[Vol. 6 



18 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



stored in a tin-lined copper tank and from here distributed to 

 the laboratories by a system of block tin tubes. The redis- 

 tilled water was prepared by distilling this water over acid 

 permanganate. The connection between the block tin con- 

 denser and the distilling flask was made by wadding with ab- 

 sorbent cotton. The results obtained are reported in table 

 in below. The data on redistilled water are, it will be noted, 

 those reported before in table i as cultures 1, 2, and 3. 



TABLE III 



INFLUENCE OF DIFFERENT WATERS USED IN PREPARATION OF MODIFIED 



ASHBY'S NUTRIENT SOLUTION 



No. 



Water used 



Treatment 



N 

 found 

 (mgs.) 



N 



fixed 



(mgs.) 



1 



Double distilled 

 Double distilled 

 Double distilled 



Stock distilled 

 Stock distilled 

 Stock distilled 



Tap 

 Tap 

 Tap 



Sterile 



Inoc. 



Inoc. 



Sterile 



Inoc. 



Inoc. 



Sterile 



Inoc. 



Inoc. 



1.31 

 2.59 

 1.98 



1.03 



3.58 

 2.78 



1.26 

 3.49 

 4.45 





2 

 3 



13 



1.28 

 0.67 



14 

 15 



16 



2.55 

 1.75 



17 

 18 



2.23 

 3.19 



The differences observed are not wide but serve to show the 

 order of magnitude of the effect produced by different waters, 

 and also illustrate the fact that something is lacking in Ash- 

 by 's solution. 



As mentioned above, very great difficulty was experienced 

 in digesting the cultures according to the Kjeldahl-Gunning 

 method. We decided next to see to what extent this difficulty 

 could be overcome by using a very much smaller amount of 

 material, a principle that finds extended use in the " micro" 

 methods of biological chemistry. Accordingly, 10-cc. portions 

 of Ashby's solution were placed in 60-cc. Erlenmeyer flasks. 

 The medium was made as above except that tap water was 

 used. Calcium carbonate was omitted in one-half the culture 

 flasks, since, as mentioned above, it seemed to exert a quite 

 marked precipitating effect on the colloidal ferric oxide which 

 was added in the amounts of 0.01, 0.05, and .1 mg. per 10 cc. of 

 culture solution. The flasks were inoculated with a spiral 



