INFLUENCE OF THE SIZE OF THE INOCULUM ON THE GROWTH OF CHLORELLA
VULGARIS IN FRESHLY PREPARED CULTURE MEDIUM 1
Robertson Pratt
THE IMPORTANCE of the unicellular green alga,
Chlorella vulgaris, to plant physiologists engaged in
studies of cellular metabolism and especially of pho-
tosynthesis is well known. Relatively few detailed
studies have been made of its growth, however. The
present paper deals with the growth curve of Chlo-
rella in cultures inoculated with different numbers
of cells taken from parent cultures of the same age.
Multiplication, or increase in cell number, was used
as an index of growth in this work, although it is
realized that a period of cell enlargement precedes
each division, and that growth is the result of these
two processes.
MATERIALS AND METHoDs.-Cells for the experi-
ments were withdrawn from four-day-old, rapidly
growing stock suspensions of Chlorella vulgaris cul-
tured as previously described (Craig and Trelease,
1937; Pratt and Trelease, 1935) in a solution that
contained KN0 3 , 0.025M; MgS0 4 "7H 20, 0.02M;
KH 2P04, O.OISM; FeS04"7H20, O.OOOOIM; po-
tassium citrate, O.OOOOIM; and Zn, Cu, B, and Mn
in approximately the concentrations employed by
Trelease and Trelease (1935). The initial pH of
this solution was 4.45. New stock cultures were
started daily so that there was always available a
supply of cells of approximately the same physio-
logical age and activity.
Cells were separated from the culture medium
and were washed in three changes of distilled water
by repeated centrifugation and decantation. They
were then suspended in distilled water and the den-
sity of the population in the suspension was esti-
mated from haemacytometer counts. Inocula con-
taining the desired number of cells were pipetted
into 500 ml. Pyrex glass Florence flasks, each of
which contained 150 ml, of the standard nutrient
solution. The flasks were continuously illuminated
from below by a water-cooled battery of twenty-
four 50-watt frosted Mazda lamps. The light in-
tensity, measured by means of a Weston photoelec-
tric-celllight meter, Model 603, varied from 21,000
lux to IS,OOO lux. The lamps were replaced by a
complete new set when the intensity fell below this
value. The temperature at the level of the flasks va-
ried from ISo to 22°C.
A gas mixture containing 5 per cent CO2 and 95
per cent air was bubbled continuously through the
solutions. The cultures were shaken vigorously twice
daily to insure separation and uniform suspension of
the cells.
Growth measurements were made periodically by
withdrawing small samples from each flask and esti-
mating the cell population from haemacytometer
counts. Triplicates of each culture were prepared,
1 Received for publication November 97, 1939.
The author is indebted to Miss Jane Fong for valuable
assistance in the laboratory.
52
and cell counts were based on the averages of haema-
cytometer counts of four different samples from
each flask. Each point in the figures represents,
therefore, the average of twelve samples. The maxi-
mum deviation from the mean value occurred in the
lower portions of the growth curves and never ex-
ceeded 5 per cent. Over the major portion of the
curves in all cultures the deviation of individual cul-
tures from the mean value was less than 3 per cent.
EXPERIMENTS AND RESULTs.-Figure 1 shows the
growth in cultures inoculated with different numbers
of cells withdrawn from parent cultures of the same
age.
Since the points describe sigmoid curves, it seemed
of interest to examine the data to ascertain whether
they could be represented by the equation charac-
teristic of autocatalyzed monomolecular reactions
that has been found, in many cases, to describe rea-
sonably well the growth of populations and of in-
dividual organisms (Ostwald, 1905; Robertson,
1908a, 1905b, 1923; Reed and Holland, 1919;
Reed, 1920, 1921a, 1921b, 1925a, 1925b, 1932;
Gaines and Nevens, 1925; Porterfield, 1928; Pratt,
1936).
The differential form of this equation expresses
the rate of growth, and may be written
dx
dt = k x (A-x) (1)
Upon integration, it becomes
x
log A-x = K (t-t]) (2)
where x represents population per cubic millimeter
at time, t; A represents the maximum number of cells
attained in the same unit volume; t, is the time when
x = A/2; and K = k A/2.3. K is inversely propor-
tional to the duration of the growth period (2 t]).
The final density of population that was attained
was found to be independent of the size of the inocu-
lum. The value 97,300 was obtained for A by com-
putation of the mean maximum number of cells at-
tained per cubic millimeter of culture medium.
Values for t] were computed by the procedure sug-
gested by Robertson (1923, p. 325), and those for
K were determined by computing the slopes of the
curves that were obtained when values of log
[x / (A -x)] were plotted against those of (t-t,).
The curves in figures 2 and 3 represent values of
x at different times (t) and were calculated by sub-
stituting known values of A, K, and t, in equation 2
and solving for ai, During the greater part of the
growth period, they afford reasonably close ap-
proximations of the actual experimental observa-
tions represented by the points.
It should be noted, however, that in order to se-
cure conformity of the calculated curves with the
Jan .. 1940] PRATT-GROWTH OF CHLORELLA VULGARIS 53

experimental points over the major portion of the

100 '.ggg «!;Lsi",!'"
, 00
'0
growth periods, it was necessary to slide the curves
I, CEL.L/IO ~M. on the time axis. Therefore, the initial points of the
~7S
different curves do not coincide with the experimen-
is tal zero time except in one instance-i.e., when the
(j;- initial concentration was 100 cells/cu. mm, It is
..J
..J
W noteworthy, and may be of biological significance,
USO
III
o
Z
~ +2
::>
§? 25
f-
+1

0
320 400
-I
o P)() CELLS/C.MM.
.500 .. ..
xl1 -2
o 100 19 INITIAL NUMBER
• '0 0 CELLS/ C,MM.
::i • 10 ..
--l -3
~7S 0-----------1000
U e- - - -----. ·500
"-
III -4
~-- - - - •••. 100
.- - - - - . - 50
..J c- - - - . - _. 10
GJ .-------- I
uSO 0- - - - - - - I IN 10
-5

~
a
0 80
t
160 240
(HOURS)
320
i: 25
Fig. 4. Experimental values of log [x/(A-x)] as func-
-2 tions of time, reckoned from the time of inoculation. The
LOG INITIAL. CELLS I c.MloA;
o 2 +J
ordinates are correct for cultures initially containing
-40 0 160 240 320 1,000, SOD, and 100 cells/cu. mm, To avoid confusion the
HOU:<S
other curves have been lowered as follows: SO/cu. mm.
100L-~~-~~--'-"--'---'-~-"'------' -0.3; 10/cu. mrn, -0.3; I/cu. mm. -0.7; O.l/cu. mm, -1.0. The
time scale is correct for all curves in this figure.

~
::<75 that for the different curves the deviation of the
tJ
<, - theoretical starting point from the actual time of
III
:J inoculation varied in a regular manner with the ini-
w
vso tial population density over a relatively wide range.
This relationship is shown in the inset in figure 2
i
,=o 25
where the deviation in hours is plotted as a function
of the logarithm of the initial cell concentration.
o 100 CELLS le""M. Equation 2 may be written
o I .. ..
• I CELL/IO CMM
x n
log A-x -log A-n = K t (3)

Fig. I (above). Growth of Chlorella vulgaris in cultures

inoculated with different numbers of cells.
Fig. 2 (center). Growth of Chlorella vulgaris in cultures
inoculated with different numbers of cells. The curves have
the equation log [x/(97,300-x)] =0.0133 (t-I71). The
time scale is correct for the culture started with 100 cells/
cu. mm. For all other cultures the curves have been shifted
along the time axis. Points represent experimental ob-
servations. Inset. Deviation of origins of calculated curves
from the experimental zero time as a function of the
logarithm of the initial cell concentration.
Fig. 3 (below). Growth of Chlorella vulgaris in cultures
inoculated with different numbers of cells. The curves
have the equations log [x/(97,30D-;v)] 0.0133 (t-I71)
and log [x/(97,300-x)] =0.0162 (t-140) for cultures ini-
tially containing 100 and I or 0.1 cell/cu. mm., respectively.
where si, A, K, and t have the same significance as
above, and n represents the value of x when t = o.
The intercepts of the curves in figure 4 with the
ordinate then represnt values of log [n/ (A-n)]
when t =
0, and values of n, or the number of cells
theoretically initially present immediately after
The time scale is correct for the culture started with 100
cells/cu. mm. For the other cultures the curves have been
shifted along the time axis. Points represent experimental
observations. Inset. Same curves drawn with a common
= origin. Values of tl and J( are based on the duration of the
time during which measurable growth occurred, not on
the elapsed time from inoculation to cessation of growth.
54 AMERICAN JOURNAL OF BOTANY [Vol. 27,

inoculation, may easily be computed for the different however, to consider growth not as a function of the
cultures.f colony but of the individual cells. Figures 6 and 7
Invariably the calculated value of n exceeded the show that the rate of growth considered in terms of
experimental value (table 1) and neal e. increased as the density of population, or reproduction per cell
a power function of n ex p . (Fig. 5). This indicates ("capacity of the individual cells to reproduce"),
constantly decreased. Experiments showed that the
decline in rate of growth was not due to depletion of
3 nutrients in the culture medium.

900 36
./ - <,6. CELLS/C.MM./HR.
-,

~
. /
2 800 ~32
~
-c w / "-
u 700 U28 / \
z
/ \
o
o
..J
~600~4 / \
........ ri. I \
:i500~0 I
\
3 ~ / \
o ~oo lJI6 /
L_':-,----!:-----l---+----:c!;-'
o +1 " +3
+2 ~ ........ \
LOG NEXp. d300~12 /
\
U d I
Fig. 5. Values of log neal e• as functions of. log nexp.
<]200 U 8 l \
(See text for explanation.) <] \
100 4 h ~
that in the early stages of growth the rate of multi-
plication was more rapid than that indicated by the o 10 30 50 70
THOOSANDS CELLS !C)J.M.
idealized form (equation 2) and that the deviation
, ,
1. Experimental and calculated values of n in
,,
TABLE 800 32
Ohlorella cultures started with different densities of
population. (See text for explanation.) 700*8 t:. CELLS /C.MM.
-I
-I
ncal c. 600tj24
n ex p . ncal c . .
(cells [ea. mm.) (cells/cu. mrn.) n'exp.
SOO§20
1,000 2,344 2.34 ~ <;
1,413 2.83 ~ ~
500
_lJ400:::E16
<.J
100 501 5.01
50 316 6.32 <I) -
::J300~12
10 133 13.30 w ..J
I 25 25.00 U w
U
200 8
0.1 5 50.00 <I <I
100 4
from expectations increased with the dilution. Ex-
pressed in another way, in a series of cultures, the o 80 160 240
one with the least dense initial population had the HOURS
highest rate of growth per cell. Fig. 6 (above). Hourly rates of growth in cultures of
The rate of multiplication per cell varied in- Chlorella vulgaris started with different numbers of cells,
versely with the density of population, not only plotted as functions of the population. Initial populations
among different cultures in the early stages of equal I cell/cu. rnm. (discontinuous curve) and 100 cells/
growth, but throughout the growth period in each cu. mrn, (continuous curve).
culture. This is shown graphically in figures 6 and Fig. 7 (below). Increase in population in Chlorella vul-
7, in which hourly rates of increase in Chlorella are garis cultures as a function of time. These curves are cor-
rect for cultures started with 100 cells/cu. mm., but apply
plotted. approximately to those started with 1,000, 500, 50, or 10
The data yield the familiar "grand-period of cells/cu. mm. if zero time is considered as the origin of
growth" type of curve, that has been interpreted to the ideal curve (fig. 2).
mean that "the capacity of the individual cells to re-
produce "(within a given period) increases up to a
One interesting result of these experiments was
certain maximum with the population of. the cul-
the discovery that the growth rate for a given den-
ture" (Robertson, 1923, p. 83). It seems logical,
sity of population was always greater in cultures
2 It should be noted that t in equation 3 refers to total
elapsed time from inoculation. Therefore, it has a different started with a small number of cells than in those
numerical value than t in equation 2. J{ in equation 3 is inoculated with a larger number. This is shown
identical with J{ in equation 2. graphically in figures 4 and 6.
Jan., 1940] PRATT-GROWTH OF CHLORELLA VULGARIS
In figure 4 ordinates and abscissas represent log
[x/(A-x)] and time, respectively, and the slopes
represent values of the velocity constant, K. The
slope of the curve for each dilution is slightly great-
er than that of the curve for the preceding dilution
(table 2). Although the differences among the five
upper curves and between the two lower ones are
small, they may be considered significant, since they
do not vary in a random manner but indicate a defl-
TABLE 9. Velocity constant J{ for cultures started with
diff erent densities of population.
Initial No.
Cells/cu. mm.
1,000... . . ..
500..
100......
50.... .. .. ..
10.... . .. . .
1. . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . ..
0.1 . . . . . .. . . .. .. . . . ..
nite trend. Although there is a gradual and constant
change in K from the most concentrated to the least
concentrated initial cell suspension, the curves may
conveniently be considered as falling into two groups
-i.e., those for cultures initially containing 10 or
more cells per cu. mm, and those containing less than
10 cells per cu. mm, The curves were treated in this
manner, and the mean values of K for each group
were used in calculating the values of x plotted in
figures 2 and 3. For convenience of comparison, typi-
cal calculated curves for cultures with small and
large inocula are drawn with the same point of
origin, and the values of A, tI, and K for each are
given in the. inset of figure 3. 3
The curves in figure 4 show that the rate of in-
crease throughout the entire growth period was
greater in cultures with the more dilute initial sus-
pensions.
The distinctly higher growth rate is shown graphi-
cally in another way in figure 6, from which it may
be seen that for a given density of population, the
growth rate, as measured by increase in number of
cells per hour, was greater in cultures started with a
very small number of cells than in those inoculated
with a larger number.
DISCUSSION.-It was found that in a series of cul-
tures in the early stages of growth, under the ex-
perimental conditions employed, the culture with the
least dense population exhibited the highest rate of
reproduction per cell. This may be interpreted to
mean that a toxic or growth-inhibiting substance, the
amount of which varied directly with the number of
cells, was carried into the cultures with the original
inoculum. Since the cells were always thoroughly
3 Note that the values of t and t l that were used in
calculating the values of x plotted in figures 9 and 3 are
not based on total elapsed time from inoculation to cessa-
tion of growth. (See legend.)
55
washed in distilled water before transfer, it seems
unlikely that a significant amount of the old culture
medium adhered to the cells. I t may be suggested,
therefore, that a growth-inhibiting factor was con-
tained within the cells. Further evidence to support
the view that the cells contained a growth-inhibiting
agent and that it was produced by the cells during
their growth is furnished by the fact that in any
given culture, the rate of reproduction per cell stead-
ily decreased as the density of population increased.
It was also found that in a series of cultures inocu-
lated with different numbers of cells, the rate of
multiplication throughout the growth period varied
inversely with the initial density of population-
J{ i.e., the rate of increase at any given level of popu-
lation was greater for cultures initially containing a
0.0131 small number of cells than for those started with
0.0130
0.0139
more dense suspensions. This is in harmony with the
0.0134 ideas suggested above, and may indicate that the
0.0136 rate of multiplication in Chlorella is determined, in
0.0159 part at least, by the concentration of the hypotheti-
0.0165 cal growth-inhibiting agent in the cells. According
to this idea, the concentration of inhibiting agent in
the external solution increases more rapidly in cul-
tures started with larger numbers of cells and tends
to check further loss of the substance from the cells,
thus facilitating its intra-cellular accumulation and
thereby tending to suppress division.
Thus, the concentration of "auto-inhibitor" with-
in the cells, and hence the rate of reproduction for
a given density of population in any culture, may
depend, among other things, upon the age of the
parent culture from which the inoculum for the
daughter suspension is withdrawn, the size of the
inoculum with respect to the volume of the new cul-
ture, and the size of the inoculum used to seed the
parent culture. Preliminary results from experi-
ments in which cells have been transferred from ac-
tively growing cultures to fresh media and to cell-
free media in which some growth had already oc-
curred tend to confirm this hypothesis. It should be
emphasized, however, that more data are required
before the full significance of the present experi-
ments can be determined and that the suggested
hypothesis is purely tentative.
Discussion of the literature pertinent to this phase
of the growth problem will be deferred to a later
paper in which it is hoped to correlate the data given
here with those of experiments now in progress. Im-
portant reviews dealing with problems of mutual
growth acceleration and inhibition among living or-
ganisms are given by Allee (1931, 1934), Johnson
(1933), Rahn (1932), and Robertson (1923). In
addition, the work of Peebles (1929) on echinoderm
larvae, Phelps (1935, 1936) and Mast and Pace
(1938) on protozoa, and of Pearsall and Loose
(1937) on Chlorella should be mentioned.
SUMMARY
The multiplication of Chlorella vulgaris furnished
with a gas mixture of 5 per cent CO 2 and 95 per
56 AMERICAN JOURNAL OF BOTANY
cent air in the nutrient solution employed in con-
tinuous light may be empirically described by the
equation characteristic of an autocatalyzed mono-
molecular reaction.
Although the equation furnishes a reasonably ac-
curate picture of the course of multiplication over
the greater portion of the growth period, it was
found to be inadequate in the earliest stages of
growth, and the discrepancy between calculated and
experimental values when the cell count was small
increased with the dilution.
The maximum population attained was independ-
ent of the size of the inoculum, but the rate of multi-
plication throughout the growth period, indicated by
the velocity constant (K), was greater for cultures
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