1. Description
Bulky white powder.2. Solubility
Practically insoluble in water and in alcohol, soluble in dilute acids.3. Identification
Reaction of magnesium
Reagent required3N Hydrochloric acid
Ammonium chloride
Ammonium Carbonate
6N Ammonium Hydroxide
Procedure: Dissolve about 1 gm sample in 20 ml of 3N Hydrochloric acid. This solution in the presence of ammonium chloride yield no more than a slightly hazy precipitate when neutralized with ammonium carbonate, but on the subsequent addition of dibasic sodium phosphate, a white, crystalline precipitate, which is insoluble in 6 N ammonium hydroxide, is formed
4. Loss on drying
Limit: Not more than 2.0 %.Procedure: Weigh 1.000 g of substance in a clean and dried pre-weighed LOD Bottle. Cover the stopper and gently shake to distribute material to not more than 10 MM height. Place the LOD Bottle in oven and remove cover and leave it also inside the oven. Dry the sample at 105° C for 2 hr. On opening the chamber, immediately close the LOD Bottle, transfer it to desiccators and bring it to room temperature. Weigh up to constant weight.
Calculation
W2 – W1
% Loss on Drying = --------------- X 100
W2 – W3
Where,
W1 = Weight of empty clean and dried LOD Bottle.
W2 = Weight of LOD Bottle + sample.
W3 = Weight of LOD Bottle + sample. (After drying)
5. Loss on Ignition
Limit: Between 30.0 and 33.0%Procedure: Ignite a clean platinum crucible at 900°c for 30 minutes, cool and weigh the crucible. Transfer about 500 mg. of the sample into the crucible and record the weight. Ignite the crucible along with the sample at 900°c for 2 hours. Cool the crucible to room temperature in a desiccator and record the weight.
Calculation
W2 – W3
%Loss on ignition = --------------- X 100
W2 – W1
Where,
W1 = Weight of empty platinum crucible.
W2 = Weight of crucible + sample.
W3 = Weight of crucible + sample. (After ignite)
6. Soluble Salts
Limit: Not more than 10 mg of residue remains.Procedure: Boil 2.0 g sample with 100 ml of water for 5 minutes in a covered beaker, filter while hot, cool, and dilute the filtrate with water to 100 ml. Titrate 50 ml of the diluted filtrate with 0.10N sulfuric acid, using methyl red as the indicator: not more than 2.0 ml of the acid is consumed. Evaporate 25 ml of the diluted filtrate to dryness, and dry at 105° for 3 hours: not more than 10 mg of residue remains.
7. Carbonates
Boil a mixture of 0.10 g sample with 5 ml of water, cool, and add 5 ml of 6N acetic acid: not more than a slight effervescence is observed.8. Calcium
Limit: Not more than 1.5%Dilute hydrochloric acid: Dilute 100 ml of hydrochloric acid with water to 1000 ml.
Lanthanum solution: To 58.65 g of lanthanum oxide add 400 ml of water, and add, gradually with stirring, 250 ml of hydrochloric acid. Stir until dissolved, dilute with water to 1000 ml, and mix.
Test preparation: Transfer 250 mg of Magnesium Hydroxide, previously dried, to a beaker, add 30 ml of Dilute hydrochloric acid and stir until dissolved, heating if necessary. Transfer the solution so obtained to a 200-ml volumetric flask containing 4 ml of Lanthanum solution, dilute with water to volume, and mix.
Standard preparations: Transfer 249.7 mg of calcium carbonate, previously dried at 300° for 3 hours and cooled in desiccators for 2 hours, to a 100-ml volumetric flask, dissolve in a minimum amount of hydrochloric acid, dilute with water to volume, and mix. Transfer 5.0, 10.0 and 15.0 ml of this stock solution to separate 1000-ml volumetric flasks, each containing 20ml of Lanthanum solution and 40 ml of dilute hydrochloric acid, add water to volume and mix.
Blank solution: Transfer 4 ml of Lanthanum solution and 10 ml of dilute hydrochloric acid to a 200-ml volumetric flask, dilute with water to volume and mix
Procedure: Concomitantly determine the absorbance of the Standard preparations and the Test preparation at the calcium emission line at 422.7 nm with a suitable atomic absorption spectrophotometer equipped with a Calcium hollow-cathode lamp and an air-acetylene flame, using the Blank solution as the blank. Plot the absorbance of the Standard preparations versus their concentrations of calcium, in µg per ml, and draw a straight line through the three points. From the line so obtained and the absorbance of the Test preparation, determine the concentration, in µg per ml, of calcium in the test preparation.
9. Heavy metals
Limit: Not more than 20 ppmTest Preparation: Dissolve 1.0 g sample in 15 ml of 3 N hydrochloric acid, and evaporate the solution on a steam bath to dryness. Toward the end of the evaporation, stir the residue frequently, disintegrate it so that finally a dry powder is obtained, dissolve the residue in 20 ml of water, and filter. To the filtrate, which should be neutral to litmus, add 2 ml of 1 N acetic acid, and dilute with water to 25 ml. Into a 50-ml color-comparison tube place 25 ml of above solution. Adjust with 1 N acetic acid or 6 N ammonium Hydroxide to a pH between 3.0 and 4.0, using short-range pH indicator paper as an external indicator, dilute with water to 40 ml, and mix.
Standard solution: Into a 50-ml color-comparison tube pipette 2 ml of Standard Lead Solution (20 µg of Pb), and dilute with water to 25 ml. Adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and 4.0, using short-range pH indicator paper as an external indicator, dilute with water to 40 ml, and mix
Procedure: To each of the three tubes containing the Standard Preparation, and the Test Preparation add 2 ml of pH 3.5 Acetate Buffer, then add 1.2 ml of Thioacetamide –glycerin base TS, dilute with water to 50 ml, mix, allow to stand for 2 minutes, and view downward over a white surface: the color of the solution from the Test Preparation is not darker than that of the solution from the Standard Preparation.
Test Preparation: Dissolve 1 g of Magnesium Hydroxide to a suitable flask add 20ml of 3 N hydrochloric acid. and a few glass beads, and digest on a hot plate in a hood until charring begins. Other suitable means of heating may be substituted. Add, dropwise and with caution, 30 percent hydrogen peroxide, allowing the reaction to subside and again heating between drops. Add the first few drops very slowly, mix carefully to prevent a rapid reaction, and discontinue heating if foaming becomes excessive. Swirl the solution in the flask to prevent the unreacted substance from caking on the walls of the flask. [ NOTE — Add peroxide whenever the mixture turns brown or darkens. ] Continue the digestion until the substance is completely destroyed, copious fumes of sulfur trioxide are evolved, and the solution is colorless. Cool, cautiously add 10 ml of water, evaporate until sulfur trioxide again is evolved, and cool. Repeat this procedure with another 10 ml of water to remove any traces of hydrogen peroxide. Cautiously dilute with 10 ml of water, and cool.
Procedure: Transfer the Test Preparation, rinsing with 10 ml of water; add 6 ml of Ammonium Citrate Solution and 2 ml of Hydroxylamine Hydrochloride Solution. Add 2 drops of phenol red, and make the solution just alkaline (red in color) by the addition of ammonium hydroxide. Cool the solution if necessary, and add 2 ml of Potassium Cyanide Solution. Immediately extract the solution with 5-ml portions of Dithizone Extraction Solution, draining off each extract into another separator, until the dithizone solution retains its green color. Shake the combined dithizone solutions for 30 seconds with 20 ml of dilute nitric acid (1 in 100), and discard the chloroform layer. Add to the acid solution 5.0 ml of Standard Dithizone Solution and 4 ml of Ammonia-Cyanide Solution, and shake for 30 seconds: the color of the chloroform layer is of no deeper shade of violet than that of a control made with 10ml of Diluted Standard Lead Solution (10 ppm) equivalent to the amount of lead permitted in the sample under examination, and the same quantities of the same reagents and in the same manner as in the test with the sample.
Procedure: Transfer about 75 mg of Magnesium Hydroxide, previously dried and accurately weighed, to a conical flask. Add 2 ml of 3 N hydrochloric acid, and swirl to dissolve. Add 100ml of water, adjust the reaction of the solution to a pH of 7 with 1 N sodium hydroxide, add 5 ml of ammonia- ammonium chloride buffer TS and 0.15 ml of Eriochrome black TS, and titrate with 0.05 M Edetate Disodium VS to a blue endpoint. Each ml of 0.05 M Edetate Disodium is equivalent to 2.916 mg of Mg(OH)2
V x M x F x 100 100
% Assay on dried basis. = ----------------------- x ------------------
0.05 x W (100- LOD)
Where:
V = Consumed volume of 0.05 M Edetate Disodium
M= Molarity of 0.05 M Edetate Disodium
F= Factor
W= Weight of substance
Procedure: Test Solution: Dissolve 5.0 g in a mixture 50 ml of 5M acetic acid and 50 ml of distilled water; not more than a slight effervescence is produced. Boil for 2 minutes, cool and dilute to 100 ml with 2M acetic acid. Filter, if necessary, through a previously ignited and weighed porcelain or silica crucible of a suitable porosity to give a clear filtrate (solution A). Reserve any residue (residue R) for the test for Substances insoluble in acetic acid.
Ferric Chloride Colorimetric Solution (FCS): Dissolve about 55 g of ferric chloride hexahydrate in enough of a mixture of 25ml of hydrochloric acid and 975 ml of water to produce 1000ml. Pipette 10ml of this solution into a 250-ml iodine flask, add 15 ml of water, 3 g of potassium iodide and 5ml of hydrochloric acid and allow the mixture to stand for 15 minutes. Dilutes with 100 ml of water and titrate the liberated iodine with 0.1M sodium thiosulphate using 0.5ml of starch solution, added towards the end of the titration, as an indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1M sodium thiosulphate is equivalent to 0.02703 g of FeCI3,6H2O. Adjust the final volume of the solution by the addition of enough of the mixture of hydrochloric acid and water so that each ml contains 0.045 gm of FeCI3,6H2O.
Cobaltous chloride Colourimetric Solution (CCS): Dissolve about 65 g of cobaltous chloride in enough of a mixture of 25ml of hydrochloric acid and 975 ml of water to produce 1000 ml. Pipette 5 ml of this solution into a 250 ml iodine flask, add 5 ml of hydrogen peroxide solution (10 volume) and 15 ml of sodium hydroxide solution, boil for 10 minutes, cool and add 2g of potassium iodide and 60 ml of dilute sulphuric acid. Dissolve the precipitate by gentle shaking, if necessary, and titrate the liberated iodine with 0.1 m sodium thiosulphate using 0.5 ml of starch solution, added towards the pink end-point, as an indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1 m sodium thiosulphate is equivalent to 0.02379g of CoCl2,6H2O. Adjust the final volume of the solution by the addition of enough of the mixture of hydrochloric acid and water so that each ml contains 0.0595g of CoCl2,6H2O.
Cupric Sulphate Colorimetric Solution (CSS): Dissolve about 65g of cupric sulfate in enough of a mixture of 25 ml of hydrochloric acid and 975 ml of water to produce 1000 ml. Pipette 10 ml of this solution into a 250 ml iodine flask, add 40 ml of water,4 ml of acetic acid, 3g of potassium iodide, and 5 ml of hydrochloric acid and titrate the liberated iodine with 0.1m sodium thiosulphate using 0.5 ml of starch solution, added towards the pale brown end-point, as indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1M sodium thiosulphate is equivalent to 0.02497g of CuSO4,5H2O. Adjust the final volume of the solution by the addition of enough of the mixture hydrochloric acid and water so that each ml contains 0.0624 g of Cu Reference Solution BS3: Take 11.2 ml of FCS, 11.2 ml of CCS, 9.0 ml of CSS in 100 ml volumetric flask and make up the volume with 1.0% w/v HCl.
Transfer to a flat-bottomed test tube of neutral glass 15 to 25 mm in diameter, a suitable volume of a liquid being examined such that the test tube is filled to a depth of 40mm. Into another match, test tube add the same volume of water or of the solvent used for preparing the solution being examined or of the reference solution stated in the individual monograph. Examined the column of liquid in diffused light by viewing down the vertical axis of the tubes against a white background.
Sample Preparation: Dissolve 2.5 g in 18 ml of brominated hydrochloric acid and 42 ml of water and remove the excess of bromine with a few drops of stannous chloride solution AsT. Into the bottle or conical flask introduce the test solution; add 5 ml of 1M potassium iodide and 10 g of zinc AsT. Immediately assemble the apparatus and immerse the flask in a water-bath at a temperature such that a uniform evolution of gas is maintained. After 40 minutes any stain produced on the mercuric chloride paper is not more intense than that obtained by treating in the same manner 1.0 ml of arsenic standard solution (10 ppm As) diluted to 50 ml with water.
Procedure: Dilute 5 ml of solution A to 15 ml with distilled water Add 10 ml of dilute nitric acid, except when nitric acid is used in the preparation of the solution, dilute to 50 ml with water and add 1 ml of 0.1 M silver nitrate. Stir immediately with a glass rod and allow standing for 5 minutes protected from light. When viewed transversely against a black background any opalescence produced is not more intense than that obtained by treating a mixture of 10.0 ml of chloride standard solution (25 ppm Cl) and 5 ml of water in the same manner.
Procedure: Test Solution: 0.6 ml of solution A diluted to 15 ml with distilled water.
To 1.0 ml of a 25.0 % w/v solution of barium chloride in a Nessler cylinder add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO4), mix and allow standing for 1 minute. Add 15 ml of test solution and 0.15 ml of 5M acetic acid. Add sufficient water to produce 50 ml, stir immediately with a glass rod and allow standing for 5 minutes. When viewed transversely against a black background any opalescence produced is not more intense than that obtained by treating in the same manner 15 ml of sulphate standard solution (10 ppm SO4) in place of the solution being examined.
Procedure: Dissolve 0.2 g sample in 7 ml of 2M hydrochloric acid and dilute to 20 ml with water. Add 2 ml of 20 % w/v solution of iron-free citric acid and 0.1 ml of thioglycolic acid, mix make alkaline with iron-free ammonia solution, dilute to 50 ml with water and allow standing for 5 minutes. Any color produced is not more intense than that obtained by treating in the same manner 2.0 ml of iron standard solution (20 ppm Fe) in place of the solution being examined.
Reagents required
Nutrient Broth
Mac Coney’s broth
Peptone water
Kovac’s reagent
Procedure: Transfer 1 g of the sample to a screw-capped container containing 50 ml of sterile nutrient broth and shake. Loosen the cap and allow standing for 1 hrs and again shaking. Loosen the cap and incubate at 37°C for 18 to 24 hrs. This is enrichment culture.
Primary test - Add 1.0 ml of the enrichment culture to a tube containing 5 ml of MacConkey broth. Incubate in a water-bath at 36°C to 38°C for 48 hours. If the contents of the tube show acid and gas carry out the secondary test.
Secondary test - Add 0.1 ml of the contents of the tubes containing (a) 5 ml of MacConkey broth, and (b) 5 ml of peptone water. Incubate in a water-bath at 43.5°C to 44.5°C for 24 hours and examine tube (a) for acid and gas and tube (b) for indole. To test for indole add 0.5 ml of Kovac’s reagent, shake well, and allow to stand for 1 minute; if a red color is produced in the reagent layer indole is present. The presence of acid and gas and of indole in the second test indicates the presence of Escherichia coli. Carry out a control test by repeating the primary and secondary tests adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli (NCTC9002) organisms, prepared from a 24-hour culture in nutrient broth, to 5 ml of MacConkey broth. The test is not valid unless the results indicate that the control contains E. coli.
10. Lead
Limit: Not more than 0.001%Test Preparation: Dissolve 1 g of Magnesium Hydroxide to a suitable flask add 20ml of 3 N hydrochloric acid. and a few glass beads, and digest on a hot plate in a hood until charring begins. Other suitable means of heating may be substituted. Add, dropwise and with caution, 30 percent hydrogen peroxide, allowing the reaction to subside and again heating between drops. Add the first few drops very slowly, mix carefully to prevent a rapid reaction, and discontinue heating if foaming becomes excessive. Swirl the solution in the flask to prevent the unreacted substance from caking on the walls of the flask. [ NOTE — Add peroxide whenever the mixture turns brown or darkens. ] Continue the digestion until the substance is completely destroyed, copious fumes of sulfur trioxide are evolved, and the solution is colorless. Cool, cautiously add 10 ml of water, evaporate until sulfur trioxide again is evolved, and cool. Repeat this procedure with another 10 ml of water to remove any traces of hydrogen peroxide. Cautiously dilute with 10 ml of water, and cool.
Procedure: Transfer the Test Preparation, rinsing with 10 ml of water; add 6 ml of Ammonium Citrate Solution and 2 ml of Hydroxylamine Hydrochloride Solution. Add 2 drops of phenol red, and make the solution just alkaline (red in color) by the addition of ammonium hydroxide. Cool the solution if necessary, and add 2 ml of Potassium Cyanide Solution. Immediately extract the solution with 5-ml portions of Dithizone Extraction Solution, draining off each extract into another separator, until the dithizone solution retains its green color. Shake the combined dithizone solutions for 30 seconds with 20 ml of dilute nitric acid (1 in 100), and discard the chloroform layer. Add to the acid solution 5.0 ml of Standard Dithizone Solution and 4 ml of Ammonia-Cyanide Solution, and shake for 30 seconds: the color of the chloroform layer is of no deeper shade of violet than that of a control made with 10ml of Diluted Standard Lead Solution (10 ppm) equivalent to the amount of lead permitted in the sample under examination, and the same quantities of the same reagents and in the same manner as in the test with the sample.
11. Assay
Limit: Not less than 95.0 percent and not more than 100.5 percent of Mg(OH)2 on the dried basis.Procedure: Transfer about 75 mg of Magnesium Hydroxide, previously dried and accurately weighed, to a conical flask. Add 2 ml of 3 N hydrochloric acid, and swirl to dissolve. Add 100ml of water, adjust the reaction of the solution to a pH of 7 with 1 N sodium hydroxide, add 5 ml of ammonia- ammonium chloride buffer TS and 0.15 ml of Eriochrome black TS, and titrate with 0.05 M Edetate Disodium VS to a blue endpoint. Each ml of 0.05 M Edetate Disodium is equivalent to 2.916 mg of Mg(OH)2
V x M x F x 100 100
% Assay on dried basis. = ----------------------- x ------------------
0.05 x W (100- LOD)
Where:
V = Consumed volume of 0.05 M Edetate Disodium
M= Molarity of 0.05 M Edetate Disodium
F= Factor
W= Weight of substance
12. Color of Solution
Limit: Solution A is not more intensely colored than reference solution BS3Procedure: Test Solution: Dissolve 5.0 g in a mixture 50 ml of 5M acetic acid and 50 ml of distilled water; not more than a slight effervescence is produced. Boil for 2 minutes, cool and dilute to 100 ml with 2M acetic acid. Filter, if necessary, through a previously ignited and weighed porcelain or silica crucible of a suitable porosity to give a clear filtrate (solution A). Reserve any residue (residue R) for the test for Substances insoluble in acetic acid.
Ferric Chloride Colorimetric Solution (FCS): Dissolve about 55 g of ferric chloride hexahydrate in enough of a mixture of 25ml of hydrochloric acid and 975 ml of water to produce 1000ml. Pipette 10ml of this solution into a 250-ml iodine flask, add 15 ml of water, 3 g of potassium iodide and 5ml of hydrochloric acid and allow the mixture to stand for 15 minutes. Dilutes with 100 ml of water and titrate the liberated iodine with 0.1M sodium thiosulphate using 0.5ml of starch solution, added towards the end of the titration, as an indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1M sodium thiosulphate is equivalent to 0.02703 g of FeCI3,6H2O. Adjust the final volume of the solution by the addition of enough of the mixture of hydrochloric acid and water so that each ml contains 0.045 gm of FeCI3,6H2O.
Cobaltous chloride Colourimetric Solution (CCS): Dissolve about 65 g of cobaltous chloride in enough of a mixture of 25ml of hydrochloric acid and 975 ml of water to produce 1000 ml. Pipette 5 ml of this solution into a 250 ml iodine flask, add 5 ml of hydrogen peroxide solution (10 volume) and 15 ml of sodium hydroxide solution, boil for 10 minutes, cool and add 2g of potassium iodide and 60 ml of dilute sulphuric acid. Dissolve the precipitate by gentle shaking, if necessary, and titrate the liberated iodine with 0.1 m sodium thiosulphate using 0.5 ml of starch solution, added towards the pink end-point, as an indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1 m sodium thiosulphate is equivalent to 0.02379g of CoCl2,6H2O. Adjust the final volume of the solution by the addition of enough of the mixture of hydrochloric acid and water so that each ml contains 0.0595g of CoCl2,6H2O.
Cupric Sulphate Colorimetric Solution (CSS): Dissolve about 65g of cupric sulfate in enough of a mixture of 25 ml of hydrochloric acid and 975 ml of water to produce 1000 ml. Pipette 10 ml of this solution into a 250 ml iodine flask, add 40 ml of water,4 ml of acetic acid, 3g of potassium iodide, and 5 ml of hydrochloric acid and titrate the liberated iodine with 0.1m sodium thiosulphate using 0.5 ml of starch solution, added towards the pale brown end-point, as indicator. Perform a blank determination and make any necessary correction. Each ml of 0.1M sodium thiosulphate is equivalent to 0.02497g of CuSO4,5H2O. Adjust the final volume of the solution by the addition of enough of the mixture hydrochloric acid and water so that each ml contains 0.0624 g of Cu Reference Solution BS3: Take 11.2 ml of FCS, 11.2 ml of CCS, 9.0 ml of CSS in 100 ml volumetric flask and make up the volume with 1.0% w/v HCl.
Transfer to a flat-bottomed test tube of neutral glass 15 to 25 mm in diameter, a suitable volume of a liquid being examined such that the test tube is filled to a depth of 40mm. Into another match, test tube add the same volume of water or of the solvent used for preparing the solution being examined or of the reference solution stated in the individual monograph. Examined the column of liquid in diffused light by viewing down the vertical axis of the tubes against a white background.
13. Arsenic
Limit: Not more than 4 ppmSample Preparation: Dissolve 2.5 g in 18 ml of brominated hydrochloric acid and 42 ml of water and remove the excess of bromine with a few drops of stannous chloride solution AsT. Into the bottle or conical flask introduce the test solution; add 5 ml of 1M potassium iodide and 10 g of zinc AsT. Immediately assemble the apparatus and immerse the flask in a water-bath at a temperature such that a uniform evolution of gas is maintained. After 40 minutes any stain produced on the mercuric chloride paper is not more intense than that obtained by treating in the same manner 1.0 ml of arsenic standard solution (10 ppm As) diluted to 50 ml with water.
14. Chloride
Limit: Not more than 0.1%Procedure: Dilute 5 ml of solution A to 15 ml with distilled water Add 10 ml of dilute nitric acid, except when nitric acid is used in the preparation of the solution, dilute to 50 ml with water and add 1 ml of 0.1 M silver nitrate. Stir immediately with a glass rod and allow standing for 5 minutes protected from light. When viewed transversely against a black background any opalescence produced is not more intense than that obtained by treating a mixture of 10.0 ml of chloride standard solution (25 ppm Cl) and 5 ml of water in the same manner.
15. Sulfate
Limit: Not more than 0.5%Procedure: Test Solution: 0.6 ml of solution A diluted to 15 ml with distilled water.
To 1.0 ml of a 25.0 % w/v solution of barium chloride in a Nessler cylinder add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO4), mix and allow standing for 1 minute. Add 15 ml of test solution and 0.15 ml of 5M acetic acid. Add sufficient water to produce 50 ml, stir immediately with a glass rod and allow standing for 5 minutes. When viewed transversely against a black background any opalescence produced is not more intense than that obtained by treating in the same manner 15 ml of sulphate standard solution (10 ppm SO4) in place of the solution being examined.
16. Iron
Limit: Not more than 0.08%Procedure: Dissolve 0.2 g sample in 7 ml of 2M hydrochloric acid and dilute to 20 ml with water. Add 2 ml of 20 % w/v solution of iron-free citric acid and 0.1 ml of thioglycolic acid, mix make alkaline with iron-free ammonia solution, dilute to 50 ml with water and allow standing for 5 minutes. Any color produced is not more intense than that obtained by treating in the same manner 2.0 ml of iron standard solution (20 ppm Fe) in place of the solution being examined.
17. Substance insoluble in acetic acid
Procedure: Any residue (residue R) obtained during the preparation of solution A in the test for the color of the solution, when washed, dried and ignited at 600°C, weighs not more than 5.0 mg.18. Microbial Limits
Limit: Escherichia coli: AbsentReagents required
Nutrient Broth
Mac Coney’s broth
Peptone water
Kovac’s reagent
Procedure: Transfer 1 g of the sample to a screw-capped container containing 50 ml of sterile nutrient broth and shake. Loosen the cap and allow standing for 1 hrs and again shaking. Loosen the cap and incubate at 37°C for 18 to 24 hrs. This is enrichment culture.
Primary test - Add 1.0 ml of the enrichment culture to a tube containing 5 ml of MacConkey broth. Incubate in a water-bath at 36°C to 38°C for 48 hours. If the contents of the tube show acid and gas carry out the secondary test.
Secondary test - Add 0.1 ml of the contents of the tubes containing (a) 5 ml of MacConkey broth, and (b) 5 ml of peptone water. Incubate in a water-bath at 43.5°C to 44.5°C for 24 hours and examine tube (a) for acid and gas and tube (b) for indole. To test for indole add 0.5 ml of Kovac’s reagent, shake well, and allow to stand for 1 minute; if a red color is produced in the reagent layer indole is present. The presence of acid and gas and of indole in the second test indicates the presence of Escherichia coli. Carry out a control test by repeating the primary and secondary tests adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli (NCTC9002) organisms, prepared from a 24-hour culture in nutrient broth, to 5 ml of MacConkey broth. The test is not valid unless the results indicate that the control contains E. coli.
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