METHODS OF ISOLATION 



103 



connexion is the use of Petri dishes containing a solid medium instead of 

 tubes of a liquid medium. It will be necessary, of course, to have a glass 

 tripod or a thick sheet of cork on which to stand the dishes, in order to 

 prevent the lower ones being flooded with the pyrogallate solution. It is 

 advisable also to stand some water in a Petri dish on the top of the upper- 

 most plate.] 



2. Tube method. 



A. Esmarch's tubes. Frsenkel, Roux have adapted the Esmarch tube 

 method of isolating aerobic organisms to the isolation of 



anaerobic species. The technique recommended by these 

 authors is somewhat complicated, and is now very rarely used. 



Frsenkel's method. Fraenkel prepares an Esmarch tube (p. 81), 

 and after sowing the medium in air, displaces the latter by hydrogen 

 by means of an arrangement similar to that described in Bombicci's 

 method (p. 101). After passing the hydrogen through the medium 

 for 5 or 10 minutes the tube is rolled as in the ordinary Esmarch 

 method. 



Roux's method. Roux sterilizes the medium in a test-tube the 

 upper part of which has been narrowed by drawing it out in the 

 flame (fig. 95, left-hand figure). When cool but still liquid the 

 medium is sown. The narrowed part of the tube is then constricted 

 at two points and the wool plug pushed down between them (fig. 

 95, right-hand figure.) Attach the tube to a water pump, exhaust 

 and wash with hydrogen, seal at the upper constriction, and roll FlG h' 95 't~b ES " 

 the gelatin. To remove the colonies, cut off the upper part of the applied to anae- 

 tube and pass a platinum wire through the opening. robic cultivation. 



B. Vignal's tube. Method recommended. 1. Take a piece of glass tubing 

 about 1 metre long and 3 or 4 mm. in diameter. Draw out one end in the 

 flame and plug the other with wool. Make a constriction in the tube 3 or 

 4 cm. below the wool plug (fig. 96). Heat the tube thoroughly in the flame 



to sterilize it. 



2. Heat a tube of sterile gelatin to boiling point (the medium 

 may, if desired, be coloured with sulphindigotate of sodium). 

 Let the gelatin cool in a current of hydrogen (p. 100), but sow it 

 before it sets, also in a current of hydrogen. Rotate the tube 

 between the hands to distribute the organisms through the 

 medium. 



3. Flame the sealed end of the tube, and break off the point. 

 Dip the end into the gelatin, and aspirate the medium into the 

 tube up to the constriction A. (It is necessary to take care that 

 no bubbles of gas enter the tube.) Seal the pointed end and 

 then close the tube at A (fig. 96, A'). 



Colonies soon appear scattered through the gelatin. The 

 growth can be removed by carefully flaming the tube or washing 

 it, first in perchloride then in alcohol, in the neighbourhood of the 

 colony which it is desired to examine. Cut the tube at the steri- 

 lized part and remove the growth with a needle. 



C. Method of Liborius-Veillon. Method recommended. Liborius' agar 

 (p. 99) which is used for deep stab culture is also available for the isolation 

 of anaerobic micro-organisms. The tubes are sown by the dilution method 

 (p. 79) and cooled rapidly. For the examination and sub-cultivation of the 

 colonies, Liborius recommended turning out the agar on to the inside of the 

 lid of a sterile Petri dish and cutting out the colonies with a sterile knife, 

 a process which was not only rather difficult but exposed the colonies to 



FIG. 96 

 Vignal's tube. 



