26 MATERIALS AND PROCESSES OF SLIDE-MAKING 



8. Take each slide individually and dip it up and down in absolute alcohol 

 until it is differentiated. This may be seen clearly with the naked eye, for 

 the slide, which is a muddy purple when differentiation starts, suddenly 

 clears to show bright blue and bright red areas. 



9. Transfer the slide to xylene which stops differentiation. 



When using this stain for the first time, it is well to transfer sections to 

 xylene and examine them under the microscope before differentiation is com- 

 plete and then to put them back into absolute alcohol to complete the differ- 

 entiation. A successful slide will show nuclei in red, cartilage and white 

 fibrous connective tissue in blue, nerves and glands in various shades of 

 violet, muscle in red, and erythrocytes and keratin in orange. The only disad- 

 vantage of the technique is the rapidity with which the blue color is removed 

 in absolute alcohol, so that differentiation must be watched very carefully. The 

 dyes used are extremely sensitive to alkali, so that the slides, if they are to be 

 kept for some time, should be mounted in a very acid medium. A procedure 

 which has been recommended is to keep a saturated solution of salicylic acid 

 in xylene and to dip each coverslip in this before applying it to the finished 

 preparation. This is much less trouble than, and just as effective as, making 

 up a special salicylic acid balsam. 



Another complex contrast stain, in which the nuclei first must be stained 

 with hematoxylin, is: 



Patay's Triple Stain: 

 First staining solution 



1% Ponceau 2R 

 Differentiating and mordanting solution 



1% Phosphomolybdic acid 

 Second staining solution 



0.5% Light green in 90% alcohol 



METHOD OF USE: 



1. Stain the sections in Carazzi's hematoxylin (see page 18) and differen- 

 tiate until not only the nuclei but also the cartilage remains blue. Wash 

 the sections in alkaline tap water until they are blue and then wash 

 thoroughly in distilled water to get rid of all the alkali. 



2. Put the sections in the first stain for 2 minutes. 



3. Rinse them briefly in water. 



4. Place them in the differentiating and mordanting solution for 2 minutes. 



5. Rinse sections briefly in water. 



6. Transfer them to 95 per cent alcohol until dehydrated. 



7. Transfer each section individually to the second staining solution for 30 

 seconds. 



