346 AUGUSTO BONAZZI 



It is unnecessary to present more data on this point since it 

 would all corroborate the above statements without adding 

 new facts of importance. It is nevertheless important to obtain 

 a crucial test of the actual storing and utilization of the stores 

 of the sugar carbon; such evidence follows. 



Autophagy of Azotohacter 



That some of the sugar which disappears from solution during 

 the first few days or hours of growth is actually stored in the 

 cells is obvious in view of the fact that the carbon of the sugar 

 contributes to the synthesis of the compounds of the cell sub- 

 stance, but in addition as will be seen, Azotobacter presents an 

 interesting case of what Duclaux designates with the term 

 ''phenomene de vie continuee." This phase is one in which the 

 organism is really li\ing on its own reserves and the by-products 

 of its previous life activities, just as yeast will continue to live 

 in a fermented mixture at the expense of the glycogen, glycerol 

 and succinic acid which it formed during its early stages of 

 development and active fermentation. 



Experiment 19. Pure cultures of Azotobacter chroococcum 

 were made on Ashby's mannitol-washed-agar plates and allowed 

 to incubate for twenty-four hours. 



The growths thus obtained were emulsified in 0.75 per cent 

 NaCl solution and aseptically placed in sterile test tubes. Slides 

 of this bacterial suspension were prepared immediately and after 

 forty-two and one hundred and fourteen hours' standing in the 

 incubator. They were stained by means of the Giemsa solution 

 which stains well the peculiar granulations studied in a previous 

 communication (Bonazzi, 1915). Examining fifty microscopic 

 fields at random on each of the slides so prepared, and counting 

 the number of cells containing granules (granulated), those 

 free of granules and those in which the granules have partially 

 disappeared (transitional) the following data were obtained. 



The cells here classed as transitional are those in which the 

 granules had nearly disappeared or were greatly diminished in 

 size and could well be classed among the ungranulated. If this 

 were done the following table would be obtained. 



