ESMARCH'S METHOD. 119 



may be accomplished by allowing the hydrogen to 

 bubble through the gelatin for about ten minutes. The 

 rubber stopper is then painted around with melted 

 paraffin and the tube rolled in the way given for ordi- 

 nary Esmarch tubes. 



During the operation the tube containing the liquefied 

 gelatin should be kept in a water-bath of a temperature 

 which will prevent its solidifying and at the same time 

 not kill the organisms with which it has been inoculated. 



Method of Liborius. Another method, that of Libo- 

 rius, is to fill a test-tube about three-quarters full of 

 gelatin. This is kept at the temperature of boiling water 

 for ten minutes to expel all air from it. It is then to be 

 rapidly cooled in ice-water, and, when between 30 C. 

 and 40 C., is to be inoculated, and the gelatin rapidly 

 solidified. It is then to be sealed up in the flame. 



Method of Kitasato and Weil. For favoring the 

 anaerobic 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. 



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

 the usual way an Esmarch tube of the organisms, and 

 this is to be subjected to a low temperature, and while 

 quite cold is filled with liquefied gelatin, and the whole 

 allowed 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 gela- 

 tin, as in the method of Liborius. 



By some workers the oxygen is removed by actual 

 pumping with the air-pump. 



Many other methods exist for this special purpose, but 

 for the beginner those given will suffice. 



