62 METHODS OF CULTIVATION OF BACTERIA. 



resting on the edges of the dish, or a beaker containing culture 

 tubes can be placed in it. The bell-jar is then placed in posi- 

 tion so that the longer glass tube is situated over that part of 

 the bottom of the shallow dish farthest away from the pyrogallic 

 acid, and the bottom and stoppers are luted. The air in the bell- 

 jar is now expelled by passing a current of coal-gas through 

 the short glass tube, and both stoppers are closed. A partial 

 vacuum is then effected in the jar by connecting the short tube 

 up with an air-pump, opening the tap, and giving a few strokes 

 of the latter. A solution of 7 grammes solid caustic potash dis- 

 solved in 145 c.c. water is made, and into the vessel containing 

 it a rubber tube connected with the long glass tube is made ta 

 dip, and the stopper of the latter being opened, the fluid is forced 

 into the chamber, spreads over the bottom of the shallow dish, 

 and, coming in contact with the acid, sodium pyrogallate is 

 formed which absorbs any free oxygen still present. Before the 

 whole of the fluid is forced in, the rubber tube is placed in a 

 little water, and this, passing through the glass tubes, washes- 

 out the soda and prevents erosion of the glass. The whole 

 apparatus may be placed in the incubator till growth occurs. 



It is often advisable in dealing with material suspected tO' 

 contain anaerobes to inoculate an ordinary deep glucose agar 

 tube with it, and incubating for 24 or 48 hours, to then apply an 

 anaerobic separation method to the resultant growth. Some- 

 times the high powers of resistance of spores to heat may be 

 taken advantage of in aiding the separation (vide "Tetanus"). 



Cultures of Anaerobes. — When by one or other of the above 

 methods separate colonies have been obtained, growth may be 

 maintained on media in contact with ordinary air. The media 

 generally used are those which contain reducing agents, and the 

 test-tubes containing the medium must be filled to a depth of 4 

 inches. They are sterilised as usual and are called "deep" 

 tubes. The long straight platinum wire is used for inoculating, 

 and it is plunged well down into the " deep " tube. A little air- 

 gets into the upper part of the needle track, and no growth 

 takes place there, but in the lower part of the needle track 

 growth occurs. From such " deep " cultures growths may be 

 maintained indefinitely by successive sub-cultures in similar 

 tubes. Even ordinary gelatin and agar can be used in the same 

 way if the medium is heated to boiling-point before use to expel 

 any absorbed oxygen. 



