THE ROMANOWSKY STAINS 105 



for nerve fibrillse, has been applied for this purpose by Levaditi 

 and gives excellent results. 



(1) The tissues, which ought to be in thin slices, about 1 mm. 

 in thickness, are best fixed in 10 per cent formalin solution for 

 twenty-four hours. 



(2) They are washed for an hour in water and then brought 

 into 96 per cent alcohol for twenty-four hours. 



(3) They are then placed in 1'5 per cent solution of nitrate 

 of silver in a dark bottle, and are kept in an incubator at 37 C. 

 for three days. 



(4) They are washed in water for about twenty minutes, and 

 are thereafter placed in the following mixture, namely : 



Pyrogallic acid, 4 parts. 



Formalin, 5 parts. 



Distilled water up to 100 parts. 



They are kept in this mixture in a dark bottle for forty-eight 

 hours at room temperature. 



(5) They are then washed in water for a few minutes, taken 

 through increasing strengths of alcohol, and embedded in 

 paraffin in the usual way. The sections ought to be as thin as 

 possible. In satisfactory preparations the spirochaetes appear of 

 an almost black colour against the pale yellow background of 

 the tissues. The latter can be contrast stained by weak carbol- 

 fuchsin or by toluidin blue. 



(For the staining of spirocheetes in films see p. 107.) 



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. It is not certainly known to what particular 



