EFFECTS OF INJURY UPON SPERM. 217 



by keeping them so long that they were nearly dead. Since it 

 proved to be so difficult to injure the sperm without destroying 

 their motility it seemed to be desirable to try the effects of the 

 X-rays or of the radium emanations; but as the proper appliances 

 were not procurable at the time, I was unable to carry out this 

 part of the work. An attempt was made to get results by using 

 sunlight but the sperm cells gather together in such a way that 

 they protect each other. No attempt was made to carry this 

 method far although it might prove useful with proper technique. 



G. Cytological Methods. 



For later cytological study of the early stages, a large number 

 of samples were taken from the experimental cultures and fixed 

 in Meves's modification of Flemming's fluid, made as follows: 

 3.5 c.c. osmic acid, 2 per cent.; 15 c.c. chromic acid, 0.5 per 

 cent.; 3 drops glacial acetic acid. The eggs were exposed to 

 the action of this solution for about 40 minutes. Iron haematoxy- 

 lin was used as a stain. The larval stages were preserved either 

 in Gilson's mercuro-nitrate fixing fluid or in Bouin's mixture 

 of formalin and picric acid. 



The fact should be mentioned at this point that it was found 

 to be impossible for one person to make as careful observations 

 and notes upon the living material, when preserving a series for 

 later study, as were made when doing nothing else except 

 following the developing eggs under the microscope. As a 

 consequence the preserved samples are of two kinds, viz.: (i) a 

 long close series with as much recorded data as the time allowed, 

 and (2) irregular preservations made during extended periods of 

 observation. Usually in getting a long series fixed it was neces- 

 sary, especially in the earlier stages, to run at least two series at 

 one time, since it is impossible to tell with certainty just how 

 much the sperm cells used for inseminating have been injured. 

 Again it should be noted that in the preservation of a long series, 

 those samples first fixed often contain a greater proportion of 

 the more normal eggs than those preserved later. This is due 

 to the fact that the most normal sperm cells seem to initiate 

 development earlier than do the others. Since the eggs which 

 have formed jelly are more readily picked up by the pipette, 



