VITAL STAINING AND REDUCTION OF VITAL 

 STAINS BY PROTOZOA. 



ELERY R. BECKER, 

 IOWA STATE COLLEGE. 



It is well known that fresh animal and plant tissues and milk 

 have the power of reducing methylene blue to the leuco-base. 

 This is supposed to be brought about by the hydrogen liberated 

 in the decomposition of water by a reducase. The reducase in 

 turn requires the presence of Schardinger's enzyme, an oxidizable 

 substance, and water. (See Bayliss, p. 588.) 



It is not generally known that protozoa, too, may possess the 

 reducase. The demonstration of its presence requires that they 

 be kept in a weak solution of the dye in an oxygen free medium. 

 The method used by the writer was a hanging drop of ciliates 

 and dye solution in an Engelmann chamber, through which a 

 stream of hydrogen was passed. The drop was observed through 

 the compound microscope. The ciliates used were Opalina sp.? 

 from the bullfrog tadpole and Paramecium caudatum, representing 

 both parasitic and free-living modes of life. Fifteen dyes were 

 tested out on Opalina, and one on Paramecium. The best 

 reductions are obtained if the hydrogen gas is first passed 

 through heated platinized asbestos to remove the oxygen. 



Janus Green. Opalina in suspension of tadpole feces from 

 posterior intestine of tadpole were suspended in a drop of Janus 

 green (i to 15,000) made up in 0.3 salt solution. Apparently 

 the dye did not stain any substance in the ciliate. After dis- 

 placing the air in the chamber with hydrogen, a number of the 

 Opalina contain pink granules, due to reduction of the Janus 

 green (diethylsafraninazodimethylanilin) to diethyl safranin. 

 On exposure to air the pink substance did not change its color. 

 Further reduction to the leuco-base could not be effected. Not 

 over one fourth of the Opalina at any one time could reduce the 

 dye. The reduction was also accomplished in a muscle chamber. 



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