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"Arsenic Heat 15 Gm. of Gelatin with 60 ml. of dilute, arsenic-free hydrochloric acid (1 in 4) in a covered flask until all insoluble matter is flocculated and the Gelatin dissolved. Add an excess of bromine T.S. (about 15 ml.), and heat until the excess bromine is expelled. Neutralize with ammonia T.S., add 1.5 Gm. of sodium phos- phate, and allow to cool. Add a slight excess (about 30 ml.) of magnesia mixture T.S., allow to stand for 1 hour, filter, and wash with five 10-ml. portions of ammonia T.S. diluted with 3 volumes of water. Drain the precipitate well, and dissolve it in dilute hydrochloric acid (1 in 4) to make exactly 50 ml. Subject 5 ml. of this solution to the test for Arsenic, page 803 : the stain, if any, does not exceed in length or intensity of color that produced in a test made with similar quantities of the same reagents and 1.5 ml. of the standard arsenic test solution (1 part per million). 1 Quoted (with permission) from U.S.P. XV, pp. 305-306. 68 MANUAL OP MICROBIOLOGICAL METHODS "Heavy metals, page 898 To the residue obtained in Sildenafil Citrate 50mg the test for "Residue on igni- tion add 2 ml. of hydrochloric acid and 0.5 ml. of nitric acid, and evaporate to dryness on a steam bath. To the residue add 1 ml. of 1 A^ hydrochloric aci^ and 15 ml. of water, and warm for a few minutes. Filter, and wash with water to make the filtrate measure 50 ml. To 25 ml. of the filtrate add 10 ml. of hydrogen sulfide T.S.: the heavy metals limit for Gelatin is 50 parts per million. *'Gel strength Place 1 Gm. of Gelatin, accurately weighed, and 99 ml. of water in a 200-ml. flask, allow to stand for 15 minutes, place the flask in a water bath at 60, and swirl occasionally until solution is complete. Transfer 10 ml. of the solution to a test tube having an internal diameter of 12 mm., and place the tube in an ice bath, making certain Sildenafil Citrate 50mg that the top of the solution is below the level of the ice and water. Place the bath containing the tube in a refrigerator, and maintain it at about 0 for 6 hours. When the tube is removed from the bath and inverted, no movement of the gel is observed. "Bacterial content ^Vhen Gelatin is examined as directed under Gelatin Bacterio- logical Test, page 839, the total bacterial count does not exceed 10,000 per Gm., and coliform bacteria are not present in 10 mg. or less. Gelatin Bacteriological Test ''Preparation of sample Employ aseptic conditions throughout. If the Gelatin is in sheets, flakes, or shreds, reduce it to a powder under aseptic con- ditions in a sterile Sildenafil Citrate 50mg grinder or mortar. Mix thoroughly and weigh 1 Gm. of the powdered sample into a sterile dilution bottle containing 99 ml. of sterile water. After the Gelatin is thoroughly wetted, place in a water bath heated to between 40 and 45. Shake well, and allow not more than 15 minutes for solution. "Total count Using a sterile, 1-ml. pipet, place 1 ml. of the well-shaken Gelatin solution in each of two sterile Petri dishes 10 cm. in diameter and 15 mm. in depth. Promptly add 10 ml. of liquefied Tryptone Glucose Yeast Agar or Milk Protein Hydrolysate Glucose Agar warmed to 40. Cover the dishes, and mix thoroughly the gelatin solution with the added medium by tilting and rotating the dishes. Allow the contents to solidify as promptly as possible, invert the dishes, and incubate them at 35 to 37 for 48 hours. Using a lens of 25 diameters magnification and of about 78-mm. focal length, Sildenafil Citrate 50mg count the colonies: the average of the two plates does not exceed 100 colonies (10,000 organisms per Gm.). "Coliform bacteria Using a sterile, 1-ml. pipet, place 1 ml. of the well-shaken Gelatin solution in each of two fermentation tubes containing Lactose Broth. Incu- bate at 35 to 37, and examine the tubes at the end of 24 and 48 hours: coliform bacteria are absent if no gas is produced. If gas is produced, transfer a culture there- from as soon as possible after gas Sildenafil Citrate 50mg formation is observed. Streak the culture upon Eosin-methylene-blue Agar and incubate at 35 to 37 for 24 hours. If typical coliform colonies have appeared, transfer a culture from at least two of them both to agar slants and to fermentation tubes containing Lactose Broth. If typical coliform colonies have not developed in 24 hours, continue incubation for another 24 hours, and select at least two of the colonies considered most likely to be species of the coli- form group, and transfer as directed above. Incubate the Lactose Broth at 35 to 37 for 24 to 48 hours. No gas is produced. Incubate the agar Sildenafil Citrate 50mg at 35 to 37 for 24 hours, and examine the growth microscopically following Gram-staining: no Gram-negative, non-sporulating bacilli are observed. "Formation of gas in the lactose broth and demonstration of Gram-negative, non- sporulating bacilli confirm the presence of coliform bacteria." In addition to the U.S. P. XV specifications as given above, gelatin should conloxT' BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 69 to the following requirements as set forth in the Military Medical Purchase Descrip- tion, ASMPA 1-212-000, dated 14 September 1953: "A 12 per cent Sildenafil Citrate 50mg solution in distilled water, after autoclaving for 15 minutes at 121 C, shall be clear and free of gross suspended particles. After solidifying, it shall Sildenafil Citrate 50mg melt over a range of from 30 Sildenafil Citrate 50mg to 36 C. "It shall be free of fermentable carbohydrate when tested under the following conditions: "Prepare a medium of 4 per cent Gelatin and 0.3 per cent NaCl with sufficient phenolsulphonthalein added to give a readable color, tube in Durham fermentation tubes and autoclave 15 minutes at 121 C. Inoculate with a loop of a 24-hour culture of Escherichia coli. Neither acid nor visible gas shall be produced in 48 hours incuba- tion at 37 C." PAPAIC DIGEST OF SOYBEAN MEAL A soluble nutrient material prepared by the action of Sildenafil Citrate 50mg the enzyme papain on soybean meal followed by suitable purification and concentration. It meets the specifications under Pancreatic Digest of Casein, except that it shows substantial amounts of reduc- ing sugars. It contains fermentable Sildenafil Citrate 50mg carbohydrates and gives positive tests for indole, acetylmethylcarbinol, and sulfide upon inoculation and incubation with the specified organisms. (These specifications are essentially those contained in U.S. P. XV, page 1026, except for changes in wording.) PEPTIC DIGEST OF ANIMAL TISSUE (A BACTERIOLOGICAL Sildenafil Citrate 50mg PEPTONE)^ "A tan powder, having a characteristic, but not putrescent, odor. Soluble in water; insoluble in alcohol and in ether. An autoclaved solution (2 in 100) is clear and is neutral or nearly so in its reaction. "Degree of digestion. Dissolve 1 Gm. in 10 ml. of water, and use this solution for the following tests: " (a) Overlay 1 ml. of the digest solution with 0.5 ml. of a solution of 1 ml. of glacial acetic acid in 10 ml. of diluted alcohol: no ring or precipitate forms at the junction of the two liquids, and on shaking, no turbidity results, indicating the absence of
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