SOP for Chemical and Microbial testing of purified water
1.0 PURPOSE:
To lay down a procedure for sampling and testing of Purified water.
2.0 SCOPE:
3.0 RESPONSIBILITIES:
Chemist Officer: To perform the procedure of sampling and chemical analysis of purified water and observe the results as per SOP.
Microbiologist: To perform the procedure of sampling and Microbiological analysis of purified water and observe the results as per SOP.
Head-QC: To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0 PROCEDURE:
4.1 Sampling of Purified water for Chemical analysis:
4.1.1 Before sampling, make sure about your sampling points and its location. Take required number of sampling containers for the scheduled water sample. Water samples are usually collected in a clean, dry and separate sampling container.
4.1.2 Before start sampling remove aluminium foil wrapped on sampling point and again wrap after sampling.
4.1.3 While sampling of water, collect the water sample from end of the particular point and if the user point is connected to hosepipe, collect sample from end of the hosepipe.
4.1.4 Open the valve until steady flow of water come out. Drain water for 1-2 min just before the taking sample.
4.1.5 Open the stopper of bottle at the time of sampling only. Immediately collect the required volume of sample in the container. Flow of water should be minimum to avoid splashing of water and bubble formation.
4.1.6 After collection of water sample, label it properly like sampling point number, location, date and sign. Sample should be processed within one hour.
4.1.7 In case of delay; the sample should be stored below 8°C. Sample must be analyzed within 24 hr.
4.2 Sampling of Purified water for Microbiological analysis:
4.2.1 Water samples are usually collected in a clean and sterilized sampling container. Always do water sampling for chemical analysis and after that for Microbial analysis.
4.2.2 Before sampling, refer your sampling points and its location. Take required number of sampling containers for the scheduled water sample.
4.2.3 Before start sampling remove aluminium foil wrapped on sampling point.
4.2.4 Spray freshly prepared 70% IPA at the opening of sampling point and allows it to dry.
4.2.5 Disinfect your hands with freshly prepared 70% IPA or you can use sterile hand glows.
4.2.6 While sampling of water, collect the water sample from end of the particular point and if the user point is connected to hosepipe, collect sample from end of the hosepipe.
4.2.7 Open the valve until steady flow of water come out. Drain water for 1-2 min just before the taking sample.
4.2.8 Open the stopper of bottle at the time of sampling only. Immediately collect the required volume of sample in the container. Flow of water should be minimum to avoid splashing of water and bubble formation.
4.2.9 Wrap aluminium foil again on point. After collection of water sample, label it properly like sampling point number, location, date and sign. Sample should be processed within one hour.
4.2.10 In case of delay; the sample should be stored below 8°C. Sample must be analyzed within 24 hr.
4.3 Chemical analysis of Purified water:
4.3.1 Appearance: Take about 10 to 20 ml of sample in to a clean and dry test tube and check the appearance.
(Note: It should be clear, colorless liquid)
4.3.2 Conductivity: Conductivity is measured on a calibrated conductivity meter. Take 50-100 ml of sample in clean and dry beaker and dip the rode in it. Wait for stable reading. Note down the value displayed on meter.
(Note: Not more than 5.1 µS/cm as per BP and Note more than 1.3 µS/cm as per USP at 25°C)
4.3.3 pH (Limit-Between 5 to 7 at 25°C): pH of water sample should be checked on calibrated pH meter. Take 50-100 ml of sample in clean and dry beaker and dip the rode in it. Wait for stable reading. Note down the value displayed on meter.
4.3.4 Acidity and alkality: To 10 ml of sample add 0.05 ml of methyl red solution. Solution should not red in color for acidity test.
To 10 ml of sample add 0.1 ml of bromothymol blue solution. The solution should not blue in color for alkality test.
4.3.5 Oxidisable substances: To 100 ml of purified water, add 10 ml of dilute sulphuric acid and 0.1 ml of 0.02 M potassium permanganate and boil for 5 min.
(Note: The solution remains faintly pink.)
Preparation of Sulfuric Acid, Dilute: Add 5.5 ml of sulfuric acid to 60 ml of water, allow to cool and dilute to 100 ml with the same solvent.
Preparation of 0.02 M potassium permanganate: Dissolve 0.3168 gm of potassium permanganate in 100 ml of water.
4.3.6 Chloride: To 10 ml add 1 ml of dilute nitric acid and 0.2 ml of silver nitrate solution.
(Note: The Solution shows no change in appearance after 15 minutes.)
Preparation of Diluted nitric acid: Dilute 20 g of nitric acid to 100 ml with water.
Preparation of Silver nitrate solution: 1.7% w/v solution in water.
4.3.7 Sulfates: To 10 ml of water sample add 0.1 ml of dilute hydrochloric acid and 0.1 ml of barium chloride solution.
(Note: The Solution shows no change in appearance for 1 hour.)
Preparation of Diluted Hydrochloric acid: Dilute 20 g of hydrochloric acid to 100 ml with water.
Preparation of Barium chloride solution: 6.1% w/v solution of barium chloride.
4.3.8 Ammonium:
Test Preparation: To 20 ml of water sample, add 1 ml of alkaline potassium tetraiodomercurate solution. After 5 min, examine the solution down the vertical axis of the tube.
Standard Preparation: Add 1 ml of alkaline potassium tetraiodomercurate solution to a mixture of 4 ml of ammonium standard solution (1 ppm NH4) and 16 ml of ammonium-free water.
(Note: The test solution is not more intensely colored than a standard preparation.)
Preparation of Ammonium Standard Solution (1 ppm NH4): Immediately before use, dilute ammonium standard solution (2.5 ppm NH4) R to 2.5 times its volume with water.
Preparation of ammonium Standard Solution (2.5 ppm NH4): Immediately before use, dilute with water to 100 times its volume a solution containing ammonium chloride equivalent to 0.741 g of NH4Cl in 1000 ml.
Preparation of Potassium Tetraiodomercurate Solution, Alkaline: Dissolve 11 g of potassium iodide and 15 g of mercury (II) iodide in water and dilute to 100 ml with water. Immediately before use, mix the solution with an equal volume of a 25% w/ v solution of sodium hydroxide.
4.3.9 Calcium and Magnesium: To 100 ml add 2 ml of ammonium chloride buffer solution pH 10.0, 50 mg of mordant black 11 triturate and 0.5 ml of 0.01 M sodium edetate.
(Note: Pure blue color is produced.)
Preparation of ammonia Buffer pH 10.0 or Ammonium chloride buffer solution pH 10.0:
Dissolve 5.4 g of ammonium chloride in 20 ml of water, add 35.0 ml of ammonia and dilute to 100.0 ml with water.
Mordant Black 11 Triturate: Mix 1 g of mordant black 11 with 99 g of sodium chloride.
Preparation of 0.01 M disodium edetate: Dissolve 0.3722 gm of disodium edentate in 100 ml of water.
4.3.10 Nitrate:
Test Solution: Place 5 ml in a test-tube immersed in iced water, add 0.4 ml of a 100 gm/lit solution of potassium chloride, 0.1 ml of diphenylamine solution and, drop wise with shaking, 5 ml of nitrogen-free sulfuric acid. Transfer the tube to a water-bath at 50°C.
Standard Solution: A mixture of 4.5 ml of nitrate-free water and 0.5 ml of nitrate standard solution (2 ppm NO3).
(Note: After 15 min, any blue colour in the solution is not more intense than that in a reference solution.)
Preparation of Diphenylamine Solution: 0.1% w/v solution in sulfuric acid.
Preparation of Nitrate Standard Solution (2 ppm NO3):Immediately before use, dilute nitrate standard solution (10 ppm NO3) to 5 times its volume with water.
4.3.11 Heavy Metals:
Test Solution: In glass evaporating dish, evaporate 200 ml purified water to 20 ml on water bath.
Procedure: Take two cylinder one with 12 ml of evaporated water and other with 10 ml of lead standard solution (1 ppm pb) and 2 ml of evaporate water in it. To each of the cylinder add 2 ml of acetate buffer ph 3.5 and mix, add 1.2 ml of thioacetamide reagent. Allow it to stand for 2 minutes, then view downward over a white surface.
(Note: The colour produced with the test solution is not more intense than that produced with the standard solution.)
Preparation of Lead Standard Solution (1 ppm Pb): Immediately before use, dilute lead standard solution (10 ppm Pb) R to 10 times its volume with water.
Preparation of Lead Standard Solution (10 ppm Pb): Immediately before use, dilute lead standard solution (100 ppm Pb) to 10 times its volume with water.
Preparation of Lead Standard Solution (100 ppm Pb): Immediately before use, dilute lead standard solution (0.1 per cent Pb) to 10 times its volume with water.
Preparation of Lead Standard Solution (0.1% Pb): Dissolve lead nitrate equivalent to 0.400 g of Pb(NO3)2 in water and dilute to 250.0 ml with the same solvent.
Preparation of Acetate Buffer pH 3.5: Dissolve 25 g of ammonium acetate in 25 ml of water and add 38 ml of 7M hydrochloric acid . Adjust the pH to 3.5 with either 2M hydrochloric acid or 6M ammonia and dilute to 100 ml with water.
Preparation of Thioacetamide Reagent: Add 1 ml of a mixture of 15 ml of 1M sodium hydroxide, 5 ml of water and 20 ml of glycerol (85%) to 0.2ml of thioacetamide solution (4.0 % solution in water), heat in a water bath for 20 seconds, cool and use immediately.
4.3.12 Total Dissolved Solid: Evaporate 100 ml of sample water to dryness on a water bath and dry to constant weight at 105°C. Calculate the residue obtained by subtracting the weight of bowl from weight of bowl + Residue.
(Note: NMT 0.001 % W/V)
4.4 Microbial analysis of Purified water:
4.4.1 Test for Total Viable count:
4.4.1.1 Pour-plate method:
4.4.1.1.1 Aseptically transfer 1ml of water sample into four different petridish (2 of R2A and 2 of SDA), which is previously labeled and kept under Laminar Air Flow.
4.4.1.1.2 Pour approximately 15-20 ml of R2A (in 2 plates) and SDA (in 2 plates) media into the respective plates and rotate the plates clockwise and anti clockwise to mix the contents and allow to solidify.
4.4.1.1.3 Incubate the R2A plates at 30-35oC for 3-5 days & SDA plates at 20–25oC for 5-7 days.
4.4.1.1.4 Positive Control: Add 1 ml of culture (10-100 CFU/ml) from one of the mention organisms S. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, in each plate & pour 15-20 ml R2A. Gently rotate the plates for the even distribution of cultures and allow solidifying. Incubate the plates of bacterial cultures at 30-35oC for 2-3 days.
Add 1 ml of culture (10-100 CFU/ml) of Candida albicans ATCC 1023 and Aspergillus brasiliensis ATCC 16404 in each plate & pour 15-20 ml SDA. Gently rotate the plates for the even distribution of cultures and allow solidifying. Incubate the plates at 20–25oC for 3-5 days.
4.4.1.1.5 Negative Control: Pour the R2A / SDA Media in pre-sterilized and labeled plates without sample and allow solidifying.
4.4.1.1.6 The incubated plates must be monitored every 24 hours. Record the Observation.
4.4.1.1.7 Interpretation:
Count the colonies produced and report the results as CFU/ml. Negative Control plate must not show any CFU. Positive Control plate must show luxuriant growth within 48-72 hours of incubation for bacteria and 72-120 hours for fungi.
The mean of the two plates calculated with dilution factor and quantity of sample finally expressed as CFU/ml of water Sample.
CFU/ml = Average No. of Colonies X Dilution Factor
Volume of sample tested.
4.4.1.2 Membrane Filtration Method:
4.4.1.2.1 Rinse the membrane with minimum 100ml of 0.1% peptone water/ Buffered sodium chloride peptone (pH 7.0). Now aseptically transfer 1ml of water sample through 0.45 µm pore size membrane having 47mm diameter. Rinse the filter paper again 2 to 3 times with 0.1% peptone water/ Buffered sodium chloride peptone (pH 7.0). Number of rinses can be increased as per the validation report for the sample up to 2 to 3 times.
4.4.1.2.2 With the help of sterile forceps transfer the membrane to the surface of pre-incubated R2A plate for determination of total aerobic microbial count. Incubate the R2A plate at 30-35°C for 3 to 5 days.
4.4.1.2.3 Positive Control: Aseptically filter 100 ml of BSCP/ sterile 0.1% of Peptone water, which previously inoculated with one of mention organism (10 to 100 cells) of S. aureusATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus subtilisATCC 6633 and place the filter paper on pre incubated R2A plates. Incubate the plates of bacterial cultures at 30–35oC for 3 days.
4.4.1.2.4 Negative Control: Aseptically filter 100 ml quantity of BSCP/ Sterile 0.1% Peptone Water by the same procedure followed for the sample & incubate the R2A plate at 30 -35oC for 3-5 days.
4.4.1.2.5 The incubated plates must be monitored every 24 hours. Record the observation.
4.4.1.2.6 Interpretation: Count the colonies produced and report the results as CFU/total volume of sample tested. Negative Control plate must not show any CFU. Positive Control plate must show luxuriant growth within 48-72 hours of incubation for bacteria and 72-120 hours for fungi. The mean of the two plates calculated with dilution factor and quantity of sample finally expressed as CFU/ gm of Sample.
CFU/ml = Average No. of Colonies X Dilution Factor
Volume of sample tested.
4.4.2 Tests for Pathogens:
Take 10ml of water sample and inoculate it in to 90ml Soybean Casein Digest Medium (SCDM). Incubate at 30-35oC for 18-24 hours.
4.4.2.1 Test for Escherichia coli:
4.4.2.1.1 Primary Test:
Observe the above SCDM tube for growth. If growth is present, aseptically pipette 1mlaliquot of the inoculated SCDM into 100ml of MacConkey broth. Incubate at 42-44°C for 24-48 hours. Examine broth for the growth in terms of turbidity. If turbidity found carry out Secondary Test.
4.4.2.1.2 Secondary Test:
Using a sterile loop, subculture a portion of the inoculated MacConkey broth, onto the surface of a MacConkey agar plate. Incubate at 30-35°C for 18-72 hrs. If upon examination, the plates containing colonies having characteristic brick red in color, have surrounding zone of precipitated bile found then carry out Confirmative Test-Growth characteristics on EMB agar & Indole test.
4.4.2.1.3 Confirmative Test (Growth on EMB Agar and Indole Test):
· Streak representative colonies from MacConkey agar plate & subculture on the surface of EMB Agar plate.
· Same time inoculate 1ml enriched culture from MacConkey Broth inoculate in to 5ml of Peptone Water for indole production.
· Incubate EMB plate & Peptone water tube at 30-35°C for 24-48 hrs.
· If upon examination, EMB Agar plates containing colonies having Metallic Sheen under reflected light and Blue-Black appearance under transmitted light found, Escherichia coli is present.
· After incubation of Peptone water to test for indole, add 0.5ml of Kovac’s reagent in Peptone water.
· Shake well and allow standing for 1min.
· If red colored ring is observed then indole test is positive. If other color observed, Escherichia coli is absent.
· If the entire tests arepositive, indicate the presence of E. coli.
4.4.2.1.4 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hours old broth culture of E.coli. ATCC 8739 or NCIM 2065. The test is invalid if the results do not indicate that the control contains E.coli.
Negative Control: Confirm the above tests by carrying out the test simultaneously without exposure of any sample to check sterility. The test is invalid if the results indicate the growth.
4.4.2.2 Test for Pseudomonas aeruginosa:
4.4.2.2.1 Primary Test:
Observe the above SCDM tube for growth, if growth is present, streak on the surface of Cetrimide agar medium aseptically. Cover the Petri plates and Incubate at 30-35°C for 48 hrs.
If upon examination, the plates containing colonies having Generally Greenish colored and Greenish Florescence in Ultra Violate light found, carry out Confirmative Test (Oxidase test).
4.4.2.2.3 Confirmative Test (Oxidase Test):
· Transfer the representative colonies on Oxidase disc, or filter paper that impregnated with N, N dimethyl-p phenylene diaminedihydrochloride and wait for 10-15 second.
· If there is no development of a pink color or changing to purple, confirm the absence of the Pseudomonas aeruginosa. If the entire tests are positive, indicate the presence of Pseudomonas aeruginosa.
4.4.2.2.4 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Pseudomonas aeruginosa ATCC 9027 or NCIM 2200. The test is invalid if the results do not indicate that the control contains Pseudomonas aeruginosa.
Negative Control: Carry out a negative control in every step without exposure of any sample to check sterility. The test is invalid if the results indicate the growth.
4.4.2.3 Test for Staphylococcus aureus:
4.4.2.3.1 Primary Test:
Observe the above SCDM tube for growth, if growth is present, streak a portion of the medium on the surface of Mannitol salt agar medium aseptically. Cover the Petri plates and incubate at 30-35°C for 48 hrs. If upon examination, the plates containing colonies having Yellow color with yellow zones found carry out Confirmative Test (Coagulase test).
4.4.2.3.2 Confirmatory Test (Coagulase Test):
· Transfer representative suspected colonies from the agar surface of the media to individual tube.
· Each containing 0.5ml of mammalian, preferably rabbit or horse plasma with or without additives.
· Incubate the tube in water bath at 37°C. Examine tubes after 3 hrs to suitable intervals up 48 hrs.
· If no coagulation occurs, confirm the absence of Staphylococcus aureus.
· The entire test are positive, indicate the presence of Staphylococcus aureus.
4.4.2.3.3 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Staphylococcus aureus ATCC-6538. The test is invalid if the results do not indicate that the control contains Staphylococcus aureus.
Negative Control: Carry out a negative control in every step without exposure of any sample to check sterility. The test is invalid if the results indicate the growth.
4.4.2.4 Test for Salmonella abony:
4.4.2.4.1 Primary Test:
Observe the above SCDM tube for growth. If growth is present, inoculate 0.1ml of SCDM sample to 10mlof sterilized and pre-incubated (Rappaport Vassiliadis Salmonella Enrichment Broth) RVSEB aseptically. Incubate at 30-35°C for18 to 24 hrs. After completion of incubation period, observe the tube. If growth is found in form of turbidity, carry out secondary test.
4.4.2.4.2 Secondary Test (Growth on XLD agar):
· Secondary test is done on Xylose Lysine Deoxycholate (XLD) agar. XLD should not be autoclaved. It should be heated with frequently agitation until medium boil. Transfer immediately to a water bath at 50°C & after that pours the plates.
· Streak a portion of the medium (RVSEB) on the surface of XLD agar medium aseptically.
· Incubate all the plates at 30-35°C for 18-24 hrs. If the specific colonies having Red, with or without black center found, carry out Confirmative Test.
4.4.2.4.3 Confirmative Test:
· Subculture any colonies from XLD agar in Triple Sugar Iron (TSI) by first streaking the surface of the slope & making a stab culture with the same inoculating needle & incubate at 30-35°C for 18 to 24 hour.
· Forming alkaline (Red) slant & acid (Yellow) butt with or without blackening of butt (H2S) production, indicate the presence of Salmonella.
· If the entire tests are positive, indicate the present of Salmonella.
4.4.2.4.4 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Salmonella abony NCTC 6017 or NCIM 2257. The test is invalid if the result does not indicate that the control contains Salmonella.
Negative Control: Confirm the above tests by Carry out a negative control in every stepwithout exposure of any sample to check sterility. The test is invalid if the results indicate the growth.
4.4.2.5 Interpretation of Pathogen Test:
If all pathogenic test are negative then it shows the absence of E.coli, Salmonella, S. aureus, P. aeruginosa, then the test of pathogens expressed as Absence of pathogen per ml of water sample. (For salmonella, absent per 10ml)
5.0 ABBREVIATION:
ATCC- American Type Culture Collection
NCTC-National Culture Type Collection
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