CULTURES OF ANAEROBES 65 



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

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

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

 of the latter. A solution of 109 grms. 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 to 

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

 into the chamber and spreads over the bottom of the shallow 

 dish ; potassium pyrogallate is thus 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 boiled water, and this, 

 passing through the glass tubes, washes out the potash 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. Sometimes 

 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. 



Carroll's Method for Anaerobic Cultures. This may be used 

 with culture tubes containing any of the media suitable for 

 anaerobes, with Esmarch's roll-tubes, or with fermentation tubes. 

 There is required a dry tube of the same diameter as the culture 

 tube, a short U-shaped glass tube, and two pieces of rubber tub- 

 ing all of like diameter. The culture tube having been inoculated, 

 the plug is pushed home below the lip of the tube. The ends 

 of the U-tube are smeared with vaseline and a rubber tube 

 slipped over each ; the end of the culture tube being similarly 



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