4. BULLETIN: MUSEUM OF COMPAKATIVE ZOOLOGY. 



be allowed to act too long, it will replace the safranin entirely. This 

 (safrauin and green) method was the only one by which the archoplasm 

 was made distinct. The granular cytoplasm was stained green, and in 

 the pale green clear areas of the archoplasm were to be seen the red 

 centrosonies. In some stages the chromosomes were stained green, indi- 

 cating that a chemical change takes place in the chromatic substance. 

 But even in such cases the nucleolus was bright red. 



In using Henueguy's method, the sections were put into permanganate 

 of potash for 5 minutes, and then stained 3-20 minutes in Zwaarde- 

 maker's safranin. The mordant gives an iron-rust color to the sections, 

 and the safranin must not be too much washed out, or the sharpness of 

 outlines will be lost. It is best to wash out the mordant very thor- 

 oughly before using the stain, for the potassic permanganate makes a 

 precipitate with the safranin which renders the sections so muddy as to 

 be nearly useless. By this method the chromosomes and nucleoli are 

 stained bright red, the individual chromosomes being sharply outlined. 

 In the metamorphosis of the spermatid, the six spheroidal chromatic 

 elements are often easily distinguished, although closely massed to- 

 gether. The chromatic crescent of the spermatid is very well defined ; 

 but the small body in the neck of the spermatozoon, so conspicuous 

 after treatment by Heidenhain's method, is hardly to be seen when this 

 method is used. Centrosomes were rarely stained ; achromatic fibres of 

 ring stages were faintly stained ; the nucleus often appeared as a clear 

 lenticular space, in which were the red chromosomes. 



The best results were obtained by use of Heidenhain's method. The 

 " black " process proved more serviceable than the " blue." The only 

 mordant used was double sulphate of iron and ammonia, NH4Fe2(S04)4. 

 A 2% aqueous solution was used as mordant, and a 4% aqueous 

 solution as decolorizer. To produce the "blue" stain, the sections 

 were placed in the mordant ^1 hour, and after washing in water 

 were stained in the haematoxylin (0.5% pure hsematoxylin in HgO) 

 1-2 hours ; finally, they were washed again in water. Sometimes it is 

 necessary to decolorize a short time, say 20 minutes, in 4% NH4Fe2(S04)4. 

 The "black" stain was obtained by leaving the sections in the mordant 

 2 hours before washing in water, staining 10-12 hours in the hsema- 

 toxylin, and decolorizing 2-8 hours, finally washing as before. For 

 either process the sections should be very thin. They must be firmly 

 affixed to the slide ; for the washing is best done by a stream of tap- 

 water allowed to run over the slide. Three washings are necessary, 

 each of which should be thorough : (1) after use of the mordant, 



