\OL. 12 (1953) GROWTH OF CMorella 25 



2. To make practically all cells in the culture receive directly the measured intensity of light, 

 in other words, to avoid as much as possible the mutual shading of cells in the suspension, the 

 population density of the culture was kept alwaA^s below o.i ml packed cell volume per liter during 

 the experiment*. 



This was effected by diluting frequently** the culture with fresh medium, by which also the 

 constancy of the nutrient medium during the culture could be assured. 



The relative rate (^g) of growth was calculated according to 



l^s = l0g,n — - 



h — h ^ 1 



Avhere Fj and V^ are packed cell volume per liter at the times i^ and /g- respectively, measured in 

 terms of days. At steady states of growth, the ratio Fj/K^ is equal to the ratio of dry weights of cells 

 (PFj/IFJ or of cell number {NJN^). 



Measurement of cell size and the ratio of dark and light cells. At each stage of culture, the diameter 

 of algal cells was measured either by the projection method adopted by Ketchum et al^ or by direct 

 microscopic observation using a calibrated micrometer; the latter method was preferred in most 

 cases because it gave sufficiently accurate results in a short time when conducted by a practiced 

 observer. With each sample, the sizes of 200 to 300 individual cells were measured***, and by grouping 

 the diameters into classes of 0.55 micron span, their distribution was represented by percentage 

 frequency polygons (see, e.g.. Figs. 3 and 5). The dark and light cells were distinguished according as 

 the cell diameter was shorter or longer than 4.5 microns, and to represent the size distribution of 

 cells in quantitative terms, the ratio of the number of dark cells ([D]) to the total cell number (N) 

 was calculated. This ratio {[DJIN) will be called "dark cell ratio" and denoted by A in the following. 



Measurement of photo synthetic rate. The rates of photosynthesis and respiration were measured 

 with the Warburg-Barcroft differential manometer with a rotary motion of vessels in a circle of 

 6-10 mm in diameter and at 250-300 r.p.m. The cells were suspended in 16 ml of M/50 phosphate 

 buffer (potassium salt) of pH 5.4 containing per liter: 2.5 g MgSO^-jHgO, 0.003 g FeSO^-yHgO and 

 I ml each of Arnon's A5 and B6 solutions, and the gas space of the vessel was filled with air containing 

 5% COg. The cell suspension, i.o cm in thickness, was illuminated from below with the arrangement 

 which was virtually the same as that employed by Tamiya and Chiba^. To make the effect of mutual 

 shading of cells negligible, the concentration of cell suspension was kept below 0.4 ml per liter (= 6.5 

 cmm cells per 16 ml of suspension). The rate of photosynthetic Og-output was determined by taking 

 into consideration the rate of respiration in the dark, the ratio CO2/O2 in both processes being regarded 

 as unity. 



To compare the rate of photosynthesis with that of growth (^g) on a common basis, the former 

 was expressed by the weight (in g) of organic photosynthate CHOH produced by i g dry weight of 

 cells per day. The rate {kp) expressed in this manner may be calculated from the ordinary expression 

 of photosynthetic rate, e.g., the 02-output in mm^ per mm^ of cells per 10 minutes, by the following 

 equation : 



^p = 0.327 X (Og-output in cmm per cmm of cells per 10 minutes). 



Determination of some cell constituents. The chlorophyll content of algal cells was measured by 

 the following procedure. The pigment was extracted from measured amounts of cells with cold methanol 

 and treated with hydrochloric acid to give phaeophytin. The latter was transferred into benzene, which 

 was thoroughly washed with water, and after removing the water layer, the benzene extract was 

 further dehydrated with a small quantity of solid NajSO^, then decanted and made up to a definite 

 volume with further addition of benzene. The quantity of phaeophytin in this solution was determined 

 spectrophotometrically at 666 m^ (i mg phaeophytin corresponds to 1.026 mg of chlorophyll). 



The phosphorus and nitrogen contents of cells were determined by Allen's method® and semi- 

 micro-KjELDAHL method, respectively. 



Both by experiments and by statistical considerations it was ascertained that, under the 

 condition of our experiments (thickness of culture solution: 2.8 cm; average diameter of cells: 3-5.5 

 microns), the light-diminishing effect of mutual shading of cells may practically be neglected when 

 the population density of cell suspension is less than o.i ml packed cell volume per liter. (C/. Tamiya 

 et al.^). 



** The interval of refreshment of culture medium varied according to the rapidity of growth; 

 it was 1/2 or i day at higher temperatures and stronger light intensities, and several days when 

 both temperature and light intensity were low. 



One of the characteristics of our algal strain is that it contains some elliptic cells with the 

 ratio of axes of about 1:0.85 to 1:0.75. The percentage of elliptic cells is, however, about 10% in 

 well illuminated culture and about 20 % in weakly illuminated culture, the rest being almost perfectly 

 spherical cells as it is the case with other Chlorella strains. For eUiptic cells the mean of both axes 

 was determined and the cells were regarded as spheres having diameters of that magnitude. 



References p. 40. 



