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is determined by trial, the preparations are washed for about 15 minutes 
in gently flowing water. They are now dehydrated by treating with absolute 
aleohol, after which they may be counter stained with clove oil orange G 
or merely cleared in clove oil and cedar oil and mounted in balsam. 
In case counter staining is desired, the process should be watched in 
order to avoid over-staining with the orange. In some cases the clove oil 
orange G need remain on the sections but half a minute. This stain may be 
removed by xylol, cedar oil, or pure clove oil, when the preparation is ready 
to be mounted in balsam. 
By this method the primordia of chloroplasts and leucoplasts and other 
similar bodies are stained black or a blue-black. 
The foregoing is much simpler than Benda’s procedure, and it gives results 
that are as satisfactory. However, since it is desirable in cytological studies 
to check up one method with another, the procedure devised by Benda is 
recommended, although it is more tedious and time-consuming. The follow- 
ing modification of Benda’s method has been used with excellent results: 
1. Fix in chrom-osmiec acid of the above-mentioned composition 24 to 
48 hours. 
2. Wash in water 1 to 2 hours. 
3. Treat objects with equal parts pyroligneous acid (rectified) and 1 per 
cent. chromic acid 24 hours. 
4. Treat with 2 per cent. solution bichromate of potassium 24 hours. 
5. Wash in water 24 hours. 
6. Bring into paraffin and section in case sections are to be made, 
7. Treat with the iron mordant 12 to 24 hours. 
8. Pour off with water and treat 10 to 20 minutes with alizarin. 
9. Pour off with water and let dry in the air. 
10. Stain now with Benda’s erystal violet by warming gently to the 
point of forming vapor. Allow the preparation to cool for 5 to 10 
minutes, after which pour off with water and let the preparation 
dry in air, standing the slide on end. 
11. Destain with 5 per cent. acetic acid under microscopic control. This 
requires from a few seconds to a minute. 
12. When the desired stain is reached, pour off with water, dehydrate with 
absolute alcohol, and counter stain, if desirable, with clove oil 
orange G. This stain is now removed with xylol or cedar oil, and the 
preparation is mounted in balsam. 
