174 Nitrogen Fixation by Yeasts 



EXPERIMENTAL. 



The material tested in these experiments consisted of seven 

 species of saccharomyces, six varieties of " pseudo-yeasts," 1 

 one mycoderma (Mycoderma vim) and three molds. The cultures 

 employed were kindly given to the writer by Prof. F. T. Bioletti 

 of the California Agricultural Experiment Station to whom I 

 desire, here, to express my sincere thanks. The cultures were 

 all examined microscopically and appeared to be pure cultures 

 of the organisms named in the tables. 



Series I. 



A culture solution 2 was prepared and distributed in 100 cc. 

 portions in 500 cc. Erlenmeyer flasks and sterilized in the auto- 

 clave. Each liter of solution consisted of the following: 



15.0 grams mannite. 

 0.2 gram dipotassium phosphate. 

 0.2 gram magnesium sulphate. 

 0.02 gram calcium chloride. 



3 drops of a 10 per cent solution of ferric chloride. 

 1000 cc. tap water. 



The solution was rendered slightly alkaline to phenolphthalein 

 by means of sodium hydrate. The solutions were carefully inoc- 

 ulated to prevent contamination, by means of a sterile platinum 

 loop and placed in the incubator at 26-28 C. for one month, 

 after which they were transferred to Kjeldahl digestion flasks, 

 30 cc. concentrated sulphuric acid added and boiled on the diges- 

 tion shelf until frothing ceased 3 ; then about 12 grams of a mixture 

 of K 2 S0 4 , FeS0 4 and CuS0 4 (in the proportion of 10 to 1 to i) were 

 added and the digestion continued for another hour or more. When 

 cool the digested solutions were transferred to copper distilling 



1 These organisms are described by Holm: Bull. No. 197, Cal. Expt. Sta- 

 tion. 



2 Used byLipman and Brown: New Jersey Agr. Expt. Station, Bulletin 

 No. 210. 



3 The method for the nitrogen determination used is given in detail because 

 it presents some new modifications which allow of rapid and accurate work, 

 as shown by tests in experiments which will be published shortly. 



