100 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



a 



- FlG 9 Q _ 



Boux's'method 

 of growing an- 



isms. 



3. To open the tube when growth has taken place, break off the lower end 

 over a sterile glass plate. If the upper end were opened first the pressure 

 of the gases formed during the growth of the organism would be sufficient 

 to forcibly expel the contents of the tube. 



D. Hydrogen method (Roux). This method is more difficult than 

 those just described. 



1. Take a tube of sterile gelatin, and constrict in the blow-pipe 

 just below the plug (a, fig. 90). 



2. Select a sterile Pasteur pipette the smaller end of which will 

 easily pass through the constriction, and bend it at a right angle below 

 the wool plug. Connect the plugged end of the pipette with a 

 hydrogen apparatus. 



3. Melt the gelatin in a water bath. Flame the narrow part of 

 the pipette to sterilize it, and after breaking off the point pass it 

 between the wool and the side of the tube down to the bottom of 

 the gelatin. 



4. Pass a stream of hydrogen through the medium for some minutes, 

 and then withdraw the pipette a little so that the hydrogen passes 

 over the surface of the gelatin and prevents air gaining access to the 

 medium while it is being cooled. 



5. Take out the wool plug and sow a stab culture with a fine wire, 

 fa e curr ent of hydrogen being maintained meanwhile. 



6. When the tube is sown, take out the pipette and seal the tube 

 as quickly as possible at the constricted part a. 



[E. Bulloch s apparatus can be used equally well with solid as with liquid 

 media (p. 96).] 



(ii) Surface cultures. 



Gelatin and agar. 



| Bulloch s apparatus is available for the growth of anae- 

 Tobic organisms in surface culture (pp. 96 and 102).] 



Roux s tube. Roux's tube for stroke cultures of anaerobic 

 organisms consists of an ordinary test-tube T drawn out above 

 (A) and provided with a lateral branch B (fig. 91). 



1. Pour some gelatin into the lower, wider part of the 

 tube T, using a narrow-stemmed funnel for the purpose. 

 Seal the tube at the constriction a in its upper part. Plug 

 the side tube B with wool between the two constrictions b 

 and b'. Sterilize in the autoclave. 



2. Attach B to the water pump, stand the tube in a water 

 bath at a temperature just sufficient to keep the gelatin 

 melted while the tube is exhausted and washed two or three 

 times with hydrogen. 



3. When the air has been displaced by hydrogen, leave the 

 tube in a slanting position while the gelatin sets. 



4. Then flame the upper part of A, break off the point a, 

 and sow a stroke culture through the opening ; the tube B 

 must at the same time be connected with the hydrogen- 

 generating apparatus and a stream of hydrogen passed 



into the tube to prevent the access of air. Seal the top of the tube A 

 again. 



5. It now only remains to seal B at the constriction b'. Growth then 

 takes place in an atmosphere of hydrogen. If necessary the tube can be 

 again exhausted after sowing and sealing a, before sealing b'. 



B 



FIG. 91. Roux's 

 tube for stroke cul- 

 tures of anaerobic 

 organisms. 



