CULTIVATION WITHOUT OXYGEN. 199 



same time not high enough to kill the organisms with 

 which it has been inoculated. 



One of the obstacles to the successful performance of 

 this method is the bubbling of the gelatin, the foam 

 from which will often fill the exit tube and sometimes 

 be forced from it. This may be obviated by reversing 

 the order of proceeding, viz. : roll the Esmarch tube 

 in the ordinary way with the organisms to be studied, 

 using a relatively small amount of gelatin, so as to 

 have as thin a layer as possible when it is rolled. 

 Theu replace the cotton plug with the sterilized rubber 

 stopper carrying the glass tubes through which the 

 hydrogen is to be passed, and allow the hydrogen to 

 flow through just as in the method first given. The 

 gas now passes over the gelatin instead of through it, and 

 consequently no bubbling results. In all other respects 

 the procedure is the same as that given by Frankel. 



Method of Kitasato and Weil. For favoring the an- 

 aerobic conditions Kitasato and Weil have suggested 

 the addition to the culture media of some strong re- 

 ducing agent. They recommend formic acid in 0.3 to 

 0.5 per cent.; glucose in 1.5 to 2 per cent.; or blue 

 litmus tincture in 5 per cent, by volume. This is, of 

 course, in addition to an atmosphere from which all 

 oxygen has been expelled. 



Esmarch' s method. Esmarch' s plan is to prepare in 

 the usual way a roll tube of the organisms; subject it 

 to a low temperature, and while quite cold fill it with 

 liquefied gelatin, which is caused to solidify rapidly. 

 In this method the colonies develop along the sides of 

 the tubes, and can more easily be studied than where 

 they are mixed through the gelatin, as in the method of 

 Liborius. 



