(5) The supernatant fluid from a bacterial suspension in a NaCl solu- 

 tion, the bacteria having been removed by the spatula method. 



(6) The supernatant fluid from a bacterial suspension in distilled 

 water, the growth having been removed as in (5). 



(7) The NaCl solution used to wash a glass slide manipulated over a 

 fresh agar surface as though growth was being removed. 



(8) The distilled water wash obtained as in (7) . 



The tabulation below gives the results of the qualitative tests for 

 phosphate, chloride, protein and protein split products, and dextrose. 

 The numbers in parentheses refer to the several descriptions listed above. 



Note: With medium containing I per cent, dextrose (i) to (4) gave 

 for sugar. 



result 



These experiments show that: 



(1) Substances are transferred from the medium to the wash solution . 



(2) Bacteria suspended in distilled water or NaCl solution may give 

 up some of their substance to the surrounding liquid. 



(3) Nothing is removed from a medium by the mere application of a 

 smooth spatula (70). 



This evidence points definitely to the second method as the superior 

 one. 



(d) Drying of growth: The technic was that recommended by Shackell 

 (61) with one exception: the bacteria were not frozen before drying. 

 Layers I to 2 mm. thick in shallow plates dried to a scaly, porous mass 

 in 24 to 36 hours. The temperature during the process varied between 21 

 and 22 C., the pressure between o and 10 mm. Hg. 



The material was then pulverized with glass mortar and pestle to a 

 very fine powder, spread in thin layers on glazed paper plates and dried 

 to constant weight. 



The dried substance was transferred to weighing bottles fitted with 

 ground glass stoppers and stored in a dessiccator over concentrated 

 H 2 SO 4 . 



IV. ANALYSIS OF THE BACTERIAL SUBSTANCE 

 i. METHODS: 



(a) Nitrogen: Nitrogen was determined by the Kjeldahl process 

 (Gunning-Arnold Dyer modification) (62) . Digestion of the substance 

 in concentrated sulphuric acid was completed with small quantities of 

 metallic mercury. Before distillation, the mercury was precipitated with 

 potassium sulphide. The titrations were made with 0.02 M NaOH. 



16 



