THE ROMANOWSKY STAINS 111 



solution (c) is dropped on, heated till steam arises, and allowed to remain 

 for another half minute. The preparation is finally washed in water and 

 dried. The spiroehsetes are of a dark brown or black colour, and are 

 easily found. This is the best method, and is easily carried out. 



(2) Indian Ink Method. — An emulsion of indian ink of fine quality is 

 sterilised by steaming and allowed to settle for a few days ; a drop of the 

 deposit diluted with an equal quantity of distilled water is well rubbed up 

 and spread on a slide with a drop of the material to be examined (exudate 

 from chancre or condyloma, scraping from congenitally affected organ, 

 etc.). The film is dried and examined, with an immersion lens without 

 the interposition of a cover. Spiroehsetes, if present, stand out unstained, 

 surrounded by the dark indian ink, and often positive results are rapidly 

 obtained by means of it. The organisms are not so readily recognised by 

 this method as by dark-ground illumination, and negative observations 

 are thus less valuable. 



(3) Congo-Bed Method (Benians). — A small drop of a 2 per cent, 

 aqueous solution of Congo red is placed on a slide, and a very small 

 quantity of the secretion or exudate to be examined is rubbed into it 

 with the platinum wire. The drop is then spread out into a tolerably 

 thick film and allowed to dry. The film is then treated with a 1 per 

 cent, solution of hydrochloric acid in absolute alcohol, and the preparation 

 is dried in the air, or with blotting-paper, though the latter is apt to tear 

 the film. Spiroehsetes and bacteria show up unstained on the dark back- 

 ground. 



(4) Giemsa's Method, see p. 113. 



The Romanowsky Stains. — Within recent years the numerous 

 modifications 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 

 organism, a 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 formulae 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 



