Bile esculina zide agar

The blood culture bottles were incubated at 37 °C and were observed daily after 48 h for 5 consecutive days for presence of turbidity, hemolysis, gas formation or color changes which are evidence of microbial growth. If the culture bottle does not show any growth within 7 days, it was reported as negative. Whenever visible growth appears, the bottle was opened aseptically; a small amount of broth was taken with a sterile loop and sub cultured on Bile-esculina zide agar (BEAA) (Hardy Diagnostics, Santa Maria, CA, United states) [12 , 14 ].
Urine samples were inoculated on BEAA media with a 10 μl calibrated loop and incubated at 37 °C for 24 h [14 ]. Presence of 10⁴ colony forming unit per ml of bacteria with black colored colony was considered as significant enterococci in the urine. Other clinical samples were directly inculcated on BEAA at 37 °C for 24 h and checked for growth of black colored colony [13 , 14 ]. The presence of enterococci were confirmed by further tests such as gram stain, catalase reaction, growth on broth containing 6.5% NaCl and growth in BHI broth, at 37 °C and 45 °C for 48 h, respectively [14 , 15 ].

The clients were provided with wide-mouthed, sterile plastic containers and informed to submit stool specimens. The collected stool specimens were streaked on Bile Esculin Azide agar (Hardy Diagnostics, Santa Maria, CA, USA) and incubated for 24 hours at 37°C. Plates were observed for appearance of characteristic colonies of growth and blackening. Typical characteristic colonies were selected randomly for characterization and presumptive identification of Enterococcci[11 ]. Each isolate was also assessed using Gram staining, catalase test and its growth in 6.5% NaCl broth. Presumptive pure colonies were picked and inoculated into Brain Heart Infusion (BHI) broth, and incubated at 45°C for 24 hours and growth in the medium was assessed by its turbidity. An isolate fulfilling the above criteria was assumed to be Enterococccus species [11 ].