82 LABORATORY EXERCISES IN BACTERIOLOGY. 



to be boiled and filtered, after which it may be distributed to tubes or flasks and ster- 

 ilized by the fractional method in steam (100 C.), or in the autoclave (120 C.). 



(6) When the bouillon is to be made from beef extract, 1000 cubic centimeters 

 of water are placed in a clean stew-pan, and five grams (various proportions have 

 been recommended, from two to five grams) of Liebig's, or other standard brand 

 of extract, added, together with ten grams of dried peptone and five grams of sodium 

 chloride. Gentle heat is applied to facilitate solution, the preparation being con- 

 stantly stirred with a clean glass rod. When solution has been effected, the liquid 

 is well boiled for from five to ten minutes, with constant stirring. It is then filtered 

 through paper, cooled, and the volume corrected to one liter, after w r hich the reaction 

 is determined and adjusted in the usual manner. It is thereafter again boiled and 

 filtered, and distributed, when it is sterilized in the usual manner. 



Exercise 25. Each student prepare one liter of bouillon from beef 

 extract as above directed, dividing the product as follows : 500 cubic centi- 

 meters for use in the manufacture of gelatine and agar; 300 cubic centi- 

 meters for manufacture of glucose-, lactose-, and saccharose-bouillon; 

 and 200 cubic centimeters for use as plain bouillon, distributing of the 

 last, sixty cubic centimeters to a dozen tubes, and placing the remainder 

 in a stock-flask for future distribution as needed. Measure out 1000 cubic 

 centimeters of distilled water into a clean granite-ware stew-pan. Add 

 five grams of Liebig's extract, which has been weighed out on a balanced 

 slip of paper (paper and contents thrown into the water), ten grams of 

 dried peptone (Witte's), and five grams of table salt. Warm and stir 

 until the above ingredients are dissolved. Boil for five to ten minutes 

 to separate any coagulable albumins. Cool to room temperature and 

 correct volume to original amount by addition of boiled distilled water. 

 Determine reaction and adjust to standard. Heat by strong boiling or 

 in autoclave for ten or fifteen minutes, and allow to cool for separation 

 of excess of phosphates. Filter and distribute, and sterilize by fractional 

 method in steam. 



(c) Glucose-, Lactose-, and Saccharose-bouillon. These preparations are used mainly 

 for the determination of fermentative qualities of various organisms, the destruction 

 of the carbohydrates setting free carbon dioxide. For their manufacture a bouillon 

 known to be free from meat sugar should be prepared, to which is added one per cent, 

 of glucose for glucose-bouillon; one per cent, of lactose for lactose-bouillon; or the 

 same proportion of saccharose for saccharose-bouillon. The presence of meat sugar 

 in a bouillon may be determined by testing with Fehling's solution or by inoculating 

 a fermentation tube filled with the sample to be tested, with a gas-forming organism, 

 like the Bacillus coli, incubating it at body temperature for twenty-four hours, when 

 gas formation will be found to have taken place if sugar be present in the bouillon. 

 Usually, bouillon made from beef extract is free from this substance and is therefore 

 of advantage in the preparation of these media. If one will inoculate a flask of ordinary 

 bouillon with the Bacillus coli communis and allow it to grow for several days at in- 

 cubator temperature, the sugar will be destroyed thereby ; and by subsequent steriliza- 



