Bile Esculin Azide Agar Plate

Principle And Interpretation

Group D Streptococci possess the group D lipoteichoic acid antigen in their cell walls. Former Group D species, which are predominant normal inhabitants of the human gastrointestinal tract, are termed as faecal Streptococci or Enterococci (10). The unique ability of Enterococci to split esculin was reported by Meyer and Schonfeld (12). Enterococci and Group D Streptococci hydrolyse esculin to esculetin and dextrose, which reacts with ferric citrate producing brownish black precipitate (11). The use of esculin hydrolysis in identification of Enterococci was first cited by Rochaix (14). Bile Esculin Agar was originally formulated by Swan (16) for the isolation and identification of Group D Streptococci from food. Facklam and Moody (4,6) further reported that using Bile Esculin Agar, Group D Streptococci could be differentiated from non-Group D Streptococci. Bile Esculin Agar was also shown to aid differentiation of Enterobacteriaceae, Klebsiella, Enterobacter, Serratia from other Enterobacteriaceae genera (3) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should be performed for identifying Enterococci (5). Bile Esculin Azide Agar is a modification of Bile Esculin Agar (6,16) as per Isenberg (7). In this medium the bile concentration is reduced and additional sodium azide is incorporated. Tryptone, proteose peptone and HM peptone B serves as sources of carbon, nitrogen, amino acids, vitamins and essential growth nutrients. Bile and sodium azide inhibits most of the other accompanying bacteria. Esculin in the medium is hydrolyzed to esculetin and dextrose. Esculetin reacts with ferric citrate to form a dark brown or black complex, visualized as a zone of black precipitate around the colonies.Also, Leuconostoc, Pediococcus, Lactococcus species causing human infections give a positive bile esculin test (13). Suspected water samples are filtered using membrane filters. These membrane filters are aseptically placed on Slanetz and Bartely Medium (M612I). Red or maroon coloured colonies observed after incubation are further confirmed by aseptically transferring the membrane filter on to Bile Esculin Azide Agar plate preheated to 44°C. Incubation at 44 ± 0.5°C for 2 hours is done following the inoculation. All typical colonies exhibiting a brown black colouration in the surrounding medium are counted as intestinal Enterococci (13). Alternatively Bile Esculin Azide Agar can also be used for direct isolation of Enterococci (without membrane filter), by incubation at 35-37°C for 18-24 hours.