160 



LABORATORY EXERCISES IN BACTERIOLOGY. 



bacteria, and it is said that hydrogen sulphide is likewise favorable; carbon dioxide 

 is restrictive. Mere reduction of atmospheric pressure by the air-pump may be em- 

 ployed for the purpose, but an actual vacuum is not conducive to their development. 

 It is probable that, like other living things, these anaerobes actually require oxygen 

 for their energies, but are able to obtain the small amounts needed from the material 

 upon which they are grown. 



Various devices and special measures have been suggested for the development 

 of anaerobic organisms. In many of these, jars of some form or other are prepared 

 so that an atmosphere of nitrogen or hydrogen may be introduced to replace the ordinary 

 air. A simple type of such jar is shown in Fig. 48. The jar, cover, and rubber ring 

 should first be disinfected and rinsed in boiled water. The inoculated tube cultures 

 (without cap or rubber stopper), small plate cultures, or open Petri dishes of small 

 size, are placed in the jar, the cover adjusted (taking care that the long supply tube 

 extending from the cover does not come in contact with plate or dish media) and 

 fastened hermetically. Hydrogen gas is generated in a Kip generator (Fig. 49) or 

 other apparatus from dilute sulphuric acid and zinc clippings (gas preferably washed 

 by being passed through one or two Woulff bottles containing water), and passed 



into the supply tube, the clamps of both the supply 

 and the escape tubes being loose. The hydrogen 

 entering the lower part of the jar gradually dis- 

 places the air which passes out through the exit tube. 

 It is possible to determine the purity of the hydrogen 

 gas after it has passed through the jar (air expelled) 

 by collecting some of the escaping gas under water in ' 

 a test-tube and applying a flame. If no explosion 

 follows, the air has all been expelled. However, if 

 one will allow the gas to flow freely for about five 

 minutes, then close the clamps for a time, and after 

 five or ten minutes more (allowed for diffusion of 

 gas in the tubes of the cultures) repeat the operation, 

 he may be confident of having obtained the desired 

 condition. The Hoffmann clamps are then tight- 

 ened, and the jar placed in the proper temperature for the best development of the 

 organisms. 



Another simple method is to rely on the removal of the oxygen from the atmosphere 

 by means of an alkalinized solution of pyrogallic acid. Buchner suggests that a large 

 test-tube be employed, the inoculated culture tube being placed in the interior. A 

 rubber stopper is obtained by which to seal the large tube. Several cubic centimeters 

 of a saturated solution of pyrogallic acid are introduced into the large tube outside 

 the culture tube, and alkalinized by adding an equal amount of a strong (forty per 

 cent.) solution of sodium hydroxide, and the rubber stopper at once applied. The 

 oxygen is absorbed by the mixture, leaving an atmosphere mainly composed of nitrogen. 

 The writer has for a number of years employed with some success the measure 

 for excluding the atmosphere from the tube cultures by filling the tube after a stab 

 or diffusion inoculation has been made with the same medium as that in the tube or 

 with paraffine still liquid, but near the temperature for solidification. Sterile oil may 

 be poured over the inoculated medium for the same purpose. On plates a sheet of 

 sterile isinglass or thin glass closely applied to the surface of the medium may suffi- 

 ciently exclude the air from the underlying medium to be followed by growth of anae- 



FIG. 48. ANAEROBIC JAR. 



