93 
of Chloroplasts and Leucoplasts. 
Staining with Crystal Violet. 
If the crystal violet is to be used, the procedure is as follows : 
a. Wash in water one to two hours. 
b. Equal parts pyroligneous acid (rect.) and i per cent, chromic acid, 
twenty-four hours. 
c. Two per cent, aqueous solution of potassium bichromate, twenty-four 
hours. 
d. Wash in water twenty-four hours. 
e. Dehydrate very carefully and bring into paraffin. 
/. Treat the sections on the slide with the iron mordant ((NH 4 ) 2 Fe 2 (S 0 4 ) 4 . 
24 H 2 0 ), twenty-four hours. Instead of a 4 per cent, solution, only 
3 per cent, was used. 
g. Slides are now rinsed with water. 
h. Treat with alizarin sodium sulphonate (Kahlbaum). This stain is 
made by adding from one to several cubic centimetres of a saturated 
solution in 70 per cent, alcohol to 80 to 100 c.c. of water. 
i. Pour off with water and let dry in the air, standing slide on end. 
j. Stain with Benda’s crystal violet by warming to the point of forming 
vapour. 
k . After cooling, pour off with water and let dry as above. 
/. Destain with a 10 per cent, solution of acetic acid under microscopic 
control. This requires from a few seconds to as many minutes, 
depending upon the character of the tissue and the thickness of the 
section. 
m. When the desired stain is reached, pour off with water and dehydrate 
quickly with absolute alcohol. The alcohol is immediately followed 
with clove oil-orange G, which is allowed to remain until the cell 
walls begin to show a slight orange colour, or until the desired 
counterstain is reached. 
11. Now pour off the clove oil-orange G with xylol and add cedar oil, which 
is followed by Canada balsam and a cover-glass. 
The primordia of leucoplasts and chloroplasts and the chondriosomes 
are stained blue. Although I have used the alizarin of several times the 
strength of Benda’s formula, this stain imparts only a pale rose colour to 
the nucleus, or no effect is noticeable. This stain is unnecessary. The 
crystal violet was prepared as follows as given by Benda: 
Saturated solution of crystal violet in 70 per cent, alcohol, one part ; 
10 per cent. HC1 in 70 per cent, alcohol, one part; aniline water, two 
parts. 
Staining zvith Iron-Alum-Haematoxylin. 
a. After fixation, as in the preceding process, the specimens are washed 
in water twenty-four hours (in flowing water, or in several changes). 
