68 MANUAL OP MICROBIOLOGICAL METHODS 



"Heavy metals, page 898 — To the residue obtained in 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 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 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, 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 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 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' 



