1. Description
Very fine, white or slightly yellowish odorless, tasteless powder or irregular white masses which are readily reducible to powder, creaks when pressed between fingers.2. Solubility
Practically insoluble in cold water and in ethanol (95%).3. Identification
A. By physical observation
Procedure: When viewed under the microscope, polyhedral granules, 2 to 23mm in size, or rounded granules, 25 to 32mm in diameter. The central helium consists of a distinct cavity or two - to five-rayed cleft; no concentric striations. Viewed between crossed Nicole prism, a distinct black cross is seen intersecting at the helium.B. By physical characteristics
Procedure: In a clean, conical flask take 1g of sample add 50 ml of water. Heat to boiling for 1 minute. Cool, thin and cloudy mucilage is produced.C. Color reaction
Reagent required0.01M iodine
Procedure: Transfer 10 ml of the mucilage obtained in “Test B” in a test-tube, add 0.05ml of 0.01M iodine, a dark blue color is produced, which disappears on heating and reappears on cooling.
4. Acidity
Limit: Not more than 2.0 ml of 0.1M sodium hydroxide is required.Reagents required
0.1M Sodium hydroxide
Phenolphthalein solution
Ethanol (70%)
Procedure: In a clean conical flask take 10 g. of the sample. Add 100 ml of ethanol (70%) previously neutralized to phenolphthalein solution. Shake for 1 hour. Filter and take 50 ml of filtrate in a dry conical flask, titrate with 0.1 M Sodium hydroxide. Not more than 2.0 ml is required to change the color of the solution.
5. pH
Limit: 4.0 –7.0Procedure: Prepare a slurry by weighing 20.0 gm of the sample with 100 ml of carbon dioxide-free water, agitate continuously at a moderate rate for 5 minutes, and then stop agitation. Immediately determine the pH to the nearest 0.1 unit.
6. Iron
Limit: Not more than 10 ppm.Reagent required
Iron standard solution (20 ppm Fe)
20% w/v citric acid (Iron free)
Thioglycollic acid
Ammonia solution (Iron free)
0.05M sulphuric acid
Hydrochloric acid
Standard preparation: Transfer 2.0 ml of iron standard solution (20 ppm Fe) into a Nessler cylinder and dilute to 50 ml with water.
Sample preparation: Dissolve the residue obtained from the test “Sulphated ash” in 4 ml hydrochloric acid with aid gentle heat dilute with water to 50 ml and mix. Transfer 25 ml of this solution in a Nessler cylinder and dilute to 50 ml with water.
Procedure: Add to each cylinder 2 ml of 20% iron free citric acid and 0.1 ml Thioglycollic acid, mix, make alkaline with iron-free ammonia solution, dilute to 50 ml with water and allow to stand for 5 minutes. View down over a white surface. Any color produced by sample solution is not more intense than that of standard solution.
7. Fluorescence
Limit: No fluorescence should be visible under screened ultra-violet light.Procedure: Spread about 1 g of the sample in a clean dry Petri dish. See under the screened ultraviolet light. No fluorescence is visible.
8. Oxidizing substance
Limit: Not more than 20 ppmReagent required
Potassium iodide
Glacial acetic acid
0.002 N Sodium Thiosulfate
Procedure: Transfer 4.0 g to a glass-stoppered, 125-ml conical flask, and add 50.0 ml of water. Insert the stopper, and swirl for 5 minutes. Decant into a glass-stoppered, 50-ml centrifuge tube, and centrifuged to clarify. Transfer 30.0 ml of clear supernatant liquid to a glass-stoppered, 125-ml conical flask. Add 1 ml of glacial acetic acid and 0.5 g to 1.0 g of potassium iodide. Insert the stopper, swirl, and allow standing for 25 to 30 minutes in the dark. Add 1 ml of starch TS, and titrate with 0.002 N sodium thiosulfate VS to the disappearance of the starch-iodine color. Perform a blank determination, and make any necessary correction. Each ml of 0.002 N sodium thiosulfate is equivalent to 34 µg of oxidant, calculated as hydrogen peroxide. Not more than 1.4 ml of 0.002 N sodium thiosulfate is required
9. Sulfated ash
Limit: Not more than 0.6%Procedure: Heat a silica crucible to redness for 10 min; allow cooling in a desiccator and weighing. Place about 1.00 g of the sample in the silica crucible, moisten with sulphuric acid, ignite gently, again moisten with sulphuric acid and ignite at about 800°C, cool, weigh again, ignite for 15 min. and repeat this procedure until two successive weighing does not differ by more than 0.5 mg.
Calculation
W3 – W1
% Sulphated Ash = --------------- X 100
W2 – W1
Where
W1 = Weight of empty platinum crucible
W2 = Weight of crucible + sample
W3 = Weight of crucible + residue (After ignition)
10. Loss on drying
Limit: Not more than 15.0%Procedure: Weigh a LOD bottle that has been dried at 105°C for one hour & cooled in a dissector. Weigh accurately about 0.2 g. of the sample in the LOD bottle. Distribute the sample as evenly as practicable by gentle sidewise shaking to a depth not exceeding 10 mm. Place the bottle is drying chamber at 130°C for 90 minutes. After drying, close the bottle and allow it to cool to room temperature in desiccators weigh again.
Calculation
W2 - W3
% Loss on drying = ------------- x 100
W2 - W1
Where
W1 = Weight of empty crucible
W2 = Weight of crucible + sample
W3 = Weight of crucible + residue
11. Sulfur Dioxide
Limit: Not more than 50 ppmProcedure: Introduce 150 ml of water into the flask and pass carbon dioxide through the whole system for 15 min at a rate of 100 ml/min. To 10 ml of dilute hydrogen peroxide solution add 0.15 ml of a 1 g/l solution of bromophenol blue in alcohol (20 percent V/V). Add 0.1 M sodium hydroxide until a violet-blue color is obtained, without exceeding the end-point. Place the solution in the test-tube. Without interrupting the stream of carbon dioxide, remove the funnel and introduce through the opening into the flask 25.0 g of the substance to be examined (mg) with the aid of 100 ml of water. Add the funnel 80 ml of dilute hydrochloric acid and boil for 1 h. Open the tap of the funnel and stop the flow of carbon dioxide and also the heating and the cooling water. Transfer the contents of the test-tube with the aid of a little water to a 200 ml wide-necked, conical flask. Heat on a water-bath for 15 min and allow cooling. Add 0.1 ml of a 1 g/l solution of bromophenol blue in alcohol (20 percent V/V) and titrate with 0.1 M sodium hydroxide until the color changes from yellow to violet-blue (V1 ml). Carry out a blank titration (V2 ml).
Calculate the content of sulfur dioxide in parts per million from the expression:
32030 x (V1-V2) x n
=----------------------------
m
12. Microbial limit
Limit: Total viable aerobic count: Not more than 1000 bacteria/gmNot more than 100 fungi/gm
E.Coli and Salmonella: Absent
A. Total viable aerobic count and fungi
Procedure: For aerobic bacteria: Dissolve or dilute 1 or 10 g./ml of the sample in 9 or 90 g./ml of sterile fluid Soybean casein digest medium containing 0.5% soy lecithin and 4% of Polysorbate 80. Allow standing for 1 hour. (4 hrs. for gelatin) at 30°c to 35°c with intermittent shaking. Plate out 1 ml of prepared sample into 90 mm Petri plates in duplicate by pour plate technique using about 15 ml of melted Soybean casein digest agar at about 45°c. or use Petri film for the total aerobic count for bacteria. Incubate the plates/Petri films at 30°c to 35°c for 5 days unless a more reliable count is obtained in shorter time. On completion of the incubation period, count number of colonies/plates or Petri film. Calculate CFU per g. or ml of the sample.For fungi: Dissolve or dilute 1 or 10g. /ml of the sample in 9or 90 g./ml of sterile fluid Soybean casein digest medium containing 0.5% soy lecithin and 4% of Polysorbate 80. Allow standing for 1 hour. (4 hrs. for gelatin) at 30°c to 35°c with intermittent shaking. Plate out 1 ml of prepared sample into 90 mm Petri plates in duplicate by pour plate technique using about 15 ml of melted Sabourad Chloremphenicol agar at about 45°c. or use Petri film for yeast and mold count for fungi. Incubate the plates/Petri films at 20°c to 25°c for 5 days unless a more reliable count is obtained in shorter time. On completion of the incubation period, count number of colonies/plates or Petri film. Calculate CFU per g. or ml of the sample.
E. Coli
Reagents requiredNutrient BrothMac Conkey’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 to stand for 1 hrs and again shake. 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° to 38° 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° to 44.5° 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.
Salmonella
Reagents required
Sterile nutrient broth
Selenite F broth
Tetrathionate bile brilliant green broth
Bismuth Sulphite agar
Brilliant green agar
Triple sugar iron agar
Urea broth
Procedure: Transfer 1g of the sample to 100 ml of sterile nutrient broth and shake. Allow to stand for 1 hour and shake again. Incubate at 37°C for 24 hours. Take in two test tubes, 1.0 ml of the enrichment culture obtained from above. To one of the tube add 10 ml of selenite F broth and to the other tube add tetrathionate bile-brilliant green broth and incubate both the tubes at 37°C for 48 hours. From each of these two culture tubes, subculture on Brilliant green agar and Bismuth sulfite agar. Incubate the plates at 37°C for 24 hours. Observe the black or green colonies on Bismuth Sulphite agar and small transparent and colorless or opaque red zone colonies on Brilliant green agar. Subculture any colony showing the above characteristics in triple sugar iron agar and at the same time into a tube of Urea broth. Incubate at 37°C for 24 hours.
The formation of acid and gas in stab culture (with or without concomitant blackening) and the absence of acidity from the surface of Triple sugar iron and absence of red color in the urea broth indicate the presence of salmonella.
Carry out the control test using 10 to 50 cells of Salmonella abony (NCTC6017), which was subcultured 24 hours before and incubated at 37°C for 24 hours.
The test is invalid if controls do not indicate the presence of salmonella.
Sterile nutrient broth
Selenite F broth
Tetrathionate bile brilliant green broth
Bismuth Sulphite agar
Brilliant green agar
Triple sugar iron agar
Urea broth
Procedure: Transfer 1g of the sample to 100 ml of sterile nutrient broth and shake. Allow to stand for 1 hour and shake again. Incubate at 37°C for 24 hours. Take in two test tubes, 1.0 ml of the enrichment culture obtained from above. To one of the tube add 10 ml of selenite F broth and to the other tube add tetrathionate bile-brilliant green broth and incubate both the tubes at 37°C for 48 hours. From each of these two culture tubes, subculture on Brilliant green agar and Bismuth sulfite agar. Incubate the plates at 37°C for 24 hours. Observe the black or green colonies on Bismuth Sulphite agar and small transparent and colorless or opaque red zone colonies on Brilliant green agar. Subculture any colony showing the above characteristics in triple sugar iron agar and at the same time into a tube of Urea broth. Incubate at 37°C for 24 hours.
The formation of acid and gas in stab culture (with or without concomitant blackening) and the absence of acidity from the surface of Triple sugar iron and absence of red color in the urea broth indicate the presence of salmonella.
Carry out the control test using 10 to 50 cells of Salmonella abony (NCTC6017), which was subcultured 24 hours before and incubated at 37°C for 24 hours.
The test is invalid if controls do not indicate the presence of salmonella.
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