CULTIVATION IN LIQUID MEDIA 



95 



4. When the apparatus has cooled, ascertain that the bung fits firmly, and 

 then lute the joints between the bung and the neck of the bottle and between 

 the tubes and the bung with Golaz's [or paraffin] wax. Dry the wool plug 

 in A by gently heating the glass tube in a Bunsen burner. 



5. In order to sow the broth, flame the external limb of B, and break off 

 the point with sterile forceps. Dip the end into the tube containing the 

 organism to be cultivated; aspirate a few drops into the bottle through A. 

 and seal the point of B in the flame. 



6. Connect A to the water pump. Exhaust and wash with hydrogen 

 several times, keeping the lower two-thirds of the bottle in a bath of water 

 at 35-40 C. 



7. Seal A in the flame in vacuo at the constriction beyond the wool plug. 

 Incubate. 



After incubating for 2 or 3 days the gas produced as the result of the growth 

 of the organism accumulates to such an extent as to prevent further multiplica- 

 tion. At this stage it is well to break off the sealed end of A (leaving the cotton- 

 wool plug in position, of course) to allow the pent-up gases to escape ; the pressure 

 of the gases remaining in the bottle and continuously generated by the growth of 

 the organism is sufficient to prevent the entrance of air. 



It is as well to add a little calcium carbonate or tricalcium phosphate to the 

 medium before sterilizing it, because with some organisms the amount of acid produced 

 is so considerable as very soon to interfere with and perhaps altogether check the 

 growth. If these salts be added, the acids will be neutralized as they are formed. 



8. To withdraw the culture from the bottle, flame the end of B and break 

 off the point. Blow through A and collect the culture in a sterile flask. 



G. Pyrogallol method. Buchner's tube. 1. Boil a tube of sterile broth, 

 cool rapidly, and sow. 



2. Place this tube as already described at p. 89 inside a larger tube containing 

 a solution of potassium pyrogallate, and incubate (fig. 85). 





FIG. 85. Buchner's 

 tube for growing anae- 

 robic organisms. 



FIG. 86. Turro's tube. 



Turro's tube. This method has advantages over Buchner's in that the oxygen is 

 much more rapidly absorbed and the culture is visible during incubation (fig. 86). 



1. Pour the medium (broth, agar or gelatin) into A through the narrow tube a. 

 Plug the upper end of the apparatus with an india-rubber stopper C and autoclave. 



