Culture Media – Page 7 – Microbe Online

Mannitol salt agar (MSA) is a selective, differential, and indicator medium used to isolate and identify Staphylococcus aureus from the clinical specimen. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. Members of the genus Staphylococcus can tolerate high salt concentration (7.5%) and grow on mannitol salt agar. Principle Mannitol salt agar (MSA) is a selective and differential medium. Yellow colonies of S. aureus in Mannitol Salt Agar (MSA). Image source: ASM Selective medium Incorporating 7.5% sodium chloride in the medium helps select only those bacteria that can tolerate high salt concentrations. Staphylococci species can 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. Differential Medium Pathogenic staphylococci, i.e. Staphylococcus aureus is able to ferment mannitol, but coagulase-negative staphylococci (CONS) are not. So, if that particular specimen contains S. aureus, it ferments mannitol 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 CONS grow, they cant ferment mannitol, so the color of the media around the bacterial colony does not change to yellow; it appears pink. 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. Other commonly used media that contain Phenol red as pH indicator are; TSI Agar, urea base agar, and XLD agar. 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 mediumSodium chloride Phenol red (pH indicator)Agar Final pH: 7.4 ± 0.2 at 25 oC Preparation of Mannitol Salt Agar You can purchase prepared mannitol salt agar from commercial suppliers, get the powder, and prepare the media in your laboratory. 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 11.1 g in every 100 ml of 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 in plastic bags to prevent moisture loss. Growth of S. aureus and CONS on MSA (Image source: Pearson Education) Shelf life: Several weeks, providing there is no change in the appearance of the medium to suggest contamination, deterioration, or alteration of pH. The medium's pH should be within the range of pH 7.3 to 7.7 at room temperature. Staphylococcus saprophyticus (coagulse-negative Staphylococci) may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus. Colony Characteristics in Mannitol Salt Agar Escherichia coli: Does not growStaphylococcus epidermidis: Colorless to pink coloniesStaphylococcus aureus: Yellow colonies; may have a yellow halo around colonies. Note: Do not perform coagulase test from the colonies isolated from mannitol salt agar. Troubleshooting When grown on mannitol salt agar, some 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 the difference between Micrococcus and Staphylococcus hereEnterococcus faecalis and Enterococcus faecium (the most common enterococcal species that have 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).