294 DYEING 



solvents, and one cannot therefore transform a vital into a perman- 

 ent preparation without taking special steps to immobilize it. In 

 most cases observations are made and photomicrographs taken if 

 necessary, and the specimen then discarded. It is sometimes con- 

 venient, however, to retain the dye permanently in the particular 

 position that it took up during life. If nerves have been coloured by 

 methylene blue, for instance, their relations with other tissues are 

 often best seen in thick permanent preparations, rendered trans- 

 parent by the use of suitable mounting media. Alternatively one 

 may want to colour vitally and then make sections. 



The way in which certain vital dyes may be fixed in the tissues 

 appears to have been discovered by chance. It was customary in 

 the eighties to make permanent preparations of small pieces of 

 tissue by putting them in a solution of ammonium carminate 

 neutralized by picric acid.^^^ The chromatin was coloured red with 

 carmine, the cytoplasm yellow by picrate. It was noticed that 

 when nerve-preparations, dyed vitally by methylene blue, were 

 treated with this picro-carmine, the colour of the axons was re- 

 tained. ^^^ It was soon shown that the trapping of the colour was 

 due to the ammonium picrate in picro-carmine, and that this salt 

 could advantageously be used alone. ^^^ These discoveries were 

 first reported in 1887.^*' The methylene blue picrate deposited in 

 the tissues is unfortunately soluble in alcohol."^ 



The trapping of methylene blue in vital preparations was com- 

 prehensively studied by Bethe.^^ '^^ It was his object to find a salt 

 of methylene blue that was insoluble in the various reagents 

 commonly used in making permanent microscopical preparations. 

 He precipitated methylene blue from its solutions by various 

 anions and studied the solubility of the precipitates. He found that 

 ferricyanide and molybdate gave precipitates with the necessary 

 insolubility. When these were tried on tissues, molybdate gave a 

 more finely granular precipitate. Bethe therefore used ammonium 

 molybdate, (NH4)gMo7024.4H20, as a fixative in the ordinary 

 sense and at the same time as a fixative or trapping agent for 

 methylene blue. He sometimes added hydrogen peroxide to 

 oxidize the dye from the leucobase, if necessary. The methylene 

 blue molybdate formed in the axons was retained through paraffin 

 or collodion embedding into permanent mounts in Canada balsam. 

 This method of trapping methylene blue, introduced in 1895, is 

 used to the present day. 



Just as methylene blue can be fixed in nerve-axons, so this and 



