Bile Esculin HiCynth™ Agar

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 (1). The unique ability of Enterococci to split esculin was reported by Meyer and Schonfeld (2). Enterococci and Group D Streptococci hydrolyse esculin to esculetin and dextrose, which reacts with ferric citrate producing brownish black precipitate (3). The use of esculin hydrolysis in identification of Enterococci was first cited by Rochaix (4). Bile Esculin Agar was originally formulated by Swan (6) for the isolation and identification of Group D Streptococci from food. Facklam and Moody (7, 8) 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 (9) on the basis of esculin hydrolysis. However, other tests such as salt tolerance should be performed for identifying Enterococci (5). Bile Esculin HiCynth™ Agar is prepared by completely replacing animal or vegetable based peptones with chemically defined peptone to avoid BSE/ TSE risks associated with animal peptones.

The medium is highly nutritious. HiCynth™ Peptone No.1 and HiCynth™ Peptone No.5 serves as sources of carbon, nitrogen, amino acids, vitamins and essential growth nutrients. Synthetic detergent No. II inhibits most of the other accompyning 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. If the media is dispensed in tubes in the form of slants, a positive reaction is indicated by blackening of more than half of the slant within 24-48 hours. If blackening is totally absent or if less than half of the slant is blackened within 24-48 hours, the test is negative. Viridans Streptococci sometimes exhibit a weak positive reaction. Also Leuconostoc, Pediococcus, Lactococcus species causing human infections give a positive bile esculin test. Inoculate and incubate the test sample in Todd Hewitt Broth (M313). After 24 hours incubation add two drops of the culture onto the surface of slant or plate media (3, 5).