Thursday, January 1, 2009


Microbiological Test of Water is provided to determine compliance with the requirements given in individual monograph/specificaitons.
Microbial testing of water includes the estimation of the number of viable aerobic bacteria present in a given quality of water.
Phosphate Buffer pH 7.2
Stock Solution
Dissolve 34 g of monobasic Potassium phosphate in about 500 mL of water contained in a 1 L 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 OC for about 15 min.
Nutrient Agar Medium
Beef Extract 10.0 g
Peptone 10.0 g
Sodium Chloride 5.0 g
Agar 12.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 to 8.4 with 5 M Sodium Hydroxide and boil for 10 min. Filter , adjust to pH 7.2 to 7.4 and sterilise by maintaining at 115 OC for 30 min.
MacConkey’s broth
Peptone 20.0 g
sodium Chloride 5.0 g
Sodium Taurocholate 5.0 g
Lactose 10.0 g
Bromocresol purple 10.0 mg
Water 1000 mL
Dissolve the Peptone, the Sodium Chloride taurocholate in the water with the aid of heat. Adjust to pH 8.0 and boil for 20 min. Cool, filter and adjust to pH 7.4. Add the Lactose and the indicator solution, mix and distribute in tubes containing inverted Durham’s tubes. Sterilize by maintaining at 115 OC for 30 min.
For double strength medium, use double the quantity of ingredients in the same amount of water.
All the above media should be incubated for 24 h at 37 OC before use. Any contaminated media should be discarded.
Instead of preparing media, use dehydrated media of Hi media / Difco can be used. Rehydrate the required quantity as per instructions on the bottle label, dispense in required quantities and sterilize at 15 psi at 121 OC for 15 min.
A. Water for Injection
Perform the Total Bacterial Count as follows :
1. Transfer aseptically 1 mL of the sample in each of two sterile petridishes.
2. Add to each dish approx. 20 mL of sterile nutrient agar previously cooled to about 45OC
3. Cover the petridishes and mix the sample with the agar by rotating the dishes 3 times both in clockwise and anti-clockwise directions.
4. Allow the agar to solidify at room temperature.
5. Invert the petridishes and incubate them at 37 OC for 48 h.
6. After incubation, examine the plates for growth and count the number of colony forming units in each plate.
7. The average of both the readings is the total bacterial count per mL.
B. Purified Water
1. Plating Method
Perform the test for Total Bacterial count as follows :
1. Prepare 1/10 dilution of the water sample by transferring aseptically 10 mL of the sample to 90 mL sterile phosphate buffer pH 7.2
2. Transfer 1 mL from this to two sterile petridishes. Add sterile nutrient agar to them in the same manner as above and incubate at 37 OC for 48 h.
3. After incubation, note the number of colony forming units in each plate and calculate the average.
4. Multiply the average count by 10. The result is the Total Bacterial Count per mL.
2. Membrane Filtration Method
Assemble the membrane filtration unit by keeping 0.22 ┬Ám filter membrane of the holder of the unit and moisten the membrane with distilled water. Autoclave the entire unit at 121 OC for 15 min. and allow to cool. Transfer 100 mL of water sample to the unit and filter it under aseptic conditions, by applying vacuum.
Transfer the membrane intended for enumeration of total bacterial count onto the surface of R2A - Agar and incubate at 28 - 30 OC for 5 - 7 days. Repeat the filtration process with 100 mL sample. Transfer the membrane to Czepak-Dox Agar and incubate at 20 - 25 OC for 5 days. Observe the plates for development of colonies and report the results as Colony forming units (CFU) per 100 mL sample.
C. Potable Water
1. Perform the test for identification of Escherichia coli as per STP No. STP-046-00.
2. Perform the test for most probable number of coliforms as follows. Transfer aseptically 10 mL sample into each of five previously sterilized 10 mL double strength MacConkey’s broth tubes Similarly transfer 1 mL and 0.1 mL sample in 10 mL single strength MacConkey’s broth tube using 5 tubes for each sample quantity. Incubate all tubes at 37 OC for 48 h. and observe for gas production.
The number of positive findings of coliform group of organisms should be computed as the combination of positives and recorded in terms of most probable number (MPN) . The MPN for a variety of combinations isgiven in the Table I.
Keep adequate conttrols of Phosphate buffer pH 7.2, Nutrient agar and MacConkey’s broth to confirm thevalidity of results.

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