CYTOPLASMIC INCLUSIONS 127 



The ability to segregate neutral red has been ascribed to a specific 

 reaction of the dye with a single material in the granule, the process 

 being called the "neutral red reaction" by Koehring (1930) and in- 

 cluded as a part of the "ferment theory of the vacuome" by Kedrowsky 

 ( 1932b) . The substance involved is supposed to be a proteolytic enzyme, 

 a conclusion based on the work of Marston (1923), who showed that 

 these enzymes are precipitated by combination with neutral red, Janus 

 green, and other azine dyes. Le Breton (1931) has reviewed the rather 

 voluminous literature resulting from this suggestion and concludes that 

 the reaction is not specific, since ordinary proteins (an important con- 

 stituent of most segregation bodies) also are precipitated. From 

 the standpoint of cytology, the theory fails to explain how neutral red 

 and Janus green have such difi^erent staining reactions in the granules 

 of living cells. Hopkins's ( 1938a) experiments with Flabellula show that 

 precipitates are formed in vacuoles after either Janus green or neutral 

 red. If Janus green was first used, and then neutral red added, a red 

 precipitate formed around the original green one. "The small neutral red 

 vacuoles are, then, the same as the vacuoles in which the condensation 

 granules are formed, but it appears that Janus green B stains a different 

 component of these vacuoles than does the neutral red." 



Not one factor, but many are responsible for the segregation of neutral 

 red by cytoplasmic granules. The role of pH in neutral red staining has 

 been demonstrated by Chambers and Pollack (1927) and Chambers and 

 Kempton (1937), who showed that neutral red tends to go from an 

 alkaline region to an acid region so that "segregation granules" would 

 be those which are acid relative to the hyaloplasm. Kedrowsky (1931) 

 demonstrated in Opalina that normally the segregation granules have 

 this relationship with the hyaloplasm and that the staining reactions 

 can be changed by altering the pW of the cytoplasm. He also showed 

 that the granules will take up acid dyes in the presence of albumoses, 

 which was confirmed by Volkonsky (1933) and included under the 

 term "chromopexie." Since neutral red has long been known as a 

 lipoid stain (Faure-Fremiet, Mayer, and Schaeffer, 1910) it is possible 

 that this may play a role in the staining of bodies which contain lipoids, 

 such as the dictyosomes of gregarinida and the digestive granules of 

 Ichthyophthhius. From these brief examples it is clear that the segrega- 

 tion of neutral red and other vital dyes is influenced by many internal 



