1. APPARATUS
1.1 Autoclave
1.2 BOD - Incubator
1.3 Bacteriological Incubator
1.4 pH – meter
1.5 Hot air Oven
1.6 Laminar Air flow
1.7 Analytical Balance
1.8 Spirit Lamp / Bunsen Burner
1.9 Micropipette
1.10 Petri dishes
1.11 Test tubes
1.12 Media bottles
2. REAGENTS AND MEDIAS
2.1 Phosphate Buffer pH 7.2
Stock Solution
Dissolve 34 g of monobasic Potassium phosphate in about 500 ml of water contained in a 1000 ml volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of 4 % w/v aqueous solution of Sodium hydroxide (about 175 ml), add water to volume, and mix. Dispense, sterilize and store under refrigeration.
For use, dilute the Stock solution with water in the ratio of 1 to 800, and sterilize in an autoclave at 121–124OC for about 30 min.
2.2 R2A agar medium
2.3 Soyabean casein digest agar
2.4 Soyabean casein digest medium
Prepare media (2.2 – 2.3)as per the current version of Respective GTP.
3. PROCEDURE
3.1 Purified Water
3.1.1 Heterotrophic bacterial count
3.1.1.1 Transfer 1 ml Water Sample to two sterile petridishes. Add about 15 to 20 ml of sterile R2A agar to them and swirl , let the media solidify and incubate at 32.5 ? 2.5 OC for 48-72 hours.
3.1.1.2 After incubation, note the number of colony forming units in each plate and calculate the average.
3.1.1.3 Keep one Negative Control ( without Sample ) and One positive Control ( With Bacteria ). The procedure shall be same as Sample.
3.1.1.4 Record the result as Total Bacterial Count per ml.
3.1.2 Plate Method
3.1.2.1 Dilute further, if necessary, the fluid so that 1 ml will be expected to yield between 30 and 300 colonies.
3.1.2.2 Pipette 1 ml of the Sample (Pretreated Sample) onto each of two sterile petri dishes.
3.1.2.3 Promptly add to each dish 15 - 20 ml of Soyabean Casein Digest Agar Medium that previously has been melted and cooled to approx. 450C.
3.1.2.4 Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature.
3.1.2.5 Invert the petri dishes, and incubate for 48 - 72 hours at 32.50C?2.50C Temperature.
3.1.2.6 Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per ml of specimen.
3.1.2.7 Record the result as Total Aerobic Microbial Count per ml.
3.1.2.8 Keep one Negative Control ( without Sample ) and One positive Control ( With Bacteria ). The procedure shall be same as Sample.
3.1.3 Pathogens Test :
Carry out the Pathogen testing as per current version of Respective GTP
3.2 Potable Water
3.2.1 Heterotrophic bacterial count
3.2.1.1 Transfer 1 ml of water Sample to two sterile petridishes. Add about 15 to 20 ml of sterile R2A agar to them and swirl , let the media solidify and incubate at 32.5 ? 2.5 OC for 48-72 hours.
3.2.1.2 After incubation, note the number of colony forming units in each plate and calculate the average.
3.2.1.3 Record the result as Total Bacterial Count per ml.
3.2.2 Pathogens
3.2.2.1 Carry out the Pathogen testing as per current version of Respective GTP
Autoclave, general testing procedure, laminar air flow, bod - incubator
