Anaerobic Egg Agar Base

Principle And Interpretation

, Clostridium species ranked behind Salmonella and Staphylococcus aureus has been the third most common etiological agent of food-borne diseases (1). Clostridium botulinum is an anaerobic, rod-shaped spore forming bacterium that produces a botulinum toxin with characteristic neurotoxicity (2). Clostridium food poisoning results from consumption of contaminated food. Anaerobic Egg Agar Base supplemented with egg yolk emulsion is recommended for detection of Clostridium botulinum in foods and also to detect the lipase activity of Yersinia sp. in accordance with FDA BAM, 1998 (3). The major virulence factor of C. botulinum is botulinum toxin, which is secreted upon invasion of the host gut, which contributes to food poisoning, gastrointestinal illnesses and even death (2).

Casein enzymic hydrolysate and proteose peptone supply amino acids and other complex nitrogenous nutrients. Yeast extract provides essential B-complex vitamins. Egg yolk emulsion (FD045F) is added to the medium by which the lipase and lecithinase activity can be observed. Agar acts as the solidification agent. Sodium chloride maintains the osmotic equilibrium of the cells. Yersinia sp. degrades lecithin of egg yolk, forming an insoluble opaque precipitate (4). Lipase breaks down free fats present in the egg yolk causing iridescent sheen to form on the colony surface. Proteolysis is indicated by clear zones in the medium surrounding the growth (5).

Representative samples of food (1-2 g solid or 1-2ml liquid) are enriched in Cooked Meat Media (M149F) up to 5days at 35°C under anaerobic conditions. Vegetative cells are destroyed either by alcohol treatment (3) or by heat treatment wherein an aliquot of 1-2ml is heated at 80°C for 10-15min. These treated cultures are streaked to either Liver-Veal-Agar Base, Modified (M1872) or Anaerobic Egg Agar Base (M902F). Typical C.botulinum colonies may be raised or has smooth or rough surfaces. They show spreading and have an irregular edge. On egg yolk medium, C.botulinum colonies usually exhibit surface iridescence when examined by oblique light. This luster zone often referred to as a pearl layer, usually extends beyond and follows the irregular contour of the colony. Colonies of C. botulinum types C, D, and E are ordinarily surrounded by a wide zone (2-4 mm) of yellow precipitate. Colonies of types A and B generally show a smaller zone of precipitation. Further biochemical tests need to be performed to differentiate it from other species of Clostridium .