5.1 Take out the working cultures from the refrigerator 30 minutes prior to the testing so as to acclimatize the culture with the working environment and place it under Laminar Air Flow (LAF).
5.2 Take a small loop of culture and inoculate it in to 50 ml of sterile 0.9% saline solution/Buffered sodium chloride peptone solution, which is the stock solution.
5.3 Gently vortex to make a uniform suspension.
5.4 Pipette 5.0 ml from stock solution and inoculate it into 45.0 ml of 0.9% sterile saline/Buffered sodium chloride peptone solution. This is 10¯1 dilution.
5.5 Transfer 5.0 ml from the above test tube to another test tube containing 45.0 ml of the 0.9% saline/ Buffered sodium chloride peptone solution. This constitutes 10¯2 dilution.
5.6 Perform further serial dilutions up to 10-8 and prepare 50 ml culture suspension at each stage of dilution.
5.7 Add 1 ml each of all the prepared dilutions in to sterile Petri plates in duplicates.
5.8 Pour sterile molten Soyabean Casein Digest Agar to all the bacterial cultures and Saboraud dextrose Agar to all the yeast or molds cultures.
5.9 Incubate the bacterial culture plates at 32.5 ± 2.5°C for 24 – 48 hrs and 22.5 ± 2.5°C for 3 – 5 days for fungi.
5.10 Preserve all dilutions after plating the cultures in refrigerator at 2-8ºC.
5.11 After specified incubation period, count the number of colonies.
5.12 Select and preserve the dilutions, which are having cells count between 10 -100 cfu/ml. Then store in refrigerator at 2 to 8°C for routine tests and discard all the other dilutions.
5.13 Perform the above exercise for all organisms, which are being used for growth promotion test.
5.14 Prepare fresh 10-100 cfu/ml suspension once in 15 days.