KARYOKINESIS. 7 



Delafield's hfematuxylin . 10 cc. 



Distilled water . 40 ce. 



Kleiiieuberg's picro-sulphuric fluid . . . . .10 drops. 



Eggs so stained are washed in alcohol, deh^vdrated and niouuted in balsam. If the eggs of C. 

 plana are mounted under a thin cover, supported at one side by a bit of glass .15 mm. thick (the eggs 

 are about .136 mm. in diameter), they can be studied under an immersion lens and the relative posi- 

 tions of nuclei, centrosomes, spheres and mid-bodies ("Zwischenkorpern"), can be determined as would 

 be impossible in sections. 



I have tried many modifications of this stain, but have found none so good as the one given. 

 The yolk is left transparent yellow, while the protoplasm is of a rosy tint, and the chromatin, centro- 

 somes and mid-bodies are blue or black. I have found by experience with the eggs of many mollusks, 

 annelids, Crustacea and insects, that this method is especially u.seful with eggs in which there is much yolk. 



Eggs fixed in Kleinenberg's fluid are always more transparent than those fixed in Boveri's, the 

 yolk stains less and the spheres, centrosomes, spindles and mid bodies stand out more clearly. On the 

 other hand the structure of the cytoplasm is not so well preserved in Kleinenberg's as in Boveri's fluid. 



For sectioning the following fixing fluids have given good results: Fleraming's, Hermann's, 

 Boveri's picro acetic, Graf's picro-forraol ; while the following were less satisfactory: Kleinenberg's 

 fluid, Pereuyi's fluid, chromo-acetic, chromo-formic, sublimate-acetic, sublimate (sat. sol. K Many 

 different stains were used, among them Heidenhain's iron alum and Delafield's haniiatoxyliu, either 

 alone or in combination with orange G., Bordeau red, eosin or acid fuchsin ; Hermann's saflranin- 

 gentian-iodine ; Biondi-Heidenhain's mixture; Auerbach's acid fuchsin and methyl green, etc. The 

 best results were obtained with material fixed in Boveri's picro-acetic and stained in Delafield's 

 hsematoxylin and orange G.' 



Many preparations which were so stained were afterwards decolorized and restained with iron 

 hsematoxylin and Bordeau red. In this way it was possible to observe certain details of structure which 

 could not otherwise be determined with certainty. Since there are always devotees of certain fixing and 

 staining media who refuse to accept results unless their favorite methods have been employed, I may 

 say here that every important result of my work has been confirmed by material fixed and stained in 

 each and every one of the methods enumerated above. These results, therefore, cannot be attributed 

 to the exclusive use of one or two media. 



In general the eggs were imbedded in parafiine in the usual vcay, but owing to the fact that they 

 contain a large quantity of yolk and are, therefore, more or less friable, some were doubly imbedded in 

 celloidin and paratfine, after the method suggested by Kultschitzky ('87) and by Ryder ('88 K This 

 method is especially valuable for tracing the movements of the pronuclei and spheres through the yolk. 

 The advantages of this method over ordinary celloidin imbedding are that thinner sections can be had, 

 and that these can be cut in series or ribbons. Its most serious disadvantage is that the sections are 

 usually much folded and cannot easily be flattened. 



MATURATION. 



A. Pre-division Stages. 



1. The Ovarian Egg. — In the stages just before its escape from the ovary, 

 the egg of C. plana is irregular or poh^gonal in outline, being pressed into this 

 shape by surrounding egg.s, and it contains a very large germinal vesicle located 

 near the center of the cell. The egg is filled with yolk spherules, which vary 

 greatly in size ; there is extremely little cytoplasm visible, and this lies close 

 around the nucleus. The latter contains a single large nucleolus, within which is a 



' The great value of Heidenhain's iron hceniatoxylin method I have had abundant occason to 

 verify. I have used it extensively in this work and for certain purposes it is without an ecjual ; hut 

 having said this, I wish to add that it is not by any means the liest stain for all purposes and with all 

 material, as some recent workers seem to suppose. It is not, as is now well known, a specific centrosome 

 stain. In all molluscau eggs which I have examined the yolk stains as denselv and retains the stain 

 almost as tenaceously as the centrosomes. For this reason I have found it impossii)le by this method to 

 recognize small centrosomes and asters in a dense mass of yolk, whereas with the piero-h;eniatoxvlin the 

 yolk is left a clear yellow, while the a.ster is red and the centrosome black. The iron luematoxlin is also 

 a less delicate stain for spheres and cytoplasm than i)icro-lueniatoxylin while, of course, it cannot be u.sed 

 at all in the preparation of entire eggs. 



