Water 45 



formation. The granules in the vacuoles oF Amoeba viira stain with Janus 

 green B; similar granules (mitochondria or mitochondria-like) occur around 

 incipient contractile vacuoles in Amoeba proteiis, and there is a rough correla- 

 tion between vacuole frequency and number of granules. '^'^ Osmium-stain- 

 ing granules, presumably Golgi bodies, occur near the vacuoles of many cili- 

 gjgg 84, 157 Weatherby--*^ has reviewed in detail the relation between granules 

 and vacuole formation. Frequently a small vacuole or vesicle contains a 

 granule which appears to dissolve as the vesicle grows.*""' 



There is little doubt, then, that oxidative secretion is involved in filling the 

 contractile vacuole in Protozoa, but whether solute or water or both are 

 secreted remains to be demonstrated. 



Kitching *■" favors the hypothesis that water or very dilute solution is 

 secreted. Nonfeeding Protozoa often continue to put out large quantities of 

 water for days in a very dilute medium. When the baling out of water is 

 stopped, the cells swell. 



An alternative theory is that solute is secreted first and that water then enters 

 the small vacuole by diftusion.^*'*^' '^^ In Amoeba m'lra the small vacuoles 

 often originate in association with food vacuoles. The time required for a 

 given increase in size of vacuoles in this species is less in 2.5 per cent than in 5 

 per cent sea water, hence Mast and Hopkins conclude that in this marine 

 species the cytoplasm is isotonic with the medium, that the vacuoles are 

 hypertonic when they start and are isotonic when fully grown. In nonfeeding 

 specimens of Amoeba m'lra many small vacuoles are seen to contain granules 

 and not to discharge water. When active feeding starts, these granules go into 

 solution, becoming osmotically active, and the vacuoles swell. When an 

 Amoeba lacerta is transferred from 5 per cent sea water to 100 per cent sea 

 water the size of the vacuole as well as the size of the amoeba decreases during 

 a period of V2 to 3 hours. ^^^ By crushing under a coverslip free vacuoles are 

 obtained; these swell in more dilute or shrink in higher concentrations of sea 

 water. The vacuole of this species, then, appears to contain osmotically active 

 material. 



The rate of growth of vacuoles has been said to be linear;-' ^^-^ however, 

 these measurements were taken at wide intervals. When very frequent 

 measurements were made '^'* on the vacuole of Amoeba proteiis it was found 

 that the c. vacuole grows in a stepwise fashion as the small vacuoles fuse with 

 the c. vacuole. No change in size of the c. vacuole occurred during quiescent 

 periods of as long as 120 seconds. This indicates that, unless the vacuolar 

 membrane is impermeable to water, the contents must be cquimolar with the 

 surrounding cytoplasm. 



The principal argument against the theory of secretion of solute has been 

 the supposed amount of solute needed. '-^^ Calculations based on the assump- 

 tion that the final concentration of the vacuolar fluid is the same as that of the 

 cytoplasm fail to support this objection. If the osmotically active material is 

 assumed to have a molecular weight and density of one of the smaller amino 

 acids (M.W.=:100), for Amoeba protens, which has a cytoplasmic concentra- 

 tion of about 0.05 M and which excretes 110 cubic micra per second (39 X 

 10"^ cm.'Vhi"-), the amount of such solute needed would be 1.1 X 10"" 

 cm.yhr., whereas the volume of the animal is 2.5 X lO-*"' cm.'* Nonfeeding 

 Peloniyxa carolinensis decrease about 8 per cent in volume every 24 hours. It 



