6 EISEN. [Vol. XVII. 



set was stained with congo, thionin, and ruthenium red, as will 

 be described below. 



The liquor-ferri-sulphurici-oxidati was used diluted about six 

 times, and the slides were kept in the solution for about twenty- 

 four hours. The haematoxylin solution was used concentrated 

 and contained about lo^o of alcohol, the solution being a year 

 old. The sections were kept in the haematoxylin solution for 

 from forty-eight to seventy-two hours, the longer time giving 

 the best results. The differentiation was made with a lo/o 

 solution of glacial acetic acid in water, to which was added a 

 small part of the liquor-ferri, sufficient to give it a very pale 

 straw color. In from fifteen to twenty minutes the differentia- 

 tion was finished. The slides were now rinsed in water and 

 counter-stained with a watery solution of congo for one or two 

 minutes, then as quickly as possible dehydrated in absolute 

 alcohol, cleared with bergamot oil, and mounted in gum-thus 

 in xylol. Several of the slides were stained over two and even 

 three times before a sufficiently satisfactory differentiation was 

 obtained. The use of congo as a counter-stain was decided on 

 only after long experiments with numerous other anilin colors, 

 and it proved to be the only stain which gave the desired 

 differentiation in the highest degree. It was the only satis- 

 factory stain for the differentiation of the spheres and their 

 secretions. 



Another combination of stains which proved useful is a triple 

 stain of congo, thionin, and ruthenium red. The slides were 

 first stained for a few seconds with a weak solution of congo 

 in water, then for about ten minutes with thionin in water, and 

 then differentiated with a watery solution of ruthenium red. 

 This latter stain was made extremely weak and of a pale rosy 

 tint ; still the differentiation was accomplished in a few minutes. 

 The ruthenium washes out the thionin, and the differentiation 

 should always be carefully watched under the microscope. 

 This combination of stains proved especially useful for differ- 

 entiating the chromoplasts, and also for the study of the out- 

 lines of the chromosomes, especially where the latter overlapped 

 each other, as the outlines of the separate chromosomes could 

 always be distinctly seen. 



