THE ROMANOWSKY STAINS 113 



The Romanowsky Stains. Within recent years the numerous 

 mollifications of the Romanowsky stain have been extensively 

 used. The dye concerned is the compound which is formed 

 when watery solutions of medicinal methylene-blue and water- 

 soluble eosin are brought together. This compound is insoluble 

 in water but soluble in alcohol the alcohol employed being 

 methyl alcohol.- The stain was originally used by Romanowsky 

 for the malarial parasite, and its special quality is that it 

 imparts to certain elements, such as the chromatin of this 

 oruiiiiHin, ;i reddish-purple hue. This was at first thought to be 

 simply due to the combination of the methylene-blue and the 

 eosin, but it is now recognised that certain changes, such as 

 occur in methylene-blue solutions with age, are necessary. In 

 the modern formula; these changes are brought about by 

 treatment with alkalies, especially alkaline carbonates, as was 

 first practised by Unna in the preparation of his polychrome 

 methylene-blue. The stains in use thus contain a mixture of 

 methylene-blue and its derivatives in combination with eosin ; 

 the differences in these bodies and the different proportions in 

 which they occur in individual stains account for the different 

 effects produced on the various constituents of a cell. The 

 underlying chemical reactions are complicated and as yet not 

 fully understood. Thus it is not certainly known to what partic- 

 ular new body the reddish hue produced in chromatin is due, 

 but the active constituent may be methyl-violet or methyl-azure 

 or thionin, all of which result from the 'action of alkali on 

 methylene-blue. The stains are much used in staining blood- 

 lilms (in which the characters of both nucleus and cytoplasm 

 in leucocytes are beautifully brought out), in staining bacteria 

 in tissues or exudates, the malaria parasite, trypanosomes, the 

 pathogenic spirochajtes (such as the spirochaete pallida), and 

 protozoa generally. 



The following are the chief formulae in use : 



1. Jcnner's Stain. This is an excellent blood stain, hut is not so good 

 for the study of parasites as the others to be mentioned. In its 

 preparation n> alkali is used. It is made by mixing equal parts of (a) 

 a 1'2 to 1*25 per cent, solution of Griibler's water-soluble eosin (yellow 

 shade) in distilled water and (b) 1 per cent. Griibler's medicinal methy- 

 leue-blue (also a watery solution). The mixture is allowed to stand 

 twenty- four hours, is filtered, and the residue is dried at 55 C. ; the 

 powder is shaken up in distilled water, filtered, washed with distilled 

 wat-r, and dried. Of the powder, '5 grin, is dissolved in 100 c.c. Merck's 

 methyl alcohol. For use a few drops are placed on the dried uniixed 

 liliu for one to three minutes, the dye is poured off, and the pre pa ration 

 wa.-Oied with distilled water till it presents a pink colour; it is then 

 dried between (liter-paper and mounted in xylol balsam. 



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