L28 



etermined by trial, the preparations ;<■ - ■'. for ;«>>'>ut 15 mil 



in gently flowing water. They are now dehydrated by treating with absolute 

 alcohol, after which they may be f-ounter stained with clove oil orang 

 or merely cleared in clove oil and cedar oil and mounted in balsam. 



In ease counter staining is desired, the pi - should be watched in 

 order to avoid - ining with the orange. In some eases 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 cytologicaJ 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-osmic 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 hour-. 



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 crystal 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. 



