, incubation time, inoculum size, and oxygen availability. Optimal conditions for mannitol fermentation are 35-37°C for 18-24 hours under aerobic conditions with a moderate inoculum size. Some strains of S. aureus may exhibit a delayed or weak fermentation of mannitol and require longer incubation or higher inoculum size to show a positive reaction. Conversely, some strains of non-fermenting bacteria may produce small amounts of acid from other sources and cause a slight color change in the medium. Therefore, careful observation and interpretation of the results are necessary to avoid false positives or negatives on MSA.

Agar is a polysaccharide extracted from red algae that has the ability to form a gel when dissolved in hot water and cooled. Agar is widely used in microbiology as a solidifying agent for culture media, as it provides a firm and stable surface for bacterial growth. Agar has several advantages over other solidifying agents, such as:

• It is not degraded by most bacteria, so it does not interfere with their metabolism or biochemical reactions.

• It has a high melting point (about 85°C) and a low gelling temperature (about 35°C), which allows easy sterilization and handling of the medium.

• It is transparent and colorless, which facilitates the observation of bacterial colonies and their pigmentation.

• It can be modified by adding different nutrients, indicators, inhibitors, or selective agents to suit different purposes.

Agar is added to Mannitol Salt Agar at a concentration of 1.5%, which gives the medium a suitable consistency and rigidity for bacterial growth. Agar also helps to maintain the pH and osmotic balance of the medium, as it buffers the acid produced by mannitol fermentation and prevents water loss due to the high salt concentration. Agar also allows the diffusion of oxygen and other gases through the medium, which is essential for aerobic bacteria. Agar is an inert and versatile ingredient that makes Mannitol Salt Agar a reliable and effective medium for the isolation and differentiation of staphylococci.

Some staphylococci can produce an enzyme called lipase, which breaks down fats into fatty acids and glycerol. Lipase activity can be detected by adding egg yolk emulsion to the mannitol salt agar medium. Egg yolk contains lecithin, a phospholipid that is hydrolyzed by lipase. The hydrolysis of lecithin results in the release of free fatty acids, which form an opaque zone around the colonies. The salt in the medium clears the egg yolk emulsion and enhances the visibility of the lipase reaction.

The addition of egg yolk emulsion to mannitol salt agar can help differentiate between different species of staphylococci based on their ability to ferment mannitol and produce lipase. For example, Staphylococcus aureus is usually mannitol positive and lipase negative, while Staphylococcus epidermidis is usually mannitol negative and lipase positive. However, some strains of S. aureus can also produce lipase, so a coagulase test is required to confirm the identity of S. aureus.

To prepare mannitol salt agar with egg yolk emulsion, sterile egg yolk emulsion (5% v/v) can be added to the medium after autoclaving and before pouring into Petri dishes. Alternatively, sterile egg yolk emulsion can be added as a drop to each inoculated plate and spread evenly with a sterile loop. The plates should be incubated at 35°C for 24-48 hours and examined for color change and lipase reaction.

Mannitol Salt Agar (MSA) is a selective and differential medium that contains the following ingredients :

• Enzymatic digest of casein: This is a source of nitrogen, vitamins, and carbon for bacterial growth.

• Enzymatic digest of animal tissue: This is another source of nitrogen, vitamins, and carbon for bacterial growth.

• Beef extract: This is also a source of nitrogen, vitamins, and carbon for bacterial growth.

• D-Mannitol: This is the only carbohydrate source present in the medium. It serves as a fermentable substrate for some bacteria, such as Staphylococcus aureus, which can produce acid from mannitol fermentation.

• Sodium chloride: This is the salt component of the medium. It has a high concentration of 7.5%, which inhibits the growth of most bacteria other than staphylococci and some halophilic or halotolerant bacteria.

• Phenol red: This is the pH indicator of the medium. It changes color depending on the acidity or alkalinity of the medium. It is red at neutral pH (6.9 to 8.4), yellow at acidic pH (below 6.9), and pink at alkaline pH (above 8.4).

• Agar: This is the solidifying agent of the medium. It provides a firm surface for bacterial growth and isolation.

The final pH of Mannitol Salt Agar (MSA) is 7.4 ± 0.2 at 25°C.

Mannitol Salt Agar is a commercially available medium that can be purchased as a dehydrated powder or ready-to-use plates. The preparation of Mannitol Salt Agar from dehydrated powder involves the following steps:

• Suspend 111 grams of Mannitol Salt Agar powder in 1000 ml of distilled water in a suitable container. Mix well to dissolve the medium completely.

• Transfer the medium to a flask or bottle and cap loosely. Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.

• If desired, sterile Egg Yolk Emulsion can be added to the medium after autoclaving to a final concentration of 5% v/v. Mix well and avoid bubbles.

• Pour the medium into sterile Petri dishes and allow it to cool to room temperature. The medium should be clear and red in color.

• Store the plates in a refrigerator until use. Do not use plates that show signs of contamination, discoloration, or deterioration.

Alternatively, ready-to-use plates of Mannitol Salt Agar can be purchased from a reputable supplier and stored in a refrigerator until use. Follow the manufacturer`s instructions for handling and storage of the plates.

To inoculate Mannitol Salt Agar plates, use a sterile loop or swab to streak the sample over the surface of the agar. Incubate the plates at 35°C for 24-48 hours in an aerobic atmosphere. Examine the plates for growth and color change of the colonies and the medium.

After inoculating and incubating the MSA plates, the results can be observed by examining the color of the colonies and the medium. The color change is due to the pH indicator phenol red, which turns yellow in acidic conditions and red or pink in neutral or alkaline conditions.

• If the bacterial species can ferment mannitol, it will produce acid as a by-product, lowering the pH of the medium. This will result in yellow colonies surrounded by yellow zones on the MSA plate. An example of a mannitol-fermenting species is Staphylococcus aureus, which is pathogenic and coagulase-positive.

• If the bacterial species cannot ferment mannitol, it will not affect the pH of the medium. This will result in red or pink colonies on the MSA plate, with no color change in the surrounding medium. An example of a non-mannitol-fermenting species is Staphylococcus epidermidis, which is non-pathogenic and coagulase-negative.

• If no bacterial growth is observed on the MSA plate, it means that the species is sensitive to the high salt concentration and cannot grow on this medium. An example of a salt-sensitive species is Escherichia coli, which is a gram-negative bacterium.

The following table summarizes the possible results of MSA:

| Bacterial Species | Growth | Colony Color | Medium Color | Mannitol Fermentation |

|-------------------|--------|--------------|--------------|-----------------------|

| S. aureus | Yes | Yellow | Yellow | Positive |

| S. epidermidis | Yes | Red or pink | Red or pink | Negative |

| E. coli | No | N/A | N/A | N/A |

Figure 1: Examples of MSA results with different bacterial species

Mannitol Salt Agar is a widely used medium in microbiology for the selective isolation and differentiation of S. aureus from clinical samples, such as wound swabs or throat swabs. S. aureus is a gram-positive bacterium that is a common cause of infections, such as skin infections and respiratory tract infections.

Mannitol Salt Agar is also used for the enumeration of staphylococci in food and dairy products. Staphylococci are bacteria that can cause food poisoning and spoilage by producing toxins and enzymes. Mannitol Salt Agar can help detect the presence and number of these bacteria in food samples.

Mannitol Salt Agar is also included in the Bacteriological Analytical Manual for cosmetics testing. Cosmetics are products that are applied to the skin or hair, such as creams, lotions, shampoos, and makeup. These products can be contaminated by bacteria during manufacturing, storage, or use. Mannitol Salt Agar can help identify and quantify bacterial contamination in cosmetics.

Mannitol Salt Agar is also used in the bacteriological examination of swimming pool water, spas, and drinking water using membrane filtration. Swimming pool water, spas, and drinking water can be contaminated by bacteria from human or animal sources, such as feces, urine, saliva, or skin. Mannitol Salt Agar can help detect and isolate the staphylococci that may be present in these water sources.

Mannitol Salt Agar is a useful medium for various applications in microbiology because it can selectively grow and differentiate S. aureus from other bacteria based on mannitol fermentation and salt tolerance. It can also detect the lipase activity of staphylococci by adding egg yolk emulsion to the medium. Mannitol Salt Agar is, therefore, a valuable tool for the diagnosis, prevention, and control of staphylococcal infections and contamination.

Mannitol Salt Agar is a useful medium for the selective isolation and differentiation of Staphylococcus aureus from clinical samples. However, it has some limitations that should be considered when interpreting the results.

• Several Staphylococcus species other than aureus are mannitol positive and produce yellow colonies surrounded by yellow zones on this medium (e.g., S. capitis, S. xylosus, S. cohnii, S. sciuri, S. simulans, and other species). Therefore, further biochemical tests are necessary for the identification of S. aureus or other species.

• Most organisms other than staphylococci are inhibited by the high salt concentration found in Mannitol Salt Agar except for some halophilic marine organisms. Accurate counting may be difficult with molds or spreading colonies. Rare, fastidious microorganisms may not grow on selective media formulations.

• A few strains of Staphylococcus aureus may exhibit a delayed fermentation of mannitol. Negative plates should be re-incubated overnight before discarding.

• Presumptive Staphylococcus aureus must be confirmed with a coagulase test. Coagulase-negative staphylococci may also cause infections and should not be overlooked.

We are Compiling this Section. Thanks for your understanding.