ANAEROBIC METHODS 229 



been 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. 38, B. 



The development that now occurs is in an atmosphere 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 the organisms with which it has been inocu- 

 lated. 



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 



outlet should then be opened and the entire volume of gas allowed to escape, 

 care being taken that no flame is in the neighborhood. This should be 

 repeated, after which a sample of the hydrogen generated should be collected 

 in an inverted test-tube in the ordinary way for collecting gases over water, , 

 viz., by filling a test-tube with water, closing its mouth with the thumb, 

 inverting it, and placing its mouth under water, when, after removing the 

 thumb, the water will be kept in it by atmospheric pressure. The hydrogen 

 which is flowing from the open generator may be conducted to the test- 

 tube by rubber tubing. When the water has been replaced test the gas 

 by holding a flame near the open mouth of the test-tube. If no explo- 

 sion occurs, the hydrogen is safe to use. Should there be an explosion, the 

 generation of hydrogen must be continued in the apparatus until it burns 

 with a colorless flame when tested in a test-tube. 



