82 CHARLES V. MOKKII.L. 



Chelinidea being intermediate; these size differences correspond 

 roughly with the difference in size of the several species. All the 

 eggs, whether in the oviduct or after laying, are enclosed in a 

 tough brown chorion. 



Several different fixing fluids were tried. Flemming's strong 

 fluid, Gilson's mercuro-nitric and Bouin's picroformol wnv found 

 very uncertain in result, as they seldom penetrate the thick 

 chorion. All these can be used, however, if the eggs are pricked 

 with fine needles before placing them in the fixing fluid, but their 

 action is such as to render the yolk very brittle and difficult to 

 cut. By far the best results were obtained by placing the eggs 

 immediately in the Gilson-Carnoy acetic-alcohol-chloroform-sub- 

 limate mixture for fifteen to thirty minutes or in a mixture of 

 glacial acetic, one part, absolute alcohol saturated with sub- 

 limate, two parts, for five to ten minutes. After either fluid, 

 the eggs w'ere transferred to iodized 95 per cent, alcohol for twelve 

 hours and preserved in 80 per cent, alcohol. The acetic-alcohol- 

 sublimate mixture was found invaluable for the earliest stages 

 of maturation which occur while the eggs are still in the lower 

 part of the oviduct and directly after laying. For later stages, 

 the Gilson-Carnoy mixture gave excellent results. After immer- 

 sion in alcohol, the egg shrinks away from the chorion which can 

 then be removed with fine forceps and cutting needle. After 

 removing the chorion, the eggs were dehydrated, cleared in ced.n- 

 oil and immersed in melted paraffin for two hours. They WITI- 

 then oriented in a drop of paraffin and embedded. Serial sec- 

 tions were cut 6-8 n thick on a sliding microtome. Very good 

 series can be obtained in this way though the yolk sometimes 

 becomes brittle and troublesome. The stain most frequently 

 employed was iron-haematoxylin with or without a counter-stain. 

 In addition to the eggs, ovaries and testes were fixed in Flem- 

 ming's strong fluid and stained in iron-haematoxylin or >al"ranin. 



About twelve hundred egg- in all were sectioned, but owing 

 to mechanical difficulties in technique, only about two hundred 

 of these were of any value for study. In the maturation and 

 fertilization stages particularly, one or two poor sections may 

 render an entire series worthless, though in later embryonic stages 

 this difficulty is not so serious. For this reason the rr-ulis an- 



