DS 11.124-DS 11.2 



DYE STAINS OF GENERAL APPLICATION 



293 



11.124 Squire 1892 calcium-hematoxylin Squire 1892, 25 



formula: water 10.5, ale. 96, hematoxylin 1, calcium chloride 8, aniinouium alum 1.2 

 preparation: Add the alum dissolved iu 6.5 water to the calcium chloride dissolved in 4 



water. Add ale, leave 1 hour, filter. Dissolve dye in filtrate. 

 note: In the early 1880's almost any alum-calcium chloride-hematoxylin was referred 

 to as "Kleinenberg," who recommended this method of preparing an aluminum 

 chloride-hematoxylin (which, in effect, this is) without recourse to the very acid salt 

 in commerce in his time. The method given by Kleinenberg 1876 (Grundsiige der 

 Entwickelungsgeschichte, Leipsig) proved impractical and a revised method (17510, 

 74:208) published in 1879, usually erroneously cited as the original, proved little 

 better. 



11.124 Thomas 1943 phosphoinolyhdic hemntoxylin 4285a, 20:49 



FOR.MULA: water 44, dioxane 40, ethylene glycol 11, phosphomolybdic acid 16. 5, hema- 

 toxylin 2.5, hydrogen peroxide 2 

 preparation: Dissolve the hematoxylin in the dioxane and add the hydrogen per- 

 oxide. Dissolve the phosphomolybdic acid in the other solvents and filter into the 

 hematoxylin. 



11.2 Carmine Stains 



As in the case of hematoxylin, carmine 

 was widely employed in the dyeing trade 

 prior to the introduction of stains into 

 microtomy. The carmine itself was com- 

 monly obtained as a tin lake, prepared by 

 boihng cochineal extracts with tin salts, 

 usually the tartrate. Though it is cus- 

 tomary to distinguish between those stain- 

 ing formulas employing carmine lakes, 

 those employing extracts of the raw cochi- 

 neal, and those employing carminic acid, 

 there is no justification for this since those 

 formulas employing the two latter re- 

 agents have always incorporated with 

 them some material which will take the 

 lake into solution. 



The six divisions of the carmine for- 

 mulas here employed are based entirely on 

 the ingredients, the most widely known 

 being the first two classes of "alcohol 

 carmines" (DS 11.22) and "alum car- 

 mines" (DS 11.21). Considerable con- 

 fusion has been occasioned by the fact 

 that Grenacher, in 1879, published for- 

 mulas for each of these two divisions and 

 early workers almost invariably employed 

 the alum carmine, while modern workers 

 seem to prefer the "alcohohc borax 

 carmine." 



The early employment of carmine for 

 staining materials before sectioning was 

 necessitated by the fact that no method 

 had been worked out for attaching sec- 

 tions to shdes, so that the fewer manipu- 

 lations which were undertaken in the sec- 

 tions, the more chance there was for 



preserving the whole. The straight alum 

 carmines are best employed for direct 

 staining from exceedingly dilute solu- 

 tions, a 1 % solution of ammonia alum 

 being the customary diluent. Alcohohc 

 carmines, particularly that of Grenacher 

 1879, are most employed for the prepara- 

 tion of wholemounts of small inverte- 

 brates. The formulas of Mayer 1881 and 

 Mayer 1892a, are the best devised for 

 small marine invertebrates. Though the 

 bora.x-carmine of Grenacher is commonly 

 made today by the method here given for 

 the "working formula direct" the original 

 method of Grenacher was to prepare the 

 dry stock and to make up working for- 

 mulas from it in various strengths of al- 

 cohol. This gives far better control of the 

 process, since the solubility of the dry 

 stock is a direct function of the concentra- 

 tion of alcohol employed. When any of 

 the alcoholic carmines are used, they are 

 differentiated with a .1% solution of 

 hydrochloric acid in 70% alcohol. 



The aceto-carmines, (DS 11.23) which 

 form the next class, are more widely em- 

 ployed in botanical than in zoological 

 techniques, and their most valuable ap- 

 phcations is the staining of unfixed nuclei. 

 They should be confined if possible to this 

 use, for their preservation as permanent 

 objects is difficult. Their only other use, 

 besides the counting of chromosomes, is 

 in the diagnostic staining of parasitic 

 platyhelminthes. 



Picro-carmines (DS 11.24) are warmly 

 recommended to the beginner, for it is 



