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To cite this article: Christine Butterwick , S.I. Heaney & J.F. Talling (1982) A comparison of eight
methods for estimating the biomass and growth of planktonic algae, British Phycological Journal,
17:1, 69-79, DOI: 10.1080/00071618200650091
A C O M P A R I S O N OF E I G H T M E T H O D S F O R
E S T I M A T I N G T H E BIOMASS A N D G R O W T H
OF P L A N K T O N I C A L G A E
Eight methods for estimating algal biomass were compared, using the colonial diatom
Asterionellaformosa as a test organism. They were based on (i) cell counts by visual microscopy
and electronic means, (ii) optical properties in vivo of scattering, attenuance and fluorescence
and (iii) chemical estimations, on filtered cell-aliquots, of reducing capacity (C-equivalent) and
solvent-extracted chlorophyll a. The main criteria were the parameters of precision, sensitivity,
limit of detection plus time taken and sample quantity required.
Most methods yielded an acceptable precision over wide ranges of algal concentrations
although for visual counting and nephelometry the coefficient of variation typically exceeded
107o. The estimations in vivo were the most rapid, but were unsuitable for very low biomass
concentrations. The chemical methods and (on undiluted samples) electronic counting generally
required the larger quantities of sample--with the notable exception of extract fluorometry.
The chemical methods were relatively slow but they allowed batches of samples to be processed
together and gave more generalized measures of algal biomass (e.g. C-equivalent, chlorophyll
a). Visual cell counts, although relatively slow and fatiguing, were unsurpassed for low limit
of detection, economy of sample, and assessment of cell condition.
1949; Tailing, 1957; Hughes &Lund, 1962; Kilham, 1975; Jaworski, Talling
& Heaney, 1981) or electronic counter (Lund, Jaworski & Butterwick, 1975)
or both (Titman & Kilham, 1976). Some comparisons have been made with
two other smaller algae.
BIOMASS ESTIMATIONS
(a) Microscope counts
Direct counts of Asterionella were carried out, after sedimentation with Lugol's iodine
solution, using the Uterm6hl inverted microscope technique as described by Lund, Kipling &
Le Cren (1958). About 100 colonies (the statistically sensitive units) were sedimented for
counting with dilution of the original sample if necessary. Individual cells were also counted,
as a better measure of biomass. Counts were made using a simple inverted microscope at
× 60 magnification. Other smaller species were counted in a slide chamber at higher power,
using a good-quality normal microscope as described by Lund (1959).
Methods for estimation of algae 71
( b ) Electronic counter
Cell volume was determined electronically by detection of the change in resistance to a
current applied across an orifice. Measurements were made using an electronic particle counter
(Model B, of Coulter Electronics Inc., 590 W. 20 Street, Hialeah, Florida, U.S.A.) following
the procedure described by Sheldon & Parsons (1967). About 150 ml of the algal suspension
was made to give a resistance of 25 kS2 by adding c. 0"5 ml of a saturated solution of NaC1.
The suspension was then passed through a 140 jzm orifice and counted in aliquots of 0"5 ml,
while mechanical stirring took place. As a culture of Asterionella contains colonies with differ-
ing numbers of cells, the total algal volume was estimated by obtaining readings of particle
numbers at a series of instrument sensitivities and calculating cumulative volumes as given in
the manufacturer's manual. A calibration factor to convert relative to absolute volume (/~m3)
was determined by estimating particles (e.g. pollen grains) of known volume. Correction was
not made for coincidence as this was < 2 ~ at the densities used.
(c) Nephelometry
The amount of light scattered at 90 ° from a beam passing through an algal suspension was
detected by a nephelometer (Model 40, of Turner Designs Inc., Mountain View, California,
U.S.A.). Measurements were made in nephelometric turbidity units (NTU) with reference to
a supplied standard of 20 NTU. To minimize observed fluctuations, readings were taken at a
standard time of 2 min after samples had been inserted.
RESULTS
T a b l e I I summarizes the most i m p o r t a n t characteristics o f the eight methods.
These are o f three basic types. G r o u p A involves a direct c o u n t of m o r p h o -
logical units (cells) present i n a given volume o f sample. G r o u p B consists of
in vivo methods in which a vessel is directly filled with sample a n d a density-
d e p e n d e n t physical property measured, a n d G r o u p C methods require filtration
of a suitable v o l u m e of sample prior to analysis.
The a p p r o x i m a t e time t a k e n to complete a n estimation was noted, a l t h o u g h
the time per sample can often be reduced by p e r f o r m i n g estimations in batches.
Some methods also include " w a i t i n g " time, such as in sedimentation a n d dark
adaptation.
T h e v o l u m e o f sample used (Table II, a - c ) for the in vivo methods a n d the
C o u l t e r c o u n t e r was determined by the size of the vessel provided with the
i n s t r u m e n t . F o r other methods the v o l u m e o f sample required depended u p o n
the culture density.
Direct c o u n t i n g o f cells with the inverted microscope was a d o p t e d as a
s t a n d a r d m e t h o d by which others were c o m p a r e d (Table II). A l t h o u g h one o f
the slower methods, liable to operator-fatigue a n d based o n a potentially
variable cell-unit, it has the advantages that it requires small volumes, gives
estimates o f statistically k n o w n precision a n d has a low limit o f detection. The
latter w o u l d be less t h a n 1 cell ml-1 if 20 m l of sample were sedimented, b u t
depends u p o n the volume used. The precision o f the estimate depends u p o n the
TABLE II. Quantitative characteristics o f the m e t h o d s as used with Asterionella
original
Sensitivity in m e a s u r e d cell
V o l u m e used Time Limit of Detection units density Coefficient o f Variation
(ml)* taken as cells as cells per 104 cells per 104 cells (m1-1 percent- as cells as cells
Methods (a) (b) (c) (min) m1-1 m1-1 x 10 -8) age m1-1
(A) C o u n t i n g m e t h o d s
(1) Microscope 1 1 7 10:~ (see text) standard -- 0.8 10-5 85 --
(2) Coulter 140 140 980 30 160 -- 17x103 CU -- 8'0 3"9 156 -- ~-
counter 44.7 2.2 490 --
(B) In, vivo m e t h o d s ~,
k~J N e p h e l o m e t r y 20 20 140 5 (25 x 103)t 1250 -- 0.56 N T U 5"3 15.2 -- 805
40.0 4.6 -- 1840
(4) Spectrophotometric 10 10 70 5 (7 x 103)t 670 -- 0-067 A 5"3 2"0 -- 148 =.
attenuance 40.0 0-6 -- 240
(5) Fluorescence 5 5 35 55 (3"5 x 10a)t 690 -- 19 F U 5"3 3,0 -- 159 ~.
in vivo 40"0 0-9 -- 360
(C) Chemical m e t h o d s o
(6) R e d u c i n g capacity 100 10 210 210 118 x 108 -- 0-052 ml titrant -- 36.2 3.5 12.8 x 108 --
(carbon equivalent) (0'32 #eq) -- t~
(7) S p e c t r o p h o t o m e t r y 100 100 210 50 1 6 x 108 -- 0.0015 A -- 15 3"3 49'5 x 108 --
(chl a extract)
(8) F l u o r o m e t r y 10 1 22 60 330 -- 39 F U -- 15 8"6 1.3 x 108 --
(chl a extract)
*(a) is the a p p r o x i m a t e m i n i m u m required for a density o f 1000 cells m l -a, (b) is t h a t used in the m e a s u r e m e n t s , (c) is t h e total necessary for the
postulated growth experiment (see text).
U n i t abbreviations: C U , Coulter units; N T U , nephelometric turbidity units; A, a b s o r b a n c e / a t t e n u a n c e ; F U , fluorescence units
t i n t h e vessels used.
:~After 1 h sedimentation (for counting) or d a r k - a d a p t a t i o n (for fluorescence).
74 C. BUTTERWICK, S. I. HEANEY AND J. F. TALLING
size of the count; thus for samples from a Poisson distribution, the 95 ~o confi-
dence limits are ~ 2 0 ~ for a count of 100 units (e.g. Asterionella colonies)
(Lund et al., 1958). An additional advantage is that the visual condition of the
cells, and possible presence of debris or precipitates, can be observed.
Direct particle counting with the Coulter counter gives a good precision and
low limit of detection. However, large volumes are normally required because
of the size of the vessel, although dilutions can be made of denser cultures. For
a colonial alga such as Asterionella, measurements of cell volume take appre-
ciably longer to estimate than with a single celled species, as several instrument
settings of the volume sensitivity are required.
More rapid estimations of biomass were obtained by the optical in vivo
methods (Group B). These use small volumes of sample which can be returned
to the growth vessel if adequate aseptic precautions are taken. They are, how-
ever, the methods least specific to algal material, and any change in an accom-
panying precipitate (e.g. due to pH increase during growth) would interfere.
The spectrophotometric and fluorometric methods showed very similar and
acceptably low limits of detection (< 1000 cells ml-1), and also good precision.
The spectrophotometer used was found to be very stable and was readable to
0-0001 A. Also, the semi-micro cuvettes of 10 cm path-length increase sensitivity
over the more usual 4 or 5 cm cells, without requiring more than 10 ml of
sample volume, and could be used in groups of four. Although measurements
of fluorescence could be made as quickly as those of attenuance, time (c. 1 h)
was necessary for the algae to become dark-adapted before fluorescence esti-
mation.
Nephelometry showed considerably higher variation than the other two
in vivo methods, even though this was minimized by taking readings after a
standardized time interval (2 min). Also, the more variable readings given by
the medium "blank" led to the high limit of detection, with the greater size of
these readings contributing to the high CV, especially that at the lower culture
density (15 ~).
The remaining methods (Group C) involved a preliminary filtration step, so
that their characteristics depended upon the volume taken-- which could be
increased for dilute suspensions. The estimation of reducing capacity (carbon
equivalent) had relatively good precision, but required a fairly large sample
volume for cultures of lower density. The relatively long time taken to complete
the analysis was offset by the possibility of storage after filtration, with later
batch-analysis.
Of moderate rapidity were the two methods for estimation of extracted
chlorophyll a. Their limits of detection were determined by instrument stability
and manipulative variation, as there was no detectable background reading
from the medium "blanks". The spectrophotometric method required a volume
of sample similar to that used for estimation of reducing capacity, but only
about one-tenth of this was needed for the fluorometric method.
Dilution series for the Coulter counter and in vivo methods (Fig. 1) show
that both fluorometry and nephelometry were linear in response up to c.
80 × 10a Asterionella cells ml-1, which was about the maximum density obtained
in batch cultures of Asterionella. Attenuance and Coulter counter methods were
linear up to c. 150× l03 cells m1-1, although at such high densities clogging of
Methods for estimation of algae 75
O0.#~r I | t
15- (b)
/
~5
.el-
•m I I t
%
300~ lc)
2O0
I00
ol
4OO
20O
0
o I 2 3
the counter orifice could occur. The precision of the remaining methods was less
dependent on sample density, as the volume filtered or sedimented was adjusted
to give estimations within a favourable range: nevertheless, the necessity for
removal of large volumes does place restrictions upon experimental design.
The significance of the differing sample volumes required between methods
can be illustrated [Table II, (c)] by comparing minimum total volumes necessary
to follow the growth of Asterionella in batch culture, from 1 × 103 to 645< 103
cells m1-1 with an unreplicated and destructive estimation at each doubling
(totalling seven estimations).
The directly density-dependent methods (nos 2, 3, 4, 5) can have the total
volumes reduced by about 30 ~ if the denser samples are diluted. The Table
shows the outstanding sample-economy of microscope counting (method 1),
and the compatibility of methods 3, 4, 5 and 8 with cultures of initial volume
150-250 ml. Larger total volumes are required for methods 2, 6, and 7.
The practical application of four methods is illustrated in Fig. 2, which
shows determinations of growth of a culture of Asterionella. Good agreement
between these methods was found, all giving a specific growth rate of 1.2 cell
divisions per day over the exponential growth phase. However, the differing
limits of detection indicate the differing abilities of the methods for following
the initial growth.
10 5 -- IOO
10 4
O'Oi
o.i ~-" l-
m
10 3 - -
I I I I | | | | I S I i i ,
2 4. 6 8 I0 0 2 4 6 I0
o 2 4 6 8 I0 0 2 4 6 8 I0
Days
FIG. 2. Estimations of the growth of an Asterionella culture, using four methods. Arrows
indicate limits of detection; that for carbon analysis applies to a sample volume of
100 ml. The symbol O indicates the initial cell density calculated from inoculum size.
(a) Cell counts (cells ml-1); (b) carbon (/zg C ml-1); (c) attenuance (A); (d) fluorescence
(arbitrary units).
Methods for estimationof algae 77
DISCUSSION
Considering the wide use of algal cultures in experimental ecology and
physiology, it is surprising that there are few (if any) extensive and rigorous
comparisons of methods for determining biomass in culture. More specialized
studies are less uncommon, such as that of Iwamura et al. (1970) on the use of
RNA, DNA and protein, and Holm-Hansen & Booth (1966) for ATP, as
indices of biomass. Several intercomparisons of methods have been made with
natural populations of phytoplankton (e.g. Hagmeier, 1961 ; Hallegraeff, 1977).
However, because of complications arising from mixed assemblages of organisms
and the presence of other particulate material, these studies may not be relevant
to work with axenic or unialgal cultures. Even with such cultures, difficulties
arise from changes in algal constitution during growth and under varied physical
and nutrient conditions. Such problems are here minimized by the use of
cultures in the exponential phase of growth.
The performance characteristics here attributed to various methods are only
illustrative as they are influenced by the quality of the instruments used and
algal species involved. Thus the inverted microscope available, although
adequate for counting Asterionella formosa, had insufficient resolution for the
smaller Cyclotella pseudostelligera and Rhodomonas minuta, which required the
higher magnifications obtainable with a normal microscope when used with a
Lund chamber. Low limits of detection and high precision were achieved for
measurements in vivo of attenuance and fluorescence using good quality,
double-beam instruments of high stability and appropriate cuvettes, some of
long (10 cm) pathlength. The relatively poor limit of detection and precision
of the present nephelometric method is disappointing; although nephelo-
metric instrumentation is varied (Vanous, 1979), it can be capable of high
sensitivity (e.g. Aurisicchio et al., 1972). Readings obtained with Asterionella
were much less stable than those with Cyclotella and Rhodomonas, suggesting
that there may be changes in orientation of the Asterionella colonies which
affect 90 ° scattering of light. Such an optical method is also liable to inter-
ference from debris and precipitates.
Less detailed studies with cultures of Cyclotella and Rhodomonas indicated
some variability between species in the sensitivities of methods based on light
scattering (nephelometry) and attenuance. Readings obtained by these measure-
ments were approximately twice as great per unit of chlorophyll a for Cyclotella
(cell volume c. 176/~ma) than for Rhodomonas of similar volume or the much
larger Asterionella cells.
The chemical methods were all relatively slow and, with the exception of the
fluorometric determination of chlorophyll a, required moderately large sample
volumes. As carbon is normally the major and most constant component of
algal matter, it has obvious advantages as an index of biomass. Although the
method of dichromate oxidation for carbon equivalent has been criticized by
Menzel & Dunstan (1973) as subject to relatively large error (> 50/~g C), the
present micro-adaptation had good precision and a limit of detection equivalent
to only 11/~g C (118 × 108 Asterionella cells).
The Coulter counter gives particle counts proportional to their volume, and
it might be regarded as giving a particularly direct measurement of biomass.
78 C. BUTTERWICK, S. I. H E A N E Y A N D J. F. TALLING
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(Accepted 21 August 1981)