118 SPECIFIC EXAMPLES OF SLIDE-MAKING 



ner described in Example 10. While these slides are drying, it is well to make 

 up the solutions necessary for Johansen's technique. 



It will be noticed by reference to page 28 that these require both methyl 

 cellosolve and tertiary-butyl alcohol. Neither of these are common laboratory 

 reagents, and their availability should be checked before commencing the tech- 

 nique. There will be required four staining solutions (safranin, methyl violet 

 2B, fast green FCF, and orange G), three differentiating solutions (which con- 

 sist of mixtures of various solvents in proportions which must be followed 

 closely), and one special dehydrating solution which must be used to avoid 

 removing the stain in the final dehydration. 



The worker thus should have in front of him the following coplin jars of 

 reagents arranged in two rows. The jars in the front row should contain xylene, 

 absolute alcohol, 95 per cent alcohol, and 70 per cent alcohol. Those in the 

 back row should contain, in order, the safranin solution, the methyl violet 

 solution, the first differentiating solution, the fast green FCF, the second differ- 

 entiating solution, the orange G, the third differentiating 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, 

 though 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 abso- 

 lute alcohol for the removal of the xylene, to 95 per cent alcohol for the re- 

 moval 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 run- 

 ning water until no more color comes away. This initial staining in safranin 

 is the point at which the technique is usually interrupted since the slides may 

 be left in the staining solution indefinitely without damage, while the subse- 

 quent operations must be conducted consecutively. 



The thoroughly washed slides are transferred to the methyl violet solution 

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

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

 entiating solution from 10 to 15 seconds. Then they are placed directly into 

 the green staining solution where they remain from 10 to 20 minutes 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 seconds) in the third differentiating solution precedes the 



