NUCLEAR STAINS, COAL TAR 181 



never overstains. The stain is permanent both in balsam and in 

 glycerin. 



This colour may be used as a chromatin stain for fresh tissues 

 in the same way as methyl green. Herla {Arch. Biol., xiii, 

 1893, p. 423) em})loys for ova of Ascaris a mixture of 0-25 gm. 

 vesuvin, 0-25 gm. malachite green, 10 of glycerin and 100 of 

 water, and washes out with weak glycerin. 



The chief use of this colour is for progressive staining ; but 

 it may be employed for staining by the regressive method (see 

 § 241), and also for intra-vitam staining (§ 769) (for this purpose 

 it is necessary to see that the colour employed be pure and neutral). 



365. Other Progressive Stains. Most of the basic tar colours used 

 for regressive staining will also give by the progressive method a nuclear 

 stain of greater or less purity if used in solutions acidified with acetic 

 acid. Amongst these may be mentioned thionin, which need not even 

 be acidified ; also, for fresh tissues especially, gentian violet, dahlia, and 

 toluidin blue. 



B. REGRESSIVE STAINS 



366. The Practice of Regressive Staining : The Staining Bath. 



Sections only, or material that is thin enough to behave like 

 sections, such as some membranes, can be stained by this method. 



The solutions employed are made with alcohol, water, or anilin, 

 or sometimes other fluids, according to the solubility of the 

 colour. There seems to be no sj^ecial object in making them with 

 alcohol if water will suffice, the great object being to get as strong 

 a solution as possible. Indeed, the solutions made with strong 

 alcohol are found not to give quite such good results as those 

 made with water or weak alcohol. Alcohol of 50 per cent, strength, 

 however, may be said to constitute a very generally desirable 

 medium. The sections must be very thoroughly stained in the 

 solution. As a general rule they cannot be left too long in the 

 staining fluid. With the powerful solutions obtained with anilin 

 a few minutes or half an hour will usually suffice, but to be on the 

 safe side it is frequently well to leave the sections twelve to 

 twenty-four hours in the fluid. Up to a certain point the more 

 the tissues are stained the better do they resist the washing-out 

 process, which is an advantage. Some workers, indeed, prefer 

 weak solutions ; so Heidenhain, Enzyk. niik. Technik, i, pp. 433, 

 434 ; but the nature of the fixing agent should be taken into 

 account. 



Material fixed in chromic or clu'omo-osmic mixtures gives a 

 sharper and more selective stain than material fixed in sublimate 

 or the like. In fact, to ensure the best results, only material fixed 

 in chromic mixtures (or Hermann's fluid) should be employed. 



During the staining the tissues become overstained, that is, 

 charged with colour in an excessive and diffuse manner. The 



