196 Theobald Smith 



saccharose. The bouillon was prepared by digesting fresh 

 beef in water at 60° C. for several hours then filtering and ad- 

 ding yl per cent, peptone, J^ per cent, sodium chloride and 

 about 3cc. of a normal solution of sodium carbonate for every 

 hundred cc. of the fluid. This suffices to make it feebly alka- 

 line. To this peptone bouillon 2 per cent, of one or the other 

 of the three sugars mentioned was added and the resulting 

 fluid sterilized in the fermentation tubes. 



These are kept, after inoculation, in the thermostat at 37° C. 

 A mark made on the sides of the closed branch at the end of 

 every 24 hours with a glass pencil furnishes an approximate 

 record of the rate of gas production. Unless this is done it is 

 impossible to know precisely when the formation of gas is at 

 an end and also whether or not the volume of gas has been 

 diminished by absorption. It is best to wait 4 or 5 days after 

 the production has ceased before making a final examination. 

 This is done by noting the condition of the growth, the re- 

 action of the fluid in the bulb* and the maximum quantity of 

 gas produced. This is most easily done by laying directly on 

 the tube a glass millimeter rule and noting the tube length 

 occupied by gas. The entire length of the closed branch is 

 also noted, making due allowance for the upper convex ex- 

 tremity and the lower constriction. This mode of measure- 

 ment is suflicient since only comparative values are desired. 

 For the same reason all barometic and thermometric correc- 

 tions are omitted in these approximate estimations. 



The examination of the gas produced was limited to the de- 

 termination of the quantity of carbon dioxide and of the ex- 

 plosive character of the gas remaining after the absorption of 

 CO2 by sodium hydrate. These facts are determined by the 

 following simple manipulations ; 



The bulb is completely filled with a 2 per cent, solution of 

 NaHO and closed tightly with the thumb. The fluid is 

 shaken thoroughly with the gas and allowed to flow back and 

 forth, from bulb to closed branch and the reverse several times 



*The reaction was uoted by placing a drop of the fluid on delicate 

 litmus paper. The cultures were occasionally boiled to drive off any 

 COj. In no case did the reaction with the litmus paper change. 



