298 Elliot R. Downing, 



Heemakn's solution has been used in place of the Meekel's with 

 almost equally good results. Stroug' Flemming. Müllee's fluid and 

 Zenkee's have all been used, with fair results, both as killing 

 ag-ents and to harden after the osmic acid; the material so preserved 

 has been valuable in checking- the results obtained from the osmic- 

 Meekel method. After the usual treatment in the fixing- fluids, the 

 animals have been dehydrated in the several grades of alcohol, 

 cleared in cedar oil and imbedded in parafin. Sections 5—8 u thick 

 answer best for the ovogenesis. 



Staining has been done on the slide. The best stains found 

 are Heidenhain's ironhaematoxylin with Bordeaux red or gentian 

 violet for counter stain or gentian violet for the nuclear stain and 

 Bordeaux red as counter stain. Safranin and gentian violet have 

 given some excellent results, particularly with the thicker sections. 

 The gentian violet is especially useful as it stains the endodermal 

 gland cells distinctly, brings out the cell walls everywhere plainly 

 and colors the developing nettle cells intensely so that their earlj^ 

 stages are readily distinguished from the oogonia which stain much 

 more lightly. As intravitam stains, methyl blue and neutral red 

 have been used; the latter is particularly satisfactory. Sudan III 

 has been used to stain the fats in fresh material. 



Many phases of the egg formation may be watched in living 

 material that is lightly stained. The earlier observations, even those 

 of Kleinexbeeg, were made on such living specimens or on macerated 

 and teased material. Hertwig's macerating fluid has been found 

 valuable for separating the tissue elements and considerable labor 

 has been expended in attempting to study the egg development in 

 such preparations; occasionally they are instructive but nothing like 

 as satisfactory views are obtained as sections give. The earlier 

 workers, by these methods, must have acquired marvellous dexterity 

 in manipulation or else unlimited patience. 



The Time of Sexual Maturity. 



The time of sexual maturity is subject to wide fluctuations even 

 in the same species; it is probably, however a seasonal phenomenon. 

 In general H. grisea and H. viridis are found sexually mature in 

 spring and summer, H. fusca and H. dioecia in fall and early winter, 

 H. dioecia rather later than H. fusca. Possible' unusual seasonal 

 changes will account for the erratic times of maturity that have 

 been recorded. It is interesting to note that H. viridis, usually 



