1810 



Journal of Applied Microscopy 



^ 



for the purpose of absorbing any traces of oxygen that may still remain in the 

 apparatus after the current of hydrogen has been passed through. This combi- 

 nation is especially desirable, where it is not convenient to use a continuous cur- 

 rent of hydrogen, in which case the apparatus is sealed when filled with the gas. 

 Traces of oxygen that may enter the apparatus as the result of 

 a diffusion of the gases through imperfect seals will then be 

 made harmless by the absorbing power of the pyrogallol. 



Again, reducing agents may be added to the medium in ad- 

 dition to the use of any one of the other principles. The 

 relative efficiency of the various reducing agents in use has 

 been treated in chapter IV and need not be discussed here. 



Deep layers of medium are also often used in combination 

 with any one of the other principles. 



In addition to these optional combinations Nenki (1880) 

 invented the apparatus shown in Fig. 54. 



Method. — Fill the tube up to (a) with inoculated medium 

 and close it with a perforated rubber stopper. Through one 

 perforation push a glass rod (b) terminating in a glass stopper 

 of ground glass. This stopper must fit well into the constric- 

 tion at (c) so that the contents of the bulb (T) are completely 

 separated from the liquid medium above. In the second per- 

 foration insert a glass tube (d) and connect the outer end with 

 an evacuater. During evacuation the bulb is placed in a 

 water bath at 37°C., and the glass rod (b) is pulled up so that 

 the stopper is located about at (a). As soon as the air is driven 

 out, a fact which is indicated by the jerky cooking — and striking 

 of the fluid against side of the apparatus — the glass rod is 

 pushed down by a careful turn until the bulb is hermetically 

 sealed. Then, while still exhausting, the glass tube (d) is hermetically sealed in 

 the flame. When cooled the sealed tube (d) is set in a concentrated solution of 

 pyrogallic acid, the seal is broken, and when the pyrogallol solution reaches (n) 

 tube (d) is again hermetically sealed. 



Vottelier's method : Prepare about four culture tubes in the ordinary way, 

 using solid media, when inoculated close them with loose cotton plugs which are 

 pushed well down into the tubes. Pour 50 c. c. of an alkaline solution of pyro- 

 gallic acid into a common glass beaker. Cover the pyrogallol with a layer of 

 liquid paraffin 2 cm. deep. Invert the culture tubes into this beaker. Introduce 

 hydrogen into each tube for about five minutes, then place the apparatus in the 

 incubator. In addition, this writer pours on top of the liquid paraffin a layer of 

 the following mixture : Paraffin, solid, 50 parts ; cera fiava, 20 parts ; vaseline, 

 30 parts. According to Vottelier this makes an absolutely air-tight seal. The 

 author recommends the use of large test tubes for this purpose, as in case of 

 small test tubes the slanted agar slides down easily. The agar must be well 

 cooked before slanting. Before inoculation the condensation water is carefully 

 poured off and a loose, sterile cotton plug is inserted ; an occasional infection 

 is made harmless by the pyrogallic acid. 



T 



Fig. 54. 



