46 Methods in Plant Histology 



stain slowly or not at all. The washing is particularly important 

 when the fixing agent contains an acid. Transfer from the stain 

 to tap water. Distilled water is neither necessary nor desirable. 

 Some writers recommend washing for 24 hours, but this is entirely 

 unnecessary; for paraffin sections on the slide, 5 or 10 minutes is 

 long enough, and even for rather thick freehand sections 20 to 30 

 minutes is sufficient. Use plenty of water and keep changing it as 

 often as it becomes in the least discolored. Precipitates are often 

 formed when slides are transferred directly to alcohol from this 

 stain, and sometimes even after washing in water. A few gentle 

 dips in acid alcohol (2 drops of HC1 to 100 c.c. of 70 per cent alcohol) 

 will usually remove the precipitates. This extracts the stain more 

 rapidly from other parts than from the nuclei, and hence gives a good 

 nuclear stain, while at the same time it removes any disfiguring 

 precipitates. Some prefer to stain for a very short time and use no 

 acid alcohol, but, as a rule, it is better to overstain and then differen- 

 tiate in this way, because sharper contrasts are obtained. Transfer 

 from acid alcohol to 70 per cent alcohol and leave here until a rich 

 purple color replaces the red due to the acid. Since small quantities 

 of the acid alcohol are carried over into the 70 per cent alcohol, it 

 is well to add a drop of ammonia now and then to neutralize the 

 effect of the acid. Too much ammonia is to be avoided, for it gives 

 a disagreeable bluish color with poor differentiation, probably on 

 account of the precipitation of alumina. The preparation is now 

 dehydrated in 95 per cent and then in absolute alcohol, cleared in 

 xylol or clove oil, and mounted in balsam. 



The following is a general schedule for staining paraffin sections 

 on the slide in Delafield's haematoxylin: 



1. Stain (from water or from 35 or 50 per cent alcohol) . . 10 minutes 



2. Rinse in water 10 minutes 



3. 35 and 50 per cent alcohol 5 minutes each 



4. Acid alcohol ' 5 seconds 



5. 70 per cent alcohol 5 minutes 



6. 85 per cent alcohol 5 minutes 



7. 95 per cent and 100 per cent alcohol 5 minutes each 



8. Xylol and 100 per cent alcohol, equal parts 5 minutes 



9. Xylol. 



10. Mount in balsam. 



