Multiple Health Pharma Ltd

Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
RM002 01 Page 1 of 7

Raw Water Testing (Method of Analysis) as per IP/BP/USP

1.0 Description
Clear, colorless, odorless and tasteless liquid.

2.0 pH
Apparatus: pH meter
Procedure: Immerse the electrodes in the solution being examined and measure the
pH.
Limit: Between 6.5 to 8.5

3.0 Heavy metals

Reagent: Acetate buffer pH 3.5, Thioacetamide
Test preparation: Heat 200 m in a glass-evaporating dish on a water-bath until the
volume is reduced to 20 ml. To 12 ml of the prescribed aqueous solution add 2 ml of
acetate buffer pH 3.5, mix, add 1.2 ml of thioacetamide reagent, mix immediately and
allow to stand for 2 minutes.
Standard preparation: Take 10 ml of lead standard solution (1 ppm Pb), and 2 ml of
the solution being examined. Add 2 ml of acetate buffer pH 3.5, mix, add to 1.2 ml of
thioacetamide reagent, mix immediately and allow to stand for 2 minutes
Conclusion: Any brown colour produced by test preparation should not be greater
than the colour produced by standard preparation.
Limit: Not more than 0.1 ppm.

4.0 Iron
Apparatus: UV spectrophotometer
Reagent: Conc. Hydrochloric acid, Hydroxylamine HCl, O-phenanthroline
Test Preparation: To the residue saved in total dissolved solids, add 1 ml of conc. HCl,
allowing the acid to flow the sides of the dish and 10 ml of distilled water. Carefully heat
to boiling. Filter into a 50 ml volumetric flask, and wash the filter several times with small
portion of distilled water.

Multiple Health Pharma Ltd

Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 2 of 7

Standard preparation: At the same time add to 0.3 ml, 0.5 ml, 0.8 ml and 1.0 ml of
Iron standard solution (100 mcg/ml) to separate 50 ml volumetric flask.
Colour development: Add 5 ml of Hydroxylamine HCl solution to each flask and heat
to boiling, or heat in a boiling water bath for 10 minutes. Cool, and add 0.5 ml of the O-
phenanthroline solution, Make the solution alkaline with ammonia. Make up the volume
with distilled water. Measure the absorbance at 515 nm against reagent blank.

Plot a graph of std abs against its conc.

From the graph find out the conc. of iron in sample.
Limit: NMT 0.3 ppm

5.0 Chlorides
Apparatus: Autotitrator.
Procedure: Add 20 ml of the sample into plastic beaker and titrate against 0.05 N silver
nitrate solution. Find out the chloride in terms of ppm.
Limit: Not more than 250 ppm
6.0 Sulphate
Apparatus: Balance, Muffle furnace
Reagent: Conc HCl, Barium Chloride
Procedure: Take 250 ml of the filtered sample in a 500 ml beaker and add to it 1 ml of
conc. HCl. Heat to boiling till about 50 ml remains in the beaker. Add to boiling solution
10% Barium chloride solution till complete precipitation takes place. Boil gently till the
precipitate settles down, allow it to stand on water bath for one hour. Filter through
No.32 filter paper. Wash the residue till it free from chloride. Dry the ppt and ignite in a
tared silica crucible to a constant weight.
Calculation:
mg of residue x 1000 x 0.608
= -------------------------------------------------
volume of sample taken

Limit: Not more than 200 ppm

7.0 Nitrate
Multiple Health Pharma Ltd
Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 3 of 7

Reagent: Potassium chloride, Diphenylamine, Sulphuric acid

Test preparation: Place 5 ml in a test tube immersed in iced water, add 0.4 ml of a
10.0 %w/v solution of potassium chloride 0.1 ml of diphenylamine solution
and, dropwise with shaking, 5 ml of nitrogen free Sulphuric acid. Transfer the tube to
a water-bath at 50°C. Allow it to heat for 15 min.
Standard preparation: Place 4.5 ml of nitrate free water and 0.5 ml of nitrate standard
solution (450 ppm NO3) in a test tube. add 0.4 ml of a 10.0 %w/v solution of potassium
chloride, 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of nitrogen
free sulphuric acid. Transfer the tube to a water-bath at 50°C. Allow it to heat for 15 min.
Conclusion: Any colour produced by sample should not be greater than that produced
by standard preparation.
Limit: Not more than 0.2 ppm

8.0 Total Dissolved Solids

Apparatus: Balance
Procedure: Evaporate to dryness 250 ml of the filtered sample of water inconvenient
portion in a tared 100 ml evaporating dish ad dry the residue at 105°C till to constant
weight.
Calculation:
Wt of residue in mg x 1000
= -----------------------------------
Volume of sample
Limit: Not more than 500 ppm

9.0 Total Hardness

Procedure: Transfer 250 ml of the sample into a 500 ml conical flask. Add 10 ml of
ammonium chloride-ammonium hydroxide buffer solution and titrate against 0.1M EDTA
solution using Erichrome Black T as indicator. The end point is from wine red to a true
blue colour.
Calculation:
B.R. x actual normality x Calcium eqv. of sol. (4.002) X 2.5 x 1000
= ------------------------------------------------------------------------------------
0.1 x volume of sample taken

Limit: Not more than 300 ppm

 Multiple Health Pharma Ltd
Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 4 of 7

Microbial contamination:

Test for Total Aerobic Microbial Count:

Pour-plate method:

If the water sample is expected to have a high bacterial load, perform serial dilutions to reduce the
number of organisms. For serial dilution Prepare a series of sterile dilution tubes with saline or
sterile water and add 1 mL of sample added to 9 mL of diluent for a 1:10 dilution and continue
diluting as needed (10^-1, 10^-2, 10^-3) . After that aseptically transfer 1ml of water sample into
each four different Petridis (2 of R2A and 2 of SDA), which is previously labeled and kept under
Laminar Air Flow. After that pour approximately 15-20 ml of R2A and SDA media (45-50
degree) into the respective plates and rotate the plates clockwise and anti clockwise to mix the
contents and allow to solidify and Incubate the R2A plates at 30-35oC for 3-5 days & SDA
plates at 20–25oC for 5-7 days.
Negative Control:
Pour the R2A / SDA Media in pre-sterilized and labeled plates without sample and allow
solidifying prior to incubate.

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.

 Multiple Health Pharma Ltd
Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 5 of 7

Membrane filtration method

Spray 70%IPA on the vacuum filtration unit to disinfectant the surface of filtration unit. Carefully
open the cellulose nitrate fil;ter ( 0.45 micron) with forcep and attach the membrane filter to the
vacuum filtration unit and pour the water sample (typically 100 mL, or less if the sample is
heavily contaminated) onto the membrane filter. Apply vacuum pressure to filter the water
through the membrane. The microorganisms in the sample will be trapped on the surface of the
membrane. Use sterile forceps or tweezers to transfer the membrane filter onto the surface of a
prepared agar plate ( Total plate count ,R2A), (For Coliform, MacConkey )

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. aureus ATCC 6538,
Pseudomonas aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633 and place the filter paper
on pre incubated R2A plates. Incubate the plates of bacterial cultures at 30–35oC for 3 days.

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.

The incubated plates must be monitored every 24 hours. Record the observation.

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.
 Multiple Health Pharma Ltd
Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 6 of 7

CFU/ml = Average No. of Colonies X Dilution Factor

Volume of sample tested.

Test for Specified Micro-organisms :

Take 10ml of water sample and inoculate it in to 90ml Soybean Casein Digest Medium (SCDM).
Incubate at 30-35oC for 18-24 hours.

Test for Escherichia coli:

Primary Test:

Observe the above SCDM tube for growth. If growth is present, aseptically pipette 1ml 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.

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.

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
 Multiple Health Pharma Ltd
Araihazar, Narayanganj
STANDARD TEST PROCEDURE
Department
Product
PURIFIED WATER Quality
Name
Control
Effective Next
Product Code STP No. Version Page No.
from Revision
01 Page 7 of 7

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.

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.