CULTIVATION WITHOUT OXYGEN. 225 



bubbling through the gelatin for about five minutes 

 (at least) one can be reasonably sure that all oxygen 

 has been expelled. The drawn-out portions of the 

 tubes can then be sealed in the gas-flame without fear 

 of an explosion. The protruding end of the rubber 

 stopper is then painted around with melted paraffin 

 and the tube rolled in the way given for ordinary 

 Esmarch tubes. A tube thus prepared and containing 

 growing colonies is shown in Fig. 40, B. 



The development that now occurs is in an atmos- 

 phere of hydrogen, all oxygen having been expelled. 

 During the operation the tube containing the liquefied 

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

 sufficiently high to prevent its solidifying, and at the 

 same time not high enough to kill thf; 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. 

 Then 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 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 an- 



15 



