146 



METHODS IN PLANT HISTOLOGY 



Fig. 26. — Diospyros discolor: section of endo- 

 sperm fixed in formalin and stained in Haiden- 

 hain's iron-alum haematoxylin. X1135. 



Diospyros virginiana, is good, but small and harder to hold. Best of all 

 is the endosperm of the Philippine Diospyros discolor (Fig. 26). 



In Diospyros discolor the best 

 view will be obtained in sections 

 cut parallel with the flat surface 

 of the seed. Imbedding is neither 

 necessary nor desirable. Clamp 

 the endosperm in the microtome 

 directly and cut sections 8-10 m 

 thick. For cutting pieces of endo- 

 sperm too small to be clamped 

 directly, fasten the piece to a con- 

 venient block with cellulose ace- 

 tate, which is easily made by dis- 

 solving a photographic film (with 

 the emulsion removed by keeping 

 it a little while in hot water) in 

 acetone. The solution should have the consistency of thick syrup. 

 Celloidin or even glue will hold the piece to the block. Put the sec- 

 tions in ether and leave them there for 24 hours, changing once or 

 twice to remove any oily or fatty substances. If oils and fats are not 

 removed completely, a good stain will he impossible. Staining is not 

 difficult, if all oils and fats have been removed and the display of proto- 

 plasmic connections is very striking (Fig. 26). 



1. Fix in formalin, formalin-alcohol, or in glycerin-alcohol for a week. 



2. Cut sections 8-12 fi thick. 



3. Chloroform or ether, 24 hours. 



4. Absolute alcohol, 95 per cent and 50 per cent alcohol, 20 minutes each. 



5. Wash in water, 5 minutes. 



6. Iron alum, 4 per cent, 8-24 hours. 



7. Wash in water, 30 minutes. 



8. Stain in ^ per cent haematoxyUn, 24 hours. 



9. Wash in water. At this stage, examine carefully, because it may not be 

 necessary to reduce the stain in iron-alum. If the connections are not 

 too deeply stained, simply dehydrate, clear, and mount in balsam. 



After the first 5 stages, a strong stain in Delafield's haematoxylin, 

 10-24 hours, followed by a very weak hydrochloric acid, has given 

 good results. A sharp stain in crystal violet, differentiated with orange 



