Triple-stained Section of Aristolochia 225 



entiating solution, and the special dehydrating reagent. A large dish 

 should be available in the sink, in which the slides can be washed in 

 running water. 



The description to follow will be based on the handling of a single 

 slide, although there is nothing to prevent racks of slides from being taken 

 through as readily as a single example. 



A slide is heated over a flame until the wax is melted. Then it is placed 

 in xylene until the whole of the wax has been dissolved. It is transferred 

 to absolute alcohol for the removal of the xylene, to 95 per cent alcohol 

 for the removal of the xylene-contaminated absolute alcohol, and then to 

 70 per cent alcohol. The slide is placed in safranin and left from 1 to 3 

 days or until the nuclei are seen to be deeply stained in red. Then the slide 

 is washed in running water until no more color comes away. This initial 

 staining in safranin is the point at which the technique is usually inter- 

 rupted since the slides may be left in the staining solution indefinitely 

 without damage, whereas the subsequent operations must be conducted 

 consecutively. 



The thoroughly washed slides are transferred to the methyl violet solu- 

 tion for 10 to 15 min. On removal, they should be rinsed briefly in running 

 water to remove the surface stain before being differentiated in the first 

 differentiating solution from 10 to 15 sec. Then they are placed directly 

 into the green staining solution where they remain from 10 to 20 min or 

 until their red-purple color is changed to a greenish hue. The slide is 

 taken from the green solution, drained from the corner, and the back of it 

 is wiped. A very brief rinse (5 to 10 sec) in the second differentiating solu- 

 tion precedes the 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 

 examined under a microscope. Staining should be discontinued when the 

 cytoplasm of the cell changes from a muddy gray color to a clear bright 

 orange. This will normally take from 3 to 5 min, 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 trans- 

 ferred to xylene, in which the special dehydrating agent should be re- 

 moved 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. 



