METHODS. 7 



solution of alum consisting of 1 gramme of alum to 1 c.c. of 

 water. 



2. Kleinenberg's hsematoxylin. Saturate some 70 per cent, 

 alcohol with calcium chloride ; let the mixture stand for twelve 

 to twenty- four hours over alum, shaking occasionally ; add eight 

 parts of 70 per cent, alcohol ; filter, and then add a solution of 

 hsematoxylin in absolute alcohol until the liquid has a purple-blue 

 colour; let it stand in a corked bottle exposed to sunlight for 

 about a month ; it is then fit for use. The liquid is to be diluted 

 as required with alum solution. 



3. Expose a few crystals of hsematoxylin to the action of gaseous 

 ammonia in a watch-glass under a bell-jar : then add water, and 

 a good colouring fluid is obtained. The disadvantage of this 

 is that it has to be freshly prepared every time it is required. 



The alum-solutions will stain all parts of the cell, including the 

 cell- wall. Their especial uses are (a) to make the cell- walls more 

 evident when they are naturally transparent and colourless ; 

 (b) to stain the protoplasm, so as to make its intimate structure 

 apparent ; (c) to stain the nucleus, so as to demonstrate its pre- 

 sence and to show up its structure. 



The ammoniacal solution is especially adapted for differentiated 

 staining. If a dilute solution be used, the first thing to become 

 stained is the chromatin of the nucleus, then, after a time, the 

 rest of the nucleus (achromatin), then the protoplasm. The cell- 

 walls do not stain with this fluid, or only slightly. Kleinenberg's 

 hsematoxylin stains in a few minutes, whereas the alum-solution 

 is much slower in its action. 



Hsematoxylin may be used either for fresh material, 

 or for sections which have been previously hardened 

 with alcohol, or with picric or chromic acid. In the 

 latter case the sections must be washed repeatedly in 

 distilled water to remove every trace of the acid, which, 

 if present, would interfere with the proper action of the 

 hsematoxylin. If the section becomes too deeply 

 stained, as sometimes happens when the alum-hasma- 

 toxylin is used, the excess of colouring-matter may 

 be removed by washing with watery solution of alum. 



