1. Description:
A clear, colorless, and odorless liquid.
2. pH: Determine the solution's pH by calibrating the pH meter. Place the pH electrode in a beaker containing 100 ml of water and wait until the pH meter shows _a stable pH value of water.
Acceptance criteria: Between 5.0 to7.5
3. Acidity: Add 0.05 ml of methyl red solution to the 10 ml freshly boiled and cooled water sample. After that, mix the solution properly. If the model is Acidic, then a red color is produced in the solution.
Acceptance Criteria: The resulting solution should not produce a red color.
4. Alkalinity: Add 0.1 ml of bromothymol blue solution to the 10 ml freshly boiled and cooled water sample; after that, mix properly. If the sample is alkaline, then a blue color is produced in the solution.
Acceptance Criteria: The resulting solution should not produce a blue color.
5. Conductivity:
Weep the conductivity electrode with distilled water and place the electrode into the sample to be tested. The instrument shows the conductivity of the respective water sample.
Acceptance criteria: NMT 5.1 uS/cm at 25°C
6. Ammonium:
Test solution: Add 1 ml of alkaline potassium mercuric-iodide solution to the 20 ml sample. Then, allow the solution to stand for 5 minutes.
Standard solution: 2.5 ml of dilute ammonium chloride solution is mixed in the 7.5 ml of the examined liquid. Then, add 1 ml of alkaline potassium mercuric-iodide solution. Observe the test solution vertically.
Acceptance Criteria: The resulting solution should not be more intensely colored than the prepared standard solution.
7. Arsenic:
Take 50 ml of water sample in the wide mouth bottle provided in the arsenic testing kit. Add 1 gm of' Arsenic reagent -1 as (AS-1), then 10 gm of Arsenic reagent-2 as (AS-2) followed by 10 ml .pf Arsenic reagent- 3 as (AS-3). The glass tube is fitted with lead acetate wool, and mercuric chloride paper is placed in a position. As soon as possible, assemble the apparatus, then immerse the flask in a water bath at a temperature such that a uniform gas evolution is maintained. After 40 minutes, no stain produced on the mercuric chloride paper was more intense than the solution. Which is prepared by treating in the same manner 1.0 ml of arsenic standard solution (10 ppm As) diluted with water to 50 ml. The strain produced is compared with the Arsenic chart and recorded arsenic value.
Acceptance criteria: The mercuric chloride paper should not be more intense than the arsenic standard solution or arsenic chart value (NMT 0.01 ppm).
8. Calcium & Magnesium:
To 100 ml of sample, add 2 ml of ammonium buffer pH 10.0, 50 mg mordant black, and 0.5 ml of 0.01 M disodium edetate. No other color should be produced.
Acceptance criteria: A pure blue color should be produced.
9. Chloride:
Add 1 ml of 2M HNO3 and 0.2 ml of 0.1 M silver nitrate in the 10 ml sample solution. The appearance of the solution stays the same for at least 15 minutes.
Acceptance criteria: For 15 minutes, the appearance of the solution will not be changed.
10. Sulphate:
Add 0.1 ml of 2 M hydrochloric acid and 0.1 ml of barium chloride solution to 10 ml of water sample.
Acceptance criteria: The appearance of the solution should remain the same for 15 minutes.
11. Heavy Metals:
In a glass-evaporating dish, evaporate 150 ml of sample to 15 ml in a water bath; 12 mL of solution complies with the limit test for heavy metals, method D (0.1 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard.
Standard solution: Take 10.0 ml of lead standard solution (1 ppm Pb) in the Nessler Cylinder.
Test solution: Pipette 12 ml into a small Nessler cylinder.
Procedure:
2.0 ml of test solution is mixed in the Nessler cylinder containing the standard solution. 2 ml of acetate buffer pH 3.5 is mixed in both Nessler cylinders. After that, add 1.2 ml of thioacetamide reagent. Leave the solution to stand for 2 minutes, then view it downwards over a white surface.
Acceptance criteria: The resulting solution should not be more intensely colored than the prepared standard solution (NMT 0.1 ppm).
12. Nitrate:
Take 5 ml of sample in a test tube immersed in ice. After that, 0.4 ml of a 10 % w/v solution of potassium chloride and 0.1 ml of diphenylamine solution are added. Then, add 5 ml of sulphuric acid dropwise with shaking. Place the tube in a water bath at 500 degrees Celcius for 15 minutes. Any blue color in the solution is not more intense than that in a solution prepared at the time and in the same manner, using a mixture of 0.5 ml of nitrate standard solution (2 ppm NO3) and 4.5 ml of nitrate-free H2O
Acceptance criteria: The resulting solution should not be more intensely colored than the prepared standard solution (NMT 0.2 ppm).
13 Oxidisable substances:
10 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate are mixed in a 100 ml water sample. After that, boil for 5 minutes.
Acceptance criteria: The test solution should remain faintly pink (NMT 0.5 ppm).
14. Total hardness:
Take 25 ml of water sample in the test tube, add 10 drops of Total Hardness reagent-2 as (TH-2), then add a few specs of Total Hardness reagent-1as (TH-1), and then add Total hardness reagent-4 (TH-4), shake well after each drop until the color .changes from pink to blue. Count the no. of drops of TH-4 required for color changes.
Total hardness = no. of drops x 2 (ppm in terms of CaCO3)
Acceptance criteria: NMT 20 ppm
15. Total Dissolved Solid:
Weep the TDS electrode with distilled water and place the electrode into the sample to be tested. The machine shows the TDS of the respective water sample. Note the reading.
Acceptance criteria: NMT 1.0 ppm
16. Total Suspended Solids (TSS):
Take the weight of a completely dried crucible with filter paper. Then, the 100 ml water sample was filtered through the filter. Dry the filter paper in a crucible at 105°C in an oven until completely dry, weigh the filter paper again, and calculate the TSS with the help of the formula.
TSS (mg/1) = (A-B)/V x 1000
Where,
A= weight of the crucible with filter paper before filtration (mg)
B= Weight of the crucible with filter paper after filtration (mg)
V= Volume of sample (ml)
Acceptance criteria: NMT 150 mg/1
17. Residue on evaporation: •
Evaporate 100 ml sample in an evaporating dish to dryness in a water bath and dry at 105°C and calc-Jlate the residue on evaporation with the help of the formula.
RE= (A-B)/V x 100 %
Where,
A= weight of the evaporating dish (g)
B= Weight of the evaporating dish and residue (g)
V= Volume of sample (ml)
Acceptance criteria: Residue weight should not exceed 1 mg (0.001%).
18. Microbial Contamination:
Test of microbial contamination for the total viable count by either A. B and C method.
Pour Plate Method
Take 100 ml of purified water and aseptically pipette 10 ml of water sample to 90 ml sterile 0.1% sterile buffered peptone solution to prepare 1:10 dilution. Shake the 1:10 dilution of the sample to homogenize the water sample. Aseptically pipette lml of 1:10 dilution of water sample into two sterile Petri plates. Pour 20-25 mi of sterilized, cooled SCDA media & SDA media into each Petri plate with a water sample. Rotate the plates clockwise & anti-clockwise to mix the sample uniformly with the media. After solidification of media
incubate the agar plates i.e., Soya bean Casein Digest Agar (SCDA) at 30-35°C for 24-48 hours & Sabouraud
Dextrose Agar (SDA) at 20-25°C for 3-5 days. After incubation, observe the colony and count it by digital colony counter. Report the average of two plates as CFU/ml of sample.
Calculation:
CFU/m1— Number of colonies x Dilution factor/ Sample volume in ml
Spread Plate Method:
Take 100 ml of water sample and aseptically pipette 10 ml of water sample to 90 ml sterile 0.1% sterile buffered peptone solution to prepare 1:10 dilution. Shake the 1:10 dilution of the sample to homogenize the water sample. Aseptically pipette 1 ml of 1:10 dilution of water sample into Soya bean Casein Agar & Sabouraud Dextrose Agar. Spread the sample uniformly over agar through an L-shaped sterilized glass rod.
Incubate Soya bean Casein Digest Agar (SCDA) at 30-35°C for 24-48 hours & Sabouraud Dextrose Agar
(SDA) at 20-25°C for 3-5 days. After incubation, observe the colony and count it by digital colony
counter. Report the average of two plates as CFU/m1 of the sample.
Calculation:
CFU/mL=Number of colonies/ Sample volume in mL
From methods A and B, pipette 1 ml of purified water to 10 mL each of Soyabean Casein Digest Medium and Sabouraud Dextrose broth media, or from method C, remove the filter aseptically and put in 10 mL each of Soyabean Casein Digest Medium and Sabouraud Dextrose broth media. Incubate the media at the required temperature for specified days and after incubation, if turbidity is found in the media, go for a test for a specific pathogen otherwise discard the media as per SOP.
Test for specific Pathogens: Purified water must be tested for the presence of specified pathogens.
a) Test for Escherichia coli:
Streak a portion of enriched soybean casein digest medium on the surface of sterile MacConkey agar and Eosin Methylene Blue agar medium. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, the presence of brick red colonies having a surrounding zone of precipitated bile on MacConkey agar and metallic sheen with dark grey colonies on EMB agar indicates presumptive identification of E. coli.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
E. coli are Gram-negative, rod-shaped organisms. If matching colonies were found on the media, proceed with identification by transferring the suspected colonies individually, using an inoculating loop, to the surface
of SIM media & incubate at 30-35°C. After 24 hrs, add 0.5ml of Kovac's reagent, shake well & allow to stand for 1 minute. If, upon examination, a red ring color is produced on the surface of the upper reagent layer, Indole is positive, and the test confirms the presence of E. Coli.
b) Test for Salmonella species:
Streak a portion of enriched soybean casein digest medium on the surface of Xylose Deoxycholate Medium and Wilson & Blair's Agar. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, red colonies with or without black centers in XLD and green colonies with black centers in Wilson & Blair's Agar indicate presumptive identification of Salmonella spp.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Salmonella spp. are Gram-negative, rod-shaped organisms. If matching colonies were found on media, proceed
with identification by transferring the suspected colonies individually, using inoculating loop, to the slope of the slant of TSIA (Triple Sugar Iron Agar) and then stabbing with inoculating straight wire in the butt.
Aseptically transfer the filter with the grid side up on a plate of Soya bean Casein Agar and sabouraud Dextrose Agar media. Incubate in an upright position at the required temperature, and after incubation, count the colony using a digital colony counter.
Calculation:
CFU/mL= Number of colonies/ Sample volume in ml
From methods A and B, pipette 1 ml of purified water to 10 mL each of Soyabean Casein Digest Medium and
Sabouraud Dextrose broth media or from method C, remove the filter aseptically and put in 10 mL each of
Soyabean Casein Digest Medium and Sabouraud Dextrose broth media. Incubate the media at the required temperature for specified days and after incubation, if turbidity is found in the media, go for a test for specific
pathogen; otherwise, discard the media as per SOP.
Test for specific Pathogens: Purified water must be tested for the presence of the following pathogens.
a) Test for Escherichia coli:
Streak a portion of enriched soybean casein digest medium on the surface of sterile MacConkey agar and Eosin Methylene Blue agar medium. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, the presence of brick red colonies having a surrounding zone of precipitated bile on MacConkey agar and metallic sheen with dark grey colonies on EMB agar indicates presumptive identification of E. coli.
From the selective media plates, pick the suspected colonies and go for Gram staining and biochemical test for further confirmation,
E. coli are Gram-negative, rod-shaped organisms. If matching colonies were found on the media, proceed with identification by transferring the suspected colonies individually, using an inoculating loop, to the surface of SIM media & incubate at 30-35°C. After 24 hrs, add 0.5ml of Kovac's reagent, shake well & allow to stand
for 1 minute, if upon examination, a red ring color is produced on the surface of the upper reagent layer; Indole is positive, and the test confirms the presence of E. Coli.
b) Test for Salmonella species:
Streak a portion of enriched soybean casein digest medium on the surface of Xylose Deoxycholate Medium and Wilson & Blair's Agar. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, red colonies with or without black centers in XLD and green colonies with black centers in Wilson & Blair's Agar indicate presumptive identification of Salmonella spp.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Salmonella spp. are Gram-negative, rod-shaped organisms. If matching colonies were found on media, proceed with identification by transferring the suspected colonies individually, using inoculating loop, to the slope of the slant of TSIA (Triple Sugar Iron Agar) and then stabbing with inoculating straight wire in the butt. Incubation should be done at 30-35°C for 24-48 hours. After incubation, examine the tube of TSIA for the presence of microbial growth and the following physical characteristics.
Slant surface: Alkaline reaction (red color)
Butt: Acidic (Yellow color) and/or gas bubble (with or without concomitant blackening)
If the butt slant of TSIA shows growth and physical characteristics conforming to the above descriptions, the test confirmed the presence of Salmonella spp.
c) Test for Pseudomonas aeruginosa:
Streak a portion of enriched soybean casein digest medium on the surface of the Cetrimide Agar Medium. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, greenish colonies indicate presumptive identification of Pseudomonas aeruginosa.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Pseudomonas aeruginosa are Gram-negative, rod-shaped organisms. If matching colonies were found on media, proceed with further identification by transferring the suspect colonies individually on the oxidase paper/disc for the Oxidase Test. If there is development of pink color changing to purple, the test confirmed the presence of Pseudomonas aeruginosa.
d) Test for Staphylococcus aureus:
Streak a portion of enriched soybean casein digest medium on the surface of Mannitol Salt Agar. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, yellow colonies indicate presumptive identification of Staphylococcus aureus.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Staphylococcus aureus is a gram-positive, cocci-shaped (in cluster) organism. If matching colonies were found on media, proceed with further identification by transferring the suspect colonies individually to the tube containing 0.5 mL of blood plasma. If there is a formation of elot, the test confirms the presence of Staphylococcus aureus.
e) Test for Shigella species:
Streak a portion of enriched soybean casein digest medium on the surface of the Xylose Deoxycholate Medium. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, red colonies without black centers in XLD indicate presumptive identification results for Shigella spp.
Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Shigella spp. are Gram-negative, rod-shaped organisms. If matching colonies were found on media, proceed with identification by transferring the suspected colonies individually, using inoculating loop, to the slope of the slant of TSIA and then stabbing with inoculating straight wire in the butt. Incubation is done for 24- 48 hours at 30-35°C. After incubation, examine the tube of TSIA for the presence of microbial growth and for the following physical characteristics.
Slant surface: Alkaline reaction (red color)
Butt: Alkaline (red color) and without concomitant blackening
If the butt slant of TSIA shows growth and physical characteristics conforming to the above descriptions, the test confirmed the presence of Shigella spp.
f) Test for Candida albicans.
Streak a portion of enriched Sabouraud Dextrose Broth on the surface of Sabouraud Dextrose Agar. The plate should be incubated for 24-48 hours at 30-35°C. After incubation, pasty, opaque, slightly domed, smooth, and cream or white colonies appeared. Pick the suspected colonies from the selective media plates and go for Gram staining and biochemical test for further confirmation.
Candida albicans are Gram-positive budding yeast cells. If matching colonies were found on media, proceed with further identification by transferring the suspect colonies individually to the tube containing 0.5 mL of blood serum. Incubate for 2-3 hours. After incubation, Place one drop of suspension onto a slide and observe under the microscope. If the formation of the germ tube was found, the test confirmed the presence of Candida albicars. Negative Control: Run the negative control of each media simultaneously with the test sample at the same incubation temperature and period. If negative control shows no growth at the end of the test, then the difficulty is valid.
Positive Control: Run the positive control of each test simultaneously with the test sample by inoculating 10- 100 CFU per ml of inoculums of reference organism for specific media. The test is valid if the positive control indicates the positive result for the respective organism.
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