METHODS OF CULTURE AND OF EXAMINATION. 79 



from twelve to twenty-four hours in a liter of water (at 

 summer-temperature in a refrigerator). The infusion is 

 squeezed through a clean cloth by means of a press, or 

 simply with the hands, until one liter is obtained. If #. 

 smaller amount results, sufficient water is added to make a 

 liter. This meat-infusion is the starting-point, not alone 

 for the preparation of Loffler's bouillon, but also for a 

 whole series of other nutritive media. Instead of meat- 

 infusion, a five per cent, solution of Liebig's meat-extract 

 may be employed. To the meat-infusion are added ten 

 grams of peptone (one per cent.) and five grams of sodium 

 chlorid (^ of one per cent.), and the whole is heated in an 

 enameled vessel upon an open fire, until the peptone and 

 the sodium chlorid have been dissolved. At the same 

 time the coagulable albuminoid substances are precipitated. 

 The coagula floating upon the surface of the fluid are re- 

 moved with a spoon, and the fluid itself is passed through 

 a folded filter previously moistened with distilled water. 

 The fluid obtained should be perfectly clear and of acid 

 reaction. The solution is now rendered slightly, though 

 distinctly, alkaline by means of sodium hydroxid, and 

 toward the close preferably with sodium carbonate. In 

 order to collect the precipitated earthy phosphates, the fluid 

 is further heated for an hour in the autoclave at a tempera- 

 ture of 110 C. (230 F.), or in the steam-chest for two 

 hours at a temperature of 100 C. (212 F.). Then the 

 still warm solution is filtered, the filtrate, after cooling, again 

 filtered, and made up to a liter by addition of distilled 

 water. The finished bouillon should be amber-yellow in 

 color and transparent, and yield a feebly alkaline reaction. 

 Should the reaction be not alkaline, it must absolutely be 

 made so. The bouillon is now introduced into the test- 

 tubes closed with cotton stoppers, or into Erlenmeyer 

 flasks, which are best sterilized in the dry chamber. 

 Each test-tube will contain from 10 to 15 cu. cm., each 

 flask from 50 to 100 cu. cm., according to size. Finally, 

 the tubes or flasks containing the bouillon are sterilized for 

 an hour in live steam. For the cultivation of special bac- 

 teria, additions of certain substances are at times made to 

 the bouillon as, for instance, grape-sugar, in proportion 

 of two per cent, (grape-sugar bouillon), or glycerin, in pro- 

 portion of from four to six per cent, (glycerin-bouillon). 

 It is useful to have constantly in readiness a sterile ten per 



