332 



METHODS AND FORMULAS 



DS 12.30 



preference to any other is based only on 

 the fact that it is a quicker process. If the 

 operator does not mind waiting the re- 

 quired 24 hours, there is no reason why 

 he should not employ the solution of 

 Heidenhaim or any other of the iron 

 mordant techniques. The solutions re- 

 quired for this staining method consist 

 first of a 5% solution of ferric alum, 

 second, of a glycerol-alcohol-hematoxylin 

 solution, and third of a picric alcohol 

 differentiating solution recommended by 

 Masson himself for use before his tri- 

 chrome methods. 



The first step is to raise both the ferric 

 alum and the hematoxylin solutions to 

 approximately 50°C. This temperature is 

 not critical, but the rate of staining is 

 dependent on temperature and decreases 

 rapidly as the temperature drops below 

 50°C. Temperatures above 50°C. may 

 cause the sections to fall off the slide. The 

 first batch of sections is now taken from 

 water and placed in the heated ferric alum 

 solution for a period of 30 minutes. It is 

 then removed from the ferric alum solu- 

 tion, rinsed very rapidly in distilled water, 

 and transferred to the heated hematoxylin 

 solution for a further period of at least 

 30 minutes. On removal from the hema- 

 toxyhn solution the sections should be 

 blue-black. If they are not deeply stained 

 enough, it is necessary to return them to 

 the hematoxylin. The slides may now be 

 rinsed briefly in distilled water and trans- 

 ferred to tap water prior to differentiation. 

 If relatively large runs are being taken 

 through, it will usually be desirable to 

 bring all the sections through their nuclear 

 staining before proceeding either with the 

 differentiation or the counterstain. 



The picric alcohol of Masson (ADS 21.1 

 Masson 1912), which is used for differenti- 

 ating, is a relatively slow medium in com- 

 parison with the ferric alum differentiation 

 frequently recommended for iron hema- 

 toxyUn stains. As, however, a somewhat 

 acid afterstain is being employed in this 

 instance, care should be taken that differ- 

 entiation does not proceed too far. It will 

 be quite sufficient to differentiate the 

 background to a pale straw color (it 

 will, of course, become blue in tap water) 

 rather than to the absolutely colorless 



background which would be desirable were 

 one using this technique to demonstrate 

 chromosomes. As soon as the required 

 degree of differentiation has taken place, 

 the sections are returned to tap water and 

 washed until no further picric acid leaves 

 the specimen. It will be convenient for 

 this purpose to have a fairly large dish in 

 the sink through which a current of tap 

 water is running continuously. A certain 

 amount of differentiation will take place 

 while this washing is going on, and the 

 washing should not be discontinued until 

 the nuclei are deep blue-black and the 

 differentiated background has changed 

 again from the very pale straw color to 

 which it was turned by the picric differ- 

 entiator to a pale blue. Failure to under- 

 take this second bluing after differenti- 

 ation is responsible for many of the 

 failures to retain hematoxylin in the 

 nucleus. 



The acid fuchsin-anilin blue stain, which 

 is here selected as a counterstain, is one 

 of the easiest to apply, provided the in- 

 structions are followed closely. The solu- 

 tions required are given under DS 12.31 

 Masson 1912a below; and it will be ob- 

 served from the technique described that 

 an acid environment must be retained 

 throughout, even to the mounting medium. 

 The first solution required is a mixture of 

 acetic acid and acid fuchsin in water, to 

 which the sections may be passed directly 

 from the tap water, and in which they 

 should not remain longer than five min- 

 utes. Each batch of sections to be stained 

 by this method must be taken through 

 individually, for there is no stage of the 

 proceedings between the original staining 

 with acid fuchsin till the sections are dehy- 

 drated in xylene at which a pause may be 

 made for the accumulation of separate 

 batches. After removal from the acid- 

 fuchsin solution the sections will be seen 

 to be stained deeply red, and they require 

 only the quickest rinse in tap water before 

 being placed in 1 % phosphomolybdic acid 

 where considerable quantities of red dye 

 will be removed. The time in this solution 

 is not as critical as in many of the others, 

 and the five minutes specified may be 

 varied from, say, tlwee to eight minutes 

 without any great damage to the speci- 



