[Vol. 10 



50 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



(6) The sots thus made ready ^vere incubated at 27-28°C. for 6 

 days, preliminary tests having shown that there was no point in incu- 

 bating the cultures for more than 5 or 6 days (cf. Chambers, '20). 



(7) The culture medium in each "wasp" tube was next diluted 6 

 times by adding distilled water to the 60-cc. mark. The contents of 

 each was thoroughly mixed. 



(8) Next, exactly 10 cc. of the diluted culture medium were re- 

 moved and placed in a 50-cc. lipped centrifuge tube (pi. 2, fig. 5), 

 which was marked to correspond to the culture tube from which the 

 medium came. These 10-cc. volumes, then, represented 1/6 of the 

 original culture solution, i.e. 1.066 cc. of the original 10; and if the 

 10 cc. of the culture solution contained 0.1 gm. of a carbohydrate a 

 10-cc. sample from the diluted culture should contain 0.0166 gm. of 

 the substance provided none had been consumed. 



Each tube in the set was handled in exactly the same way, the 

 same pipette being used throughout. 



(9) Ten cc. of freshly mixed Soxhlet-Fehling's solution were 

 promptly added to the 10-cc. volumes in the centrifuge tubes. 



(10) All were quickly placed in the special baskets and immersed 

 in a water bath (pi. 2, fig. 5) where they were exposed at the boiling 

 point for exactly 10 minutes. 



(11) Upon removal from the bath the tubes were nearly filled with 

 distilled water, pairs balanced, and centrifuged 1 at a moderate speed 

 for 3-4 minutes. 



(12) Upon removal from the centrifuge the Fehling's was cau- 

 tiously decanted over a white dish, the cuprous oxide at the bottom 

 being disturbed as little as possible, for fear of loss. 



(13) This having been done each tube was again filled with dis- 

 tilled water in order to wash the precipitate, pairs balanced, and again 

 centrifuged for about 4 minutes. 



(14) When finally removed from the centrifuge the wash water 

 was decanted as completely as possible without disturbing the cuprous 

 oxide in the bottom (if any was present). 



(15) The cuprous oxide was then promptly dissolved in as small a 

 volume of the ferrous sulphate-sulphuric acid solvent as possible. 



(16) The copper was then titrated immediately against N/20 

 potassium permanganate. No attempt w r as made to remove the dis- 

 solved cuprous oxide from the centrifuge tube, the actual amount of 

 copper present being determined by titrating directly into the tube. 



(17) Using the figures thus obtained and remembering that the 10- 

 cc. sample titrated represents 1.666 cc. of the original culture medium, 



1 The No. 1 centrifuge of the International Instrument Co. was used. 



