It is recommended to use 10 gm of test sample along with 90 ml of 0.1% Peptone salt solution for enumeration. The prepared
dilution may be blended at 15,000 to 20,000 revolutions per minute. Further a ten fold dilution may be prepared using 1 ml of
it in 9ml of sterile diluent within 15 minutes and mixed well. This is considered as 10 1
-
dilution. Sequential dilutions can be
prepared using same diluent and counts obtained by spread plate or pour plate technique. Tests may be performed in duplicates
as described in technique and checked for equivalent yields of organisms between the diluent batches.
Incubate the tubes with test organisms. At time of zero minutes and after 30 minutes and 2 hours, subculture an inoculum
(approximately 0.01ml) or a loop full onto Soyabean Casein Digest Agar (M290) using streak plate technique. If desired SCDA
may be also enriched with 5% v/v sheep blood depending on intended organisms to be isolated. Incubate plates at 35±2ºC
for 18-24 hours.