Anaerobic bacteria live in an oxygen-free environment. Some anaerobic bacteria actually die if oxygen is present while others fail to grow and multiply (1). One of the methods of cultivation of anaerobes is using the Sprays medium by using the anaerobic culture dish (2). Liver Veal Agar is formulated as per the medium of Spray (3). Liver Veal Agar is recommended by APHA (4) and the FDA Bacteriological Analytical Manual (BAM) (5). Liver Veal Agar on supplementation of 50% egg yolk is recommended for the cultivation of anaerobic organisms (4-6). The medium is highly nutritious and therefore is an excellent medium for growth of sporulating anaerobic bacteria.
Both the infusions, peptones, casein enzymic hydrolysate and gelatin serve as sources of carbon, nitrogen, amino acids and various vitamins. Dextrose serves as the carbon source. Starch enhances growth of anaerobic bacteria. Spray reported isolation of Clostridium perfringens within 6 hours of inoculation and Clostridium tetani within 8 hours. When the medium is inoculated with a small inoculum, gas production is not evident. Spray recommended that the medium should be taken directly from the sterilizer or should be boiled for 10 minutes to drive off dissolved oxygen and cooled without agitation. Serial inoculations are made and the medium is poured into plates. After solidification, 5 ml sterile Liver Veal Agar is poured over the medium as a cover layer to prevent the spreading of surface colonies.
Refer standard procedures for isolation and cultivation of anaerobic bacteria (7, 8).
C. botulinum and C. tetani are highly hazardous and extreme care should be taken while handling these cultures.
Directions: Suspend 97 grams in 1000 ml warm distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.