552 POWDERED DRUGS 



chopped lean beef with water and allowing it to stand for twenty-four hours in an 

 ice-chest, after which it is to be filtered through a muslin bag, using pressure of the 

 hands to make the filtration more complete. The filtrate is then cooked and again 

 filtered, and neutralized by the gradual addition of a solution of carbonate of soda. 

 The solution should be tested with litmus paper, and the addition of carbonate of 

 soda should cease as soon as neutralization is accomplished. To this solution is 

 added 0.5 per cent, "of common salt. Ten Gm. of peptone may be added to a liter 

 of the infusion. 



In place of the meat infusion as prepared above, meat extract may be used in 

 the ratio of 4 to 5 Gm. per liter of water. 



Bouillon is prepared by adding i liter of water to i pound of chopped lean beef. 

 This is cooked for half an hour, then filtered and neutralized with carbonate of soda, 

 then again boiled for an hour to precipitate albuminoids. After a final filtering the 

 bouillon is poured into flasks or test-tubes and sterilized. 



Infusions of hay and dried fruits may also be used for nutrient media. A hay 

 infusion for the growth of Bacillus subtilis may be prepared as follows: Chopped 

 hay is placed in a beaker and barely covered with well water; this is kept in an 

 incubator at a temperature of 36C. for four hours, after which time the extract 

 is poured off and diluted, if necessary, to a specific gravity of about 1.004. The 

 extract is now poured into a flask which, having been closed with a cotton plug, 

 is placed in a steam sterilizer and subjected to a gentle evolution of steam for 

 about an hour. The flask is then placed in an incubator at 36C. for a day or two, 

 after which time a film produced by colonies of Bacillus subtilis will have formed 

 over the surface of the extract. The spores of this bacterium are particularly 

 resistant to heat, and for this reason while the spores of other bacteria are killed 

 by the process of steaming, those of Bacillus subtilis still retain their vitality. 



Solid culture media may be prepared by adding to any of the fluid culture media 

 a sufficient amount of a gelatinous substance to keep the mixture from liquefying 

 at the temperature of the laboratory or, if desired, at the higher temperature of an 

 incubator. One of the most used of the solid media is prepared by adding to the 

 peptonized infusion of meat, as above described, 10 per cent, of the best French 

 gelatine. The gelatine may be increased up to twice this amount, as the tempera- 

 ture may require. One hundred grams of gelatine is allowed to soak in i liter of 

 the meat infusion until the gelatine becomes swollen, and then a gentle heat is 

 applied until the gelatine is completely dissolved. After the gelatine is dissolved 

 the solution should again be neutralized, if necessary, with carbonate of soda. 

 When the solution stands at a temperature of about 5OC., an egg stirred up in 100 

 Gm. of water is added while the mixture is stirred with a glass rod. The mixture 

 is then kept at the boiling-point for about ten minutes. This coagulates the egg- 

 albumen and clarifies the liquid. The clarified liquid is now filtered by means of 

 a hot-water funnel or while kept warm in an incubator, the high temperature 

 being necessary for the reason that the mixture would become stiff at a low tem- 

 perature, and so incapable of being filtered. The medium should be distributed 

 while warm in sterilized test-tubes or flasks, which are then stoppered with baked 

 cotton plugs. It should then be subjected to a temperature of 100 in the steam 

 sterilizer for 10 minutes at 4 successive intervals of 24 hours. For the reason that 

 gelatine loses its power of solidifying at ordinary temperatures after being sub- 

 jected to the temperature of boiling water for a long period, the time of each 

 sterilization is necessarily reduced to about 10 minutes and the number of steriliza- 

 tions is increased to 4; whereas with other solidifying substances, such as agar-agar, 

 the length of each sterilization may extend to I hour, and the number of steriliza- 

 tions need be only 2 or 3. 



In pouring the filtered medium into the test-tubes care should be taken not to 



