200 Eisen: Notes on fixation, stains, the alcohol metbocl, etc. XIV, 2. 



a blue precipitate will be formed. The sections should be immersed in 

 tbis hsematoxylin bath for 12 hours or more, the longer the better. 

 Differentiation may be inade with the same liqnor ferri etc., g r e a 1 1 y 

 dilnted, or with 25 per cent. (or less) formic, acetic, or other acids, 

 or with mixtures of acids and the liqnor ferri. The result is a 

 perfectly clean preparation withont any precipitates. The chro- 

 matin and other stainable parts are stained intensely black and may 

 be differentiated to almost any degree or shade. I have entirely 

 discarded the iron-alum salts used by many, as they require ex- 

 traordiuary care and waste of time, in order to prevent precipitates 

 on the slide. One point it is of importance to observe. There should 

 be no trace of alcohol in the sections wheii placed in the liqnor 

 ferri etc., as this might cause a precipitate difficiüt to remove. 



Thionin -ruthenium -red, a double stain. I have obtained 

 most admirable resnlts by the nse of thionin and rntheninm-red in 

 staining varions tissues, such as Salamander testes and pollen mother 

 cells. This combination will produce entirely opposite resnlts accor- 

 ding to the length of time occupied by the thionin staining or the 

 age of the ruthenium mixture, as will be shown below. For nxing 

 I nse my iridium chlorid-acetic mixture as described above, prefering 

 the 1 j 2 per cent. Solution. 



Ruthenium red (Grübler) is a very expensive stain , but bap- 

 pily very small quantities will suffice. The ruthenium red is 

 dissolved in a mixture of 80 per cent. distilled and filtered water, 

 10 per cent. absolute alcohol, and 10 per cent. glycerine, which latter 

 must be as absolutely pure as can be procured. Unfortjmately 

 this mixture is apt to degenerate and oxidise in a few weeks , and 

 only a very small quantity should therefore be prepared at a time, 

 preferably a day or two before using it. When spoiled the mixture 

 will stain yellowish brown instead of red. 1 will now give tlie two 

 methods. 



I. Stain first with stronger Solution of thionin of one per cent., in 

 water with 10 per cent. of alcohol. The sections must be well 

 washed in distilled water before the stain is applied or eise some 

 kiml of thionin-cristals are deposited , which can only be removed 

 with aniline oil. This stain remaius on the slide for about live 

 minutes. Remove and rinse in distilled water and immediately add 

 to the sections a few drops of the ruthenium red mixture. The 

 latter begins to quickly wash out the thionin. and the process must 



