CULTURES OF ANAEROBES. 67 



cultures, i.e., b from a and c from b. A current of hydrogen 

 being passed through the pieces of quill-tubing to expel the 

 air, the contents of each of the test-tubes are now sucked up 

 with the mouth into one of the quill-tubes and both the 

 ends of the latter sealed off in the flame. The agar will 

 solidify in situ and the tubes may be incubated. The 

 colonies will be observed as small grey specks. The tube 

 in which these are farthest apart must be chosen, the glass 

 notched with a file opposite to a colony to be examined, 

 and the quill-tube broken. A tube of a suitable medium 

 may now be inoculated with the growth. If there is any 

 appearance of gas formation in the agar the tubes must be 

 broken carefully, otherwise the contents may be ejected ; 

 for this reason it is desirable to take for examination a 

 tube containing very few colonies. It is even better to 

 use four tubes and make the dilution more complete. 



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 must be 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 from a colony on a 

 glucose gelatine roll-tube or a glucose agar quill-tube, 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. The needle may be pre- 

 vented from carrying down air by melting the upper half- 

 inch of the medium. This is easily effected with gelatine, 

 but in the case of agar care must of course be taken to 

 cool the part down to 42 C. In the case of agar it is 

 better to liquefy the whole tube and cool the lower part 

 until it is solid by putting it in water. From such " deep " 

 cultures growths may be maintained indefinitely by succes- 

 sive sub-cultures in similar tubes. 



Cultures of Anaerobes in Liquid Media. It is necessary 



