HiCombi™ Blood -Mannitol Salt Agar Plate

Principle And Interpretation

Blood Agar Base

Blood Agar Base is a highly nutritious medium generally used as a basal medium for preparing blood agar by supplementation with blood. It can also be used as general-purpose media without the addition of blood. Blood Agar Base media can be used with added phenolphthalein phosphate (1) for the detection of phosphate producing Staphylococci, with added salt and agar for assessment of surface contamination on equipment and pig carcass (2) and to determine salinity range of marine Flavobacteria (3). It can also be used for preparation of Salmonella Typhi antigens (4). Blood Agar Base is recommended by APHA (5) and Standard Methods (6,7) for testing of food samples.

HM peptone B and tryptose provides carbon, nitrogen, amino acids and vitamins. Sodium chloride helps in maintaining the osmotic equilibrium of the medium. Addition of blood makes the medium more nutritious by providing additional growth factors required by fastidious organisms. It also helps in visualizing the haemolytic reactions. However, haemolytic reactions depend on the animal blood used. Sheep blood gives best results for Group A Streptococci (8). But sheep blood fails to support growth of Haemophilus haemolyticus since sheep blood is deficient in pyridine nucleotides. However when horse blood is used H. haemolyticus colonies produce haemolysis and mimic Streptococcus pyogenes (9).

Mannitol Salt Agar

Staphylococci are widespread in nature, although they are mainly found on the skin, skin glands and mucous membranes of mammals and birds. The coagulase-positive species i.e Staphylococcus aureus is well documented as a human opportunistic pathogen. The ability to clot plasma continues to be the most widely used and accepted criterion for the identification of pathogenic staphylococci associated with acute infections (9). Staphylococci have the unique ability of growing on a high salt containing media (10). Isolation of coagulase-positive staphylococci on Phenol Red Mannitol Agar supplemented with 7.5% NaCl was studied by Chapman (11).

The resulting Mannitol Salt Agar Base is recommended for the isolation of coagulase-positive Staphylococci from cosmetics, milk, food and other specimens (5,9,12,13,15). The additional property of lipase activity of Staphylococcus aureus can be detected by the addition of the Egg Yolk Emulsion (FD045). The lipase activity can be visualized as yellow opaque zones around the colonies (16). HM peptone B and proteose peptone supply essential growth factors and trace nutrients to the growing bacteria. Sodium chloride serves as an inhibitory agent against bacteria other than staphylococci. Mannitol is the fermentable carbohydrate, fermentation of which leads to acid production, detected by phenol red indicator.

S.aureus ferment mannitol and produce yellow coloured colonies surrounded by yellow zones. Coagulase-negative strains of S.aureus are usually mannitol non-fermenters and therefore produce pink to red colonies surrounded by red-purple zones. Presumptive coagulase-positive yellow colonies of S. aureus should be confirmed by performing the coagulase test [tube or slide] (9). Lipase activity of S.aureus can be detected by supplementing the medium with egg yolk emulsion.

A possible S.aureus must be confirmed by the coagulase test. Also the organism should be subcultured to a less inhibitory medium not containing excess salt to avoid the possible interference of salt with coagulase testing or other diagnostic tests (e.g. Nutrient Broth) (M002) (16). Few strains of S.aureus may exhibit delayed mannitol fermentation. Negative results should therefore be re-incubated for an additional 24 hours before being discarded (16).