Define Procedure for Gram Staining of Bacterial Cultures?

Now carry out the exercise following the steps enumerated herewith:

1. Label the nutrient agar plates with the name of the food sample and the dilution to be plated.

2. Weigh 20 gm of each food sample and mix it with 180 ml of sterile water in the blender jar by blending for 2 minutes. This gives 1:10 (10^-1) dilution of each food sample. Physiological saline or peptone water can be used as a diluent. Homogenization can also be done in stomacher.

3. From 10^-1 dilution, prepare further dilutions in a series from 10^-2 to 10^-7. This is done by adding 1 ml of 1:10 dilution into a diluent tube marked 10^-2 and shake vigorously 1 ml of 10^-2 (1:100) is then transferred to a tube marked 10^-3 (1:1000) and so on. After each transfer shake the suspension vigorously.

4. Spread 0.1 ml of each dilution on the nutrient agar plate using sterile bent glass rod (spreader). Other media like brain heart infusion broth or plate count agar can also be used.5. Incubate the plates at appropriate temperature (about 37°C) for 24 to 48 hours.

6. Observe the plates for colony count and determine the CFU/gm of the food by using only those plates that fit the criteria of the counting rules (i.e. colonies count between 30 and 300).