94 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



E. Long-necked flask method. This is a useful method when large 

 quantities of culture are required. 



1. Take a flask with a long neck, fill it one-third full of broth, plug with wool 

 and autoclave (fig. 83). 



2. When cool take out the wool plug and sow the broth with a long Pasteur 

 pipette, taking every care to avoid introducing contaminations. Replace the 

 plug and push it half-way down the neck. 



3. Make a shallow constriction below the plug at A, and draw out the upper 

 end B. 



4. Connect the upper end B with the water pump : exhaust, and wash with 

 hydrogen : seal the neck above the plug in the flame in vacuo, and incubate. 



5. To withdraw the culture after incubation, cut the neck above the level of 

 the plug (p. 47), take the plug out, and aspirate the fluid into a pipette or 

 into a sterile distributing flask. [The culture may equally well be drawn up 

 into a Cobbett's bulb by the method used in the preparation of serum (p. 45).j 



F. Bottle method. Method recommended. The advantages of the method 

 are that (1) large quantities of broth can be used, and (2) the culture can be 

 very easily removed. 



1. Select a bottle of 1 or 2 litres' capacity with a mouth large enough to 

 take an india-rubber bung perforated with two medium-sized holes. Fill 

 the bottle two-thirds full of broth (fig. 84). 



FIG. 83. Flasks with long necks 

 for the cultivation of anaerobic 

 organisms. 



FIG. 84. Bottle arranged for anaerobic 

 cultivation. 



2. Take a piece of glass tubing of the same diameter as the holes in the 

 india-rubber bung, and bend it at right angles about its centre. In one 

 limb make two constrictions a short distance apart, and plug this space with 

 wool. Pass the other limb through one of the holes in the bung so that its 

 lower end projects a distance of 3 or 4 cm. below the bung. 



Take another piece of glass tubing and bend it as shown in the figure. 

 Pass the straight limb through the other hole so that it almost reaches the 

 bottom of the bottle, while the other limb terminates outside the bottle in a 

 solid point sealed in a flame. 



3. Fit the india-rubber bung firmly into the neck of the bottle and sterilize 

 at 115 C. for 20 minutes, but let the temperature rise gradually for fear of 

 cracking the bottle. 



