NOTES
AND
COMMENT
AN AUTOMATED TECIINIQUE FOR DETERMINING
CHAIN-FORMING
THE GROWTH RATE OF
PI~YT~IXANKTON
INTRODUCTION
METI1ODS
El-Sayed and Lee ( 1963) have described
the use of a Coulter Counter, Model A, for
the enumeration of unicellular algae, They
concluded, however, that the instrument is
not particularly suitable for counting algal
species where long chains of cells are encountered and suggested that the Model B
Coultcr Counter with an automatic cell-size
distribution plotter could be used to distinguish between a mixture of algal species of
different cell size. Their suggestion has been
investigated and verified, but what appears
to be of greater importance is the use of
this newer instrument for measuring the
growth rate of chain-forming diatoms.
In many coastal areas the predominant
diatoms are chain-forming spccics; the commonest is probably Skeletonema costatum.
On occasion a phytoplankton
bloom may
be largely composed of one species, or an
association of several species may account
for most of the standing stock. At present,
however, there is no way of determining
the growth rate of the individual species in
a plankton bloom except by the laborious
technique of microscopically counting individuals, a technique that is not sufficiently
accurate to show an increase of much less
than about twofold. In addition, the microscopic enumeration of phytoplankton
gives
little indication of changes in cell volume.
The Model B Coulter Counter, used in
conjunction with an automatic cell-size distribution plotter ( Model J ) , can be used on
mixed cultures or on natural populations to
measure the growth rates of individual
species if there is sufficient difference in
their ccl1 volumes. If two species have
similar cell volumes, the growth rate of the
two can be determined as an average.
Some parts of the work carried out here
arose from suggestions made by Dr. D.
Cushing, Lowestoft, U.K.
Cullzcres-In
one experiment, two culturcs of diatoms were grown in oceanic seawater (ca. 32% salinity) enriched with 500
lug-at. N/liter as KNOZ, 50 PI;-at. P,/liter as
KzHPO.1, and 500 pg-at. Si/liter as IICIneutralized sodium silicate. In addition, I
ml/liter of vitamins and trace metals were
added as dcseribed by Jitts et al, ( 1964).
The diatom cultures were obtained from
Dr. J. D. H. Strickland, Institute of Marinc
Resources, La Jolla, California, and arc described as follows: Skeletonema costntum
(R. Guillard,
isolate from Long Island
Sound ) ; Coscinodiscus concinnus ( 1%. W.
Holmes, AD-1 ) .
Experimental
procedure-The
general
operation of Coulter counters has been described in a number of recent publications
(El-Sayed and Lee 1963; Maloney, Donovan,
and Robinson 1962). In the following cxperiments, a Model B Coulter Counter with
a Model J automatic cell-size distribution
plotter was used for all the electronic counting. The apparatus was fitted with a 400 p
aperture and calibrated using ragweed pollcn to record 100 E,L~
per threshold unit at an
amplification of 1 and an aperture current
of %. Plots of the volume distribution
of
different species were obtained at this setting or at some multiple that allowed for a
total range of particle volumes from 5 X lo”
to 2 x lo7 /LY The actual number of particles in one volume setting was determined
from a separate count by allowing 2 ml of
culture to flow through the aperture. The
number of particles in all other volume scttings was obtained as a simple ratio of the
height of the cnumcrated setting to the
height of each of the other settings. The
total cell volume of the species was obtained from the sum of the number of
particles times the mean volume for each
volume setting. The log,,, of the total cell
598
NOTES
s
AND
COSTATUM
0
FIG.
during
1. Cell volume
7 days growth.
plot
of S. costatum
cells
volume at time t2 minus the log,, of the
total cell volume at time tl divided by the
time interval, tZ - tl, was used to obtain the
growth constant of the species.
Coincidence corrections were not applied
to the total counts, because it was assumed
that the counting error caused by coincidence would be the same for counts at
times tr and t2. This assumption is not entirely correct, because the magnitude of the
coincidence correction increases with the
number of particles counted and would be
greater for counts at time t2 than at time tl.
However, the magnitude of the coincidence
correction, which also increases with the
size of the aperture, can be reduced by
counting low numbers of cells. In these experiments, a volume setting was chosen for
enumeration in which the total count was
< 5 x 101:<
particles in 2 ml of medium. Below this level of counting, coincidence corrections should still be applied, however,
for large differences in total counts or if the
absolute count is required.
The growth constant as measured above
was compared with the growth constant obtained from the increase in cell numbers
per field as estimated by use of an inverted
biological microscope on a settled volume.
Two experiments are reported here as
examples of the technique. In the first ex-
599
COMMENT
periment, cultures of S. costatum and C.
concinnus were grown in enriched seawater
at 15C under fluorescent lights. After the
cultures began to grow, they were combined. The number of cells per microscope
field were counted on the next day (Day 1)
and on the 2nd and 7th days. Cell volumes
were measured with the Coulter Counter
for Skeletonema on days 1, 2, and 7, and
for Coscinodiscus on days 2 and 7. Cells
were kept in suspension during counting by
manual agitation of the medium with a
glass rod.
In a second experiment, natural seawater
was taken during the winter from Departure Bay, British Columbia, and incubated
for 6 days at 15C under fluorescent lights
( 16 hr light, 8 hr dark). The volume of
particulate material was measured- on the
1st and 6th days at different amplification
and aperture current settings. Growth rates
were estimated after day 6, when the
growth of phytoplankton had increased significantly
above the detrital background.
Chlorophyll a was estimated in this experiment by the procedure described in Parsons
and Strickland ( 1963).
RESULTS
Figs. 1 and 2 show the increase in cell
volumes of the mixed culture of Skeletonema
and Coscinodiscus as traced from the cellsize plotter. As the volume of Skeletonema
( Fig. 1) increased beyond the range setting
for the 7th day, a second range setting was
employed. The range setting for particles
of < 800 $ for the first two and 7th days
were not included in the estimations of the
total volume, because particles in this size
range were less than the mean cell size of
the species (ca. 1.4 x lo” $) and represented background particulate
matter in
the culture medium. The increase in the
chain length of Skeletonema in the culture
medium is reflected in Fig. 1 as an increase
in cells in different volume settings; new
volume settings were occupied as the chain
length of the species increased. In Table 1,
the growth constant of the species, determined from Fig. 1 and the cell count as
measured automatically, is compared with
600
TABLE
NOTES
1. A comparison
of growth
constants
Skeletonema
Time
AND
estimated
/‘c,, (days)-I,
Volume
Day 1 to 2
Day 2 to 7
Total
cell
volume
(pL3/2 ml)
11.4
22.3
434.0
3.74 x 10G
7.08 x 10"
158.2 x 10'
constants,
and microscopic
Coscinodiscus
Coy$ydper
1
(mean
of
10 fields)
W/2
Growth
by electronic
costatum
Total
cell
volume
ml)
Day 1
Day 2
Day 7
COMMENT
0.28
0.22
the growth constant obtained by microscopic enumeration,
The growth of Coscinodiscus is shown in
Fig. 2. This organism was too large to be
recorded using the range settings shown in
Fig. 1. Similarly, SkeZetonema was too small
to interfere with the volume estimate of
Coscinodiscus shown in Fig. 2. Skeletonema
is recorded in Fig. 2 in the first volume setting together with other particulate material in the culture medium and appears to
be larger in volume than shown in Fig. 1
because the plotter sensitivity has been
greatly increased to enumerate the relatively
low number of Coscinodiscus cells. The
growth constant of the Coscinodiscus cells,
as measured automatically and by microscopic counts, is shown in Table 1.
Fig. 3 shows the result of incubating
natural seawater for 6 days. The background of detritus was measured on the
first day when the chlorophyll n concentration was 0.46 pg/liter. At the beginning of
the light period on day 6, the size distribution of phytoplankton
and detritus was
measured. This was repeated 10 hr later,
and the difference in cell volumes was used
to determine the growth constant. From
microscopic examination, the phytoplankton were found to be a polymictic bloom of
small flagellates and diatoms, in which S.
costatum and Thalassiosira sp. predominated.
- - By changing ^_ the _volume settings
and the sensitivity of the plotter, it was possible to record the amount of growth of
enumeration
concinnus
Coy;ydPer
(mean of
10 fields)
3.5
5.6
6.15
306 x lo"
349 x 10';
as measured
by the two methods
CoTzydper
Volume
Com&per
0.204
0.0066
0.009
0.30
0.215
these two species and of the remaining mixture of small diatoms and flagellates. The
latter plot included some of the Skeletonema
cells in the size range BOO-l,900 p3. The
Thalassiosira sp. was considerably larger
than the Skeletonema and could be plotted
without any overlapping with another population. Growth constants were estimated
for the three populations shown in Fig. 3
by subtracting the detrital background in
each volume setting from the phytoplankton volumes before and after the lo-hr
The results showed that rapid
period.
growth occurred during the lo-hr period as
follows : kl, for small flagellates and diatoms, 0.026 hr-l; k10 for S. costatum, 0.041
0005
FIG.
2.
during 7
0225
051
Cell volume
days
growth.
077
J.?I 106
I02
plot of C. concinnus
I
cells
NOTES
100
AND
601
COMMENT
.
SMALL
AND
S. COSTATUM
FLAGELLATES
THALASSIOSIRA
SP.
DIATOMS
DAY
6
DAYS
a
IO
0
BACKGROUND
HOURS
6
l-J3
FIG.
3. Cell volume
water for 6 days.
plot of a mixed phytoplankton
hr-I; and kl, for Thalassiosira sp., 0.043 hrl.
The chlorophyll a content of the culture at
the end of the experiment was 26.0 pg/liter.
Thus, the total population had an average
growth constant of 0.011 hr-l during the
6.5 days. The difference between this value
and the values for the three individual populations might be explained if appreciable
growth was delayed until after the 3rd day;
this phenomenon, the “lag” phase of growth,
has been observed by others (see Strickland 1960, for discussion and references).
DISCUSSION
The growth constant of Skeletonema as
estimated by the Coulter Counter is in
close agreement with the growth constant
based on the actual cell counts (Table 1).
For Coscinodiscus, however, there is some
disagreement. It appears from Fig. 2 that
this may be explained by the change in
mean cell volume of this organism that occurred during the 6-day interval. Thus, on
day 2, a greater proportion of cells had a
volume of less than 1.02 x 10” $ than on
day 7. This change would be difficult to
DAYS
8.
IO
HOURS
)A3x IO3
A3
population
grown
after incubation
of natural
sea-
detect by microscopic examination, and it
must be considered that the automatic volume count gives a better estimate of the
growth rate of this organism.
In any study of natural populations or
cultures, it now appears possible to estimate the growth constants of individual
species in a mixture of species, provided
their cell volumes are sufficiently different
(Fig. 3). While the level of chlorophyll c1
(26 pug/liter) in the second experiment was
higher than values encountered in the
oceans, the sensitivity of the plotter in the
first size range could have been increased
eight times. Since a smaller increase in the
relative number of cells than is shown in
Fig. 3 would still be measurable, the lower
limit of sensitivity for the smallest organisms would be at a standing stock level of
at least a tenth of that employed in the experiment reported here. Using an aperture
smaller than 400 p, it would be possible to
lower this limit of detection further. For an
organism with a mean generation time of 24
hr, an increase of 1.15 in the cell volume
would be expected after 5 hr. Thus, if sam-
602
NOTES
AND
COMMENT
ples of seawater are withdrawn
and incubated (as in 14C studies), a volume plot at
zero time and after 6 to 12 hr would give
results similar to the lo-hr interval shown
in Fig. 3. By the use of different amplification settings, plots can be obtained of
species having different volumes, and the
growth rates of the individual species can
be determined in a polymictic bloom.
The difficulty of eliminating background
detritus from the total volume count appears to be less than might be supposed.
From optical studies of seawater, much of
the detritus in the oceans is composed of
particles of less than 20 ,.Lin diameter (Parsons 1963). For phytoplankton larger than
this, estimates of cell volumes can be made
by selecting an amplification to give a volume range in which these particles are confined to the first volume setting (that is,
< 3 x lo” ,1..2), For species of the same size
as the particulate detritus, the background
may be assumed to remain constant during
an incubation and, if appreciable growth of
the phytoplankton
can be measured, an
estimate of the amount of biomass produced per unit time can be made. In using
the Coulter Counter to determine biomass,
however, attention should be given to the
interpretation of what is actually measured
in terms of volume when nonspherical
shapes pass through the aperture. A com-
COLLECTION
OF SLICK-FORMING
prehensive discussion of this is given by
Hastings, Sweeney, and Mullin ( 1962).
T. R.
Fisheries Research Board of Canada,
Pacific Ocennogrnhic Group,
Nnnaimo, B.C.
REFERENCES
B. D. LEE.
1963. Evaluation of an automatic technique for counting
J. hlarine
Res., 21:
unicellular
organisms.
EL-SAYED,
S.
Z.,
AND
59-73.
AND
HASTINGS,
J. W., B. M. SWEENEY,
1962. Counting and sizing
MULLIN.
M. M.
of uniN.Y. Acad.
Ann.
cellular marine organisms.
Sci., 99: 280-289.
JITTS, H. R., C. D. MCALLISTER,
K. STEPHENS, ANII
1964. The cell division
J. D. H. STRICKLANII.
rates of some marine phytoplankters
as a
function of light and temperature.
J. Fisheries
Res. Board Can., 21: 139-157.
MALONEY,
T., E. DONOVAN,
AND E. ROBINSON.
1962. Determination
of numbers and sizes of
algal cells with an electronic particle counter.
Phycologia, 2: l-8.
PARSONS, T. R. 1963.
Suspended organic matter
in sea water, p. 205-239.
In hl. Sears [ed.],
Progress in oceanography,
v. 1. Pergamon
Press, London.
1963. Dis3 AND J. D. H. STRICKLAND.
cussion of spectrophotometric
determination
of marine-plant
pigments, with revised equations for ascertaining
chlorophylls
and carotenoids. J. Marine Res., 21: 155-163.
STRICKLAND,
J. D. H. 1960. Measuring the production of marine phytoplankton.
Bull., Fisheries Res. Board Can., 122: 172 p.
MATERIALS
A factor that can alter many properties of
the air-sea interface is a layer of adsorbed
surface-active organic matter. Such layers,
usually monomolecular films, can cause significant modifications in the capillary wave
structure and a consequent change in the
albedo of the area. According to Jarvis
( 1962), the adsorbed film may also alter
the surface temperature of the sea and interrupt the normal mass and thermal convection processes occurring just beneath
the air-water interface. A monomolecular
film inhibits the formation of small waves,
the breaking of waves, and increases the
PARSONS
FROM THE SEA SURFACE
rate of decay of capillary ripples (Garrett
and Bultman 1963; Davies and Rideal 1963).
Blanchard ( 1964) postulated that organic
films on the sea may influence the oceanic
production of cloud-forming
condensation
nuclei and modify the sea-to-air flux of
charged particles. Knowledge of the chemistry of organic films at the sea surface is
fundamental to an understanding of these
effects. The objective of this research was
to develop a technique to collect and recover the constituents of natural oceanic
slicks so that their chemical composition
could be determined.