14 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 6 



tion was carefully checked in regard to its nitrate content. 

 A nitrate determination on a 5-cc. portion by the modified 

 Devarda method did not give an amount of nitrogen detect- 

 able with N/50 acid. A blank solution was prepared contain- 

 ing saccharose and NaCl equivalent to the FeCl3 and added to 

 the different cultures in the amounts indicated below. 



Two series of cultures were prepared, the one employing 

 100 cc. of Ashby's solution in 300-cc. Erlenmeyer flasks, the 

 other the same amount in 700-cc. Erlenmeyers. The culture 

 solution was prepared as indicated above, double distilled wa- 

 ter being used. The reaction was carefully adjusted to the 

 phenolphthalein neutral point and approximately |-gm. por- 

 tions of c. p. CaC03 added to each flask. One-mg. and y i0 - 

 mg. portions of colloidal Fe 2 3 were added to certain of the 

 culture solutions. These amounts were supplied by the addi- 

 tion of the proper amounts of the iron-sugar solution or of 

 dilutions prepared from it. Corresponding amounts of the 

 NaCl sugar solutions were added to the controls. 



The culture solutions were inoculated with a suspension 

 prepared from a 72-hour slant of F 4 on Ashby's soil extract 

 agar. As much of the growth as could be removed with a 

 spiral was transferred to a 10-cc. water blank, well shaken, 

 and one spiral of the suspension used for inoculating each 

 flask of culture medium. The cultures were incubated for 

 two weeks in a warm room, after which they were analyzed 

 for total nitrogen by the Kjeldahl-Gunning method. The con- 

 tents of the cultures were transferred to 500-cc. Kjeldahl 

 flasks with ammonia-free water, the complete transfer of the 

 culture material being assisted by the addition of the 30 cc. of 

 concentrated sulphuric acid in three 10-cc. charges to the 

 Erlenmeyer flasks and subsequent washings into the Kjel- 

 dahls. Ten gms. of anhydrous sodium sulphate and 2 cc. of 

 10 per cent copper sulphate solution were added and digestion 

 carried out in the regular manner. Boiling was continued for 

 1| hours after the solutions became clear. After cooling the 

 melt was treated with 200 cc. of nitrogen-free water, 60 cc. 

 of 50 per cent alkali then added, and distillation performed 

 with an apparatus essentially the same as that used previously 



