iQii] A. Franklin Shull 177 



flagellate food lived readily in the filtrate, of all concentrations, 

 and when the records were made two days later it was always 

 abundant. Starvation, therefore, could play no röle in the results. 

 Table i shows the results. 



A comparison of the totals shows that there was a gradual de- 

 crease, not only in the percentage of male-producers, but in their 

 absolute number, from the line bred in pure spring water to that 

 bred in the concentrated filtrate. 



2. Influence of various undetermined constituents of feces 

 on the percentage of male-producers. After it had been deter- 

 mined that a Solution of horse manure could wholly prevent the 

 appearance of male-producers, the immediate problem was to dis- 

 cover what constituent or constituents of the feces had this effect. 

 Two methods of investigation were practicable. Substances which 

 were known to be present in horse manure, or in feces in general, 

 could be directly tested by experiment; or the Solution of manure 

 could be treated in such a way as to remove from it substances 

 having given properties, after which the effect of the substances 

 removed, or of those that remained, or of both, could be deter- 

 mined. By the latter method, the number of substances to be tried 

 by the former method might be considerably reduced. It is this 

 indirect method which is the subject of Experiments 19 to 22, 

 inclusive. The manure Solution was boiled ; it was evaporated 

 to dryness and the residue redissolved; it was evaporated to dry- 

 ness and the residue extracted with ether and alcohol; and it was 

 decolorized by boiling with animal charcoal. The following experi- 

 ments show in detail the results of these Operations. 



Experiment 19. Boiling the manure Solution. After the 

 manure Solution from an old food culture, made up with spring 

 water, had been filtered through a Berkefeld filter, part of the 

 filtrate was boiled gently for four minutes. The loss of volume by 

 evaporation was restored with distilled water. The remainder of 

 the filtrate was not treated. The boiled filtrate was kept in stock 

 and was boiled every day until exhausted, chiefly to prevent the 

 development of bacteria. The unboiled filtrate was obtained daily 

 just before using. Three pure lines of rotifers derived from sisters 

 were reared in April, 19 10, one in boiled filtrate, one in unboiled 



