114 MICROSCOPIC METHODS 



Indian Ink Method for Films. A method has also been 

 introduced for demonstrating spirochastes in films by throwing 

 them up in an unstained condition on a background of indian 

 ink emulsion 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. Spirochaetes, if present, stand out clear, 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. 



(For the staining of spirochaetes in films, see p. 116.) 

 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 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- 

 films (in which the characters of both nucleus and cytoplasm 



