24 



H. TAMIYA et al. 



VOL. 12 (1953) 



where D and L represent dark and light cells, respectively, and n the number of dark 

 cells produced from one light cell. The two essential aspects of growth, the increase in 

 mass and the increase in cell number, are succinctly represented by the two processes : 

 the former process occurs in the light, and the latter in the dark. 



The present work was carried out with a view to investigating more in detail the 

 properties of the two kinds of cells, the nature of their interconversion, and the correlation 

 of the two processes in the over-all phenomenon of algal growth under different con- 

 ditions. 



METHOD 



Culture. Chlorella ellipsoidea was cultured in a flat flask shown in Fig. i, using a medium which 



had the following composition unless otherwise stated for particular 

 experiments; per liter of solution: 5.0 g KNO3, 2.5 gMgS04-7H20, 

 1.25 gKH2P04, 0.003 gFeSO^-yHgO, i mleach of Arnon's A5 and 

 B6 solutions (2). (pH: 5.3-5.4). 



The flask, which had a inner thickness of 2.8 cm and a total 

 capacity of 600 ml, was filled with 500 ml of the medium, and after 

 autoclaving and seeding with algae, the main body of the flask was 

 immersed in a thermostated water bath having glass walls, and 

 illuminated from outside of the thermostat in the direction per- 

 pendicular to the flat surface of the flask. During the culture, 

 air containing 5% CO 2 was constantly bubbled through the ceU 

 suspension with a velocity of 200-300 ml per minute, by which the 

 cells were kept evenly suspended in the medium. Before entering 

 the culture flask, the COj-enriched air was saturated with water 

 vapor at the same pressure as the culture medium by being 

 scrubbed through the gas wash-bottles containing a solution 

 having the same composition and temperature as the culture 

 medium. 



The illumination was furnished by a projector lamp or reflec- 

 tor flood lamp operated at 70-85 volts using a voltage stabilizer 

 and variable transformer. The intensity of the light supplied was 

 in the range between 140 and 50,000 lux (at the position of the 

 flasks in the thermostat water), which was checked and regulated 

 2 or 3 times a day using a photometer having a submergible photo- 

 receiver. 



Measurement of growth. The growth of algae was followed 

 by measuring both the packed cell volume in ml (F) and cell num- 

 ber [N) per liter of culture. In the alga used, the ratio between the packed cell volume (in ml) and the 

 dry weight (in g) was on the average 1:0.256. This ratio varied only in a narrow range between 

 0.252 and 0.263 without showing any systematic change according to the ratio of dark and light cells. 

 Two kinds of experiments were performed : in one series, the transitory phenomena of formation 

 of dark cells from light cells or vice versa were investigated; and in the other, the steady state of growth, 

 i.e. the state in which the growth rate as well as the ratio of dark and light cells was constant during 

 the course of growth. Such a steady state obtained only when the culture was kept longer than about 

 4 days under each specified set of conditions*. 



Steady state experiments were performed with the following special precautions. 

 I. To assure the establishment of the steady state, the actual measurement of growth was 

 begun only after a preliminary culture lasting 4-7 days under each condition. 



r" UCTTl *1 H— ^ 



?.8cm. 

 Fig. I. Front and side views of 

 the culture flask used. 



* As has been described elsewhere^, our alga shows, imder certain experimental conditions, the 

 peculiar phenomenon that, in the course of culture, the change of dark cells to light cells and vice 

 versa occur almost in unison in all cells existing in the culture, and, interestingly enough, it takes place 

 at a fairly definite interval of about 30 hours. Such a periodic "bursting division" occurred especially 

 markedly when the inoculum was made from a rather old and starved culture and the culture was 

 subjected suddenly to high light intensity. However, on continuing the culture for several days with 

 frequent refreshment of medium, the phenomenon ceases to occur and the cells begin to grow and 

 divide "individually", showing as a whole a definite ratio of dark and light cells in the culture. 



References p. 40. 



