Life Cycle of Hydatina Senta 317 



culture to the next since that time. Cultures were made by 

 immersing fresh horse-manure, tied up in cheese-cloth, in about 

 three times its volume of water. Manure giving a rich brown solu- 

 tion gave much better results than paler solutions. After the 

 manure had been extracted for one to several da} s, Polytomauvella 

 from an older culture was mtroduced. In from one to five days 

 it was abundant enough to use. The quantity of water in a food 

 culture varied from one pint to several quarts; the smaller cul- 

 tures were ready to use earlier, and also passed their optimum in 

 shorter time. Cultures were made up fresh at intervals of one to 

 several da\s; no culture was found to last satisfactorily longer than 

 four or five days, usually less. 



Pipettes used in handling the )oung females were heated in a 

 gas or alcohol flame before using a second time. Pipettes used for 

 food cultures were never used in transferring rotifers; at no time 

 in all my work were any rotifers of this species found in the food 

 cultures. In all experiments performed at Columbia University, 

 the Syracuse watch-glasses were heated in an oven to a tempera- 

 ture of about 200° C. before being used again. In work done at 

 Cold Spring Harbor,L.I.,the dishes containing females from which 

 I was breeding had been placed in boiling water for several min- 

 utes after previous use. Dishes for individuals to be kept only 

 until the sex of their offspring was determined, and then discarded, 

 were washed carefully and allowed to dry thoroughly, but not 

 heated. In the course of the summer, 300 of these dishes were 

 tested by placing in them water and food, but no rotifers. Not 

 a single rotifer ever appeared in any of these dishes. Further- 

 more, had there been any adhering rotifers or eggs, these would 

 have appeared later when the records were made. Had not the 

 presence of a foreign rotifer been evident from the nearly equal 

 size of two of them, it must frequently have occurred in a series 

 of families producing many males, that the two rotifers yielded 

 offspring of different sexes. Out of over nine thousand records 

 from unheated dishes, there was not one case of this kind. I con- 

 clude, therefore, that no error has been introduced by failure to 

 heat dishes before a second using. 



