KLEINENBERG'S H^MATOXYLIN. 121 



cent, alcohol, with the addition of a little alum ; after having filtered, mix a 

 volume of this with from 6 to 8 volumes of 70 per cent, alcohol. At the 

 time of using the liquid pour into it as many drops of a concentrated solu- 

 tion of haamatoxylin in absolute alcohol as are sufficient to give the required 

 colour to the preparation, of greater or less intensity according to desire. 



MAYER (Mitth. a. d. Zool. Stat. zu Neapel, 10, 1, 1891, p. 174) has an 

 elaborate criticism on this formula, from which I extract the following : 

 The object of the chloride of calcium is explained by Kleinenberg to be fEo " 

 setting up of diffusion currents between the alcohol in the tisssues and the 

 external staining medium, so as to facilitate the penetration of the latter. 

 Mayer himself does not admit this; he holds that it acts chemically, and that 

 its effect is to make the stain more precise. I gather (though this is not 

 quite clear) that he holds that it acts in this way both as a calcium salt, and 

 also, after double decomposition, with the alum, as aluminium chloride, the 

 presence of this salt being the most important factor in the production of 

 the stain. Now Kleinenberg's formula does not efficiently provide for this 

 double decomposition and formation of aluminium chloride. For cold 70 

 per cent, alcohol dissolves hardly any alum,* so that if the alcohol preserved 

 its strength when the calcium chloride is dissolved in it, it would dissolve 

 hardly any alum, and consequently hardly any aluminium chloride could be 

 formed. But as calcium chloride contains about 50 per cent, of water of 

 crystallisation the alcohol becomes gradually weaker, and in consequence 

 dissolves more chloride of calcium, until at last you get a dense liquid con- 

 taining 70 per cent, of the salt. If now alum be added to this, you get a 

 precipitate of gypsum which envelops the crystals of alum, and protects 

 them so that they no longer dissolve. And at the end the liquid contains 

 only a mere trace of aluminium chloride. If you take hot alcohol you will 

 dissolve more alum and obtain more aluminium chloride, but you will also de- 

 compose much of your alum by the heat, and thus get a strongly acid liquid, 

 not fit for staining (the sulphuric acid set free from the alum combines 

 with the calcium chloride, forming gypsum and setting free hydrochloric 

 acid). 



A further point is the vagueness of Kleinenberg's formula as regards the 

 amount of hsematoxylin to be added to make the staining bath. If too much 

 be taken, as often happens, precipitates will be formed on the surface of the 

 objects, hindering the penetration of the stain, and obliging you to wash out 

 with acidified alcohol to remove them. 



It was suggested long ago by MAYER (Mitth. a. d. Zool. Stat. zu Neapel, 

 2, 1880, p. 13) that it might be more convenient to avoid the douple decom- 

 position by taking aluminium chloride in the first instance instead of alum. 

 And, accordingly, two formulae founded on this principle have been given 

 by DIPPEL (in his Handbuch der Mikroskopie). But these formulae both 

 omit the calcium chloride, and give in consequence a blue- violet liquid which 

 furnishes a diffuse stain (as is the case with all solutions containing a rela- 

 tively large proportion of aluminium salt [chloride or nitrate] with relatively 

 little haematei'n). This makes it necessary to over-stain strongly and wash out 



* Boiling 70 per cent, alcohol does not dissolve 0'5 per cent, of alum, 

 almost all of which falls out on cooling. 



