TRIPLE-STAINED SECTION OF ARISTOLOCHIA 119 



transfer to the clove oil-orange G solution. It is perfectly satisfactory to have 

 this solution available in a drop bottle from which it can be poured on the 

 slide, although, if any large number of sections are to be handled, a coplin jar 

 is usually more convenient. 



The slide should be removed from the orange stain at intervals and exam- 

 ined under a microscope. Staining should be discontinued when the cytoplasm 

 of the cell changes from a muddy grey color to a clear bright orange. This 

 will normally take from 3 to 5 minutes, although occasionally, a longer period 

 of time is required. As soon as the required color has been reached, the slide 

 is dipped up and down three or four times in the special dehydrating agent, 

 the primary purpose of which is to remove the excess orange from the surface 

 of the slide. Then the slide is transferred to xylene, in which the special dehy- 

 drating agent should be removed completely, before being mounted in balsam 

 in the ordinary manner. 



Summary 



1. Select a suitable Aristolochia stem and fix in Lavdowsky's fluid. 



2. Wash short lengths of stem in running water, dehydrate, clear, and embed 

 in wax. 



3. Mount individual sections or short lengths of ribbon on slides, dry, dewax, 

 and transfer to 70 per cent alcohol through the usual reagents. 



4. Stain in Johansen's safranin from 1 to 5 days. 



5. Wash thoroughly in running water and stain in 1 per cent methyl violet 2B 

 for 10 to 15 minutes. 



6. Rinse in water and differentiate 10 to 15 seconds in the first differentiating 

 solution. 



7. Stain in fast green FCF from 10 to 20 minutes. 



8. Rinse in third differentiating solution 5 to 10 seconds and stain in orange G 

 3 to 5 minutes. 



9. Rinse off orange G in special dehydrating mixture, remove this mixture 

 with xylene, and mount in balsam. 



Note: The formulae for staining, differentiating, and dehydrating solutions 

 used above are given on page 28. 



