120 CYTOPLASMIC INCLUSIONS 



detected by Bensley's mass technique. However, Faure-Fremiet (1910) 

 found staining differences within the mitochondria of a single indi- 

 vidual, and Peshkowskaya (1928) reports that the ectoplasmic chondrio- 

 somes of ClJmacostomum are resistent to fixatives which usually dis- 

 solve mitochondria, although the endoplasmic mitochondria are much 

 more typical in their reactions. Pellissier (1936) found similar dif- 

 ferences, not within the cell but between various individuals, and was 

 able to show that all the mitochondria impregnate more deeply in the 

 vegetative stages than in the stages just before reproduction. By the 

 selection of species in which all granules are in the same stage at the 

 same time, Bensley's mass technique could be used very profitably 

 in exploring the changes in mitochondrial composition. 



MacLennan and Murer (1934) found heavy deposits of ash in the 

 typical mitochondrial rods, as well as in the other cytoplasmic granules 

 of Paramecium. 



The presence of enzymes in mitochondria have been indicated in- 

 directly in many cases by the morphological association of these bodies 

 with structures in which digestive or synthetic activity is going on. 

 The only direct demonstration of the localization of cytoplasmic 

 enzymes is due to Holter and Kopac (1937) and Holter and Doyle 

 (1938), who showed that dipeptidase is not present in mitochondria, 

 but that amylase is. The method used was a combination of centrifugal 

 localization of granules and micro-methods for the measurement of 

 enzymatic activity. The mitochondria were concentrated in one end 

 of an Amoeba, which was then cut and the enzymatic activity of the 

 mitochondria-rich and the mitochondria-poor portions of the cytoplasm 

 compared. Since both the centripetal and the centrifugal portions had 

 the same amount of dipeptidase (measured by the ability to split 

 alanylglycine) per unit volume of cytoplasm, Holter and Kopac con- 

 cluded that the enzyme is in the matrix. They point out that this proves 

 nothing as to the origin of the enzyme, which might diffuse out from 

 a granule as fast as it is formed. Holter and Doyle found that the 

 middle region of the centrifuged Amoeba had the most amylase 

 (measured by the digestion of starch). The nucleus, crystals, digestive 

 vacubles, mitochondria, and matrix are found in this zone of the centri- 

 fuged Amoeba. The enzyme could not be localized in the nucleus, since 

 non-nucleated fragments show no significant diminution in amylase, 



