SPECIAL METHODS. 235 



tannin reactions described in 199-208 may, of course, he- 

 used. Especially useful is Pfeffer's staining intra vitam 

 with methylene blue ( 208), which may be used with 

 particular advantage in developmental investigations. As 

 suitable objects for study may be suggested the cells of 

 Zygnema and Mesocarpus (cf. Fig. 58), as well as those from 

 the root-cap of Pistia Stratiotes or from the base of the 

 petiole of Dcsmanthus plenus (cf. Fig. 59). 



421. A good fixation of this coloring may be obtained 

 by placing the stained objects in a concentrated aqueous 

 solution of picric acid for 2-24 hours. They are then 

 repeatedly rinsed in pure water, then placed in 10-20^ 

 alcohol, which is gradually replaced by absolute alcohol by 

 means of Schulze's dehydrating vessel ( 16), then trans- 

 ferred to a mixture of three parts xylol and one part 

 alcohol, finally to xylol, and then mounted in balsam.* Ire 

 this way I have obtained beautiful permanent preparations 

 of Zygnema, in which, after six months, the tannin-vesicles 

 alone are stained deep blue ; while threads preserved for 

 the same time in glycerine-gelatine have almost entirely lost 

 their color. 



I have not experimented as to whether a solution of 

 ammonium picrate, recommended by Dogiel (I) for fixing 

 methylene blue stains in animal objects, is to be preferred, 

 to the watery solution of picric acid for vegetable prepara- 

 tions also. 



To obtain permanent preparations of the tannin-vacuoles,, 

 af Klercker (III) fixes the objects either with Flemnning's 

 chrom-osmic acid, or with a mixture of one volume of 

 Kleinenberg's picro-sulphuric acid and one volume of a 5$ 

 solution of potassium bichromate, or with a mixture of 

 equal parts picro-sulphuric acid and cupric acetate solution. 

 After washing, the objects are imbedded in paraffine in the 

 usual way. Finally, a much darker staining of the tannin 



* Clove-oil, phenol, or aniline must not be used in the transfer to balsam, 

 as they at once wash out the methylene blue stain. 



