EEGKESSIVE STAINING 493 



of ferric alum, which can only be obtained from large chemical 

 works, and does not keep well either in substance or in solution. 



Owing to the precision and depth of the stain, preparations 

 made by this process will bear study with higher microscopic 

 powers than those made by any other means ; that is to say, it is 

 certainly found in practice that they will bear notably higher 

 eye-piecing. 



It will be observed that, as with borax-carmine, this is a 

 ' regressive ' stain. The progress of decoloration, being slow, may 

 be controlled under the microscope, and a little practice with this 

 process may serve as an introduction to the art of regressive 

 staining with safranin and other tar-colours, with which the 

 progress of decoloration is so rapid that it cannot be controlled 

 under the microscope. 



Safranin is perhaps the most beautiful stain of this class. The 

 first requisite to success in staining with this colour is to obtain a 

 good sample of the dye. This is absolutely essential. There are at 

 least a score of brands of safranin on the market, many of which 

 cannot be made to afford a good stain by any means whatever. The 

 brand ' Safranin O ' supplied by Griibler and Hollborn is an excellent 

 one. 



The dye is employed in the form of a saturated or at least very 

 concentrated solution in water or alcohol. Perhaps the best plan in 

 general is to make a saturated solution in water, and another 

 saturated solution in strong alcohol, and then mix the two in equal 

 parts. Sections are soaked in the solution until thoroughly over- 

 stained the longer the better. Good stains can often be obtained 

 after half an hour in the staining bath, but for many objects it is 

 necessary, in order to ensure good results, to stain for twenty-four 

 hours, or even for many days. 



After the staining comes the ' differentiation ' of the stain. The 

 sections are just rinsed with water and brought into strong alcohol, 

 either in a watch-glass, if they be loose sections, or in a flat-bottomed 

 tube if they be affixed to a slide. ' The sections in the watch-glass 

 are seen to give up their colour to the alcohol in clouds, which are 

 at first very rapidly formed, afterwards more slowly. The sections 

 on the slide are seen, if the slide be gently lifted above the surface 

 of the alcohol, to be giving off their colour in the shape of rivers 

 running down the glass. In a short time the formation of the clouds 

 or of the rivers is seen to be on the point of ceasing ; the sections 

 have become pale and somewhat transparent, and (in the case of some 

 objects) have changed colour, owing to the coming into view of the 

 general ground-colour of the tissues, from which the stain has now 

 been removed. At this point the differentiation is complete, and 

 the extraction of the colour must be stopped instantly! 



This may be done if desired by simply putting the sections into 

 water ; but the more usual practice is to proceed at once to mount 

 them in balsam. To this end they may be cleared bv being put into 

 clove oil (or by pouring the oil over them on the slide). This will 

 extract slowly a little more colour, and may thus serve to complete 

 the differentiation in a frequently very desirable manner. Or you 



