148 METHODS IN PLANT HISTOLOGY 



small drop of the third solution to run under the cover. Allow the stain 

 to act for about 3 minutes. Then plunge the whole preparation into 

 water. The action should be stopped before the entire section has be- 

 come blue. Now wash the section quickly. If there are annoying, 

 granular precipitates, remove them with a soft brush. Mount in glyc- 

 erin. The membrane should be a clear blue, while the protoplast and 

 connections should be a blue-black. 



The following is Strasburger's modification of Meyer's method, and 

 shows the connections with great distinctness; and the preparations 

 are permanent. 



1. Treat the fresh sections with 1 per cent osmic acid, 5-7 minutes. 



2. Wash in water 5-10 minutes. 



3. Treat with a solution of iodine in potassium iodide (0.2 per cent iodine 

 and 1.64 per cent potassium iodide), 20-30 minutes. 



4. Transfer to 25 per cent sulphuric acid, which should act for at least 

 half an hour; 24 hours may be necessary. 



5. Bring the sections into 25 per cent sulphuric acid which has been satu- 

 rated with iodine. Add a drop of Meyer's pyoktanin solution (1 g. pyok- 

 tanin coeruleum as sold by E. Merck in Darmstadt in 30 c.c. of water). 



In about 5 minutes the sections will be stained sufficiently and can 

 be examined in glycerin. If there are annoying precipitates, remove 

 them with a soft brush. 



According to Meyer, the swelling is an advantage only when the 

 walls are very thin. When the walls are thick, the connections show 

 better without any previous swelling. 



Try the following method with the seeds of Diospyros, Latania, 

 Chamerops, Phoenix, or Fhytelephas: Soak in water and cut thin sec- 

 tions. Extract the oily and fatty substances with ether; wash in 95 per 

 cent, or in absolute alcohol; stain in anilin blue (Hoffman's blue 1 g. 

 dissolved in 150 c.c. of 50 per cent alcohol) for a few minutes. Examine 

 in glycerin. This method succeeds very well with seeds of the date. 



Permanent preparations may be secured by the following method : 



1. Fix in 1 per cent osmic acid, or in absolute alcohol, 5-10 minutes. 



2. Stain for 24 hours in Delafield's haematoxylin. 



3. Wash for a few minutes in acid alcohol (5 drops of hydrochloric acid in 

 50 c.c. of 70 per cent alcohol). 



4. Wash for a few minutes in ammonia alcohol (5 drops of ammonia to 

 50 c.c. of 70 per cent alcohol). 



5. Dehydrate in absolute alcohol, clear in xylol, and mount in balsam. 



