British Phycological Journal

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A comparison of eight methods for estimating the

biomass and growth of planktonic algae

Christine Butterwick , S.I. Heaney & J.F. Talling

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

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 Br. phyeol. J. 17:69-79
1 March 1982

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

By CHRISTINE BUTTERWICK, S. I. HEANEY and J. F. TALLING

Freshwater Biological Association, Ambleside, Cumbria LA22 0LP

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.

The determination o f algal biomass is central to most studies o f p h y t o p l a n k -

ton. N u m e r o u s quantitative methods have been a d o p t e d including cell counts,
optical methods using live cells or extracted pigments, and chemical determina-
tions (e.g. Strickland & Parsons, 1972; Stein, 1973; Hallegraeff, 1977; Sournia,
1978). A l t h o u g h individual methods are frequently well described, with their
precision and limitations, there are few accounts o f their systematic inter-
comparison.
The ideal method for estimating biomass is rapid, accurate, with g o o d
precision and low limit o f detection, and uses a small volume o f sample. The
response should be linear over a suitable range o f biomass and be specific to
the plant (algal) material o f the sample. Absolute accuracy is not necessary for
the estimation o f specific g r o w t h rates, or comparisons o f growth yields (e.g.
bioassay), but is desirable for m a n y other applications.
A n intercomparison is described below, involving eight m e t h o d s - - m a n y
in c o m m o n use. Their precision, limit o f detection and sensitivity were deter-
mined at cell densities developed during growth in culture but often f o u n d in
natural waters. The results, therefore, have relevance for both laboratory and
ecological studies.
Asterionella formosa Hass. was chosen as the main test organism. This
important planktonic d i a t o m has been the subject o f m a n y quantitative studies
in culture, based u p o n cell counts obtained either by microscopy (e.g. Lund,
69
0c07-1617/82/010069+ 11 $02.00/0 © 1982 British Phycological Society

Published online 17 Feb 2007

70 C. BUTTERWICK, S. I. H E A N E Y A N D J. F. TALLING

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.

MATERIALS AND METHODS

ALGAL CULTURES
Cultures of Asterionella formosa Hass., the centric diatom Cyclotella pseudostelligera
Hustedt and the cryptomonad Rhodomonas minuta var. nannoplanctica Skuja (R. lacustris vat.
nannoplanctica (Skuja) Javornick~') were isolated from Cumbrian lakes. The greatest cellular
dimensions of these algae were c. 68, 7, and 9/xm respectively, and cells of the first were
normally associated in eight-celled colonies. The corresponding cell volumes were c. 700, 176
and 170 tzm3.
The medium used (Table I) was based on solution 10 of Chu (1942) as modified by Lund,
Jaworski & Butterwick (1975). To minimize precipitation (which could affect some methods
under test), sterile solutions of NaHCO3 and Na2SiO3 were added aseptically after boiling the
rest of the medium and allowing it to cool. Before use the medium was left to stand for 3 days
to equilibrate with air at c. pH 7"3.

TABLE I. Culture medium

Constituent Final concentration
mg 1-1 b~M
1 Ca(NO3)2'4H20 20"0 84"7
2 KH~PO4 6"2 45-6
3 MgSO4-7HzO 25"0 101-6
4 NaHCOa 15.8 188.1
5 Na2SiO3 25.0 205"0
6 EDTA'Na2 2"0 5"4
7 FeCI3-6H20 1.66 6"2
8 H3BO3 2"48 40.0
9 MnC12"4H~O 1.39 7.0
10 (NH~)6Mo~O24-4H30 1.0 0"81
11 Vitamin Bx 1 × 10-8 3.0 × 10-3*
12 Vitamin B13 10× 10-3 7.4 x10 -3*
13 Biotin 1 x 10 -3 0'41 × 10-3*

*From conversions in Stein (1973, p. 21).

Constituents 1-3 and 11-13 were added from separate stock solutions at 1 ml 1-1 to glass
distilled water. Constituents 6 and 7 were then added from a mixed stock solution (chelated
iron), as were 8-10 (trace elements). After the medium had been boiled, and cooled, consti-
tuents 4 and 5 were added from separate sterile stock solutions.

Cultures were grown in a temperature-controlled cabinet at 184-1°C, illuminated from

below with continuous light from "daylight" fluorescent lamps at an irradiance (400--700 nm)
of c. 50/~E m -2 s -s, as measured with a fiat-plate quantum-sensor.

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.

(d) In vivo attenuance

Algal suspensions were placed in semi-micro cuvettes (cells) of 10 cm pathlength, with
thickened black side walls and minimum volume 10 ml. Attenuance was measured at 680 n m
in a Shimadzu UV 150-20 double-beam spectrophotometer (of Seisa Kusho Ltd., Kyoto,
Japan), readable to 0.0001 A unit. The wavelength of 680 n m was chosen as it gave slightly
greater sensitivity ( × 1.2) than the often used 750 nm, and was less prone to interference than
shorter wavelengths. Distilled water was used in the reference cell and a correction was applied
for cell-to-cell differences (1 reference, 3 samples).
(e) In vivo fluorescence of chlorophyll a
This was determined by the method of Lorenzen (1966), using a fluorometer (model 111 of
G K Turner Associates, Palo Alto, California, U.S.A.) as described by Heaney (1978). The
instrument incorporated a red-sensitive photo-multiplier and a blue lamp. The filters were a
blue excitation filter with maximum transmission at 425 n m (half band-width 96 nm), and
a red, sharp cut-off emission filter with transmission maximal at 687 nm and negligible below
645 nm. After the instrument had been zeroed with distilled water, samples were placed in
5 ml capacity cuvettes in a high-sensitivity sample holder. Samples of cultures were dark-
adapted at dull room light for 1 h before measurements were made. Fluorescence readings
were made at the appropriate sensitivity setting and the results expressed in arbitrary units
(FU) on the × 10 sensitivity scale (full scale =100 units).
( f ) Reducing capacity (carbon-equivalent)
The reducing capacity of particulate organic matter was determined by a micro-adaptation
of dichromate oxidation due to Mackereth, Heron & Tailing (1978). Aliquots of algal sus-
pension, chosen to contain c. 50/~g C per sample, were filtered on to pre-combusted (550°C),
12-5 mm diameter, Whatman G F / C filter discs. These were placed in giass-capped tubes with
100 ~1 of the oxidizing agent (0.25 N potassium dichromate solution) acidified with 200/~1 of
concentrated sulphuric acid containing silver sulphate (0.24 g AgSO4 in 20 ml conc. H2SO4).
Samples and blank filter discs were digested for c. 2 h at 100°C in a hot block. The amount of
oxidation was estimated by amperometric titration of the remaining dichromate with ferrous
ammonium sulphate solution (FAS), of normality n (c. 0"06 N), to a standardized final potential
just beyond the chemical end-point. Titrations employed a Radiometer titration system
(Emdrupvej 72, Dk-2400, Copenhagen NV, Denmark) consisting of a motorized burette
(ABU 12) of 2.5 ml capacity with a pH meter-titrator (TTT2) in conjunction with a combina-
tion platinum-calomel electrode. The difference, in ml, between titrations of blanks (Vb) and
samples (Vs) was converted to/~Eq of reducing material as (Vb--Vs) n. 103. This was converted
to carbon equivalents (in/~g) by multiplying by a factor of three obtained from glucose stan-
dards.
(g) Extracted chlorophyll a by spectrophotometry
Extraction was by hot 90% methanol as described by Tailing (1974), and absorbance
measured with a Shimadzu UV150-20 double-beam spectropbotometer. Appropriate volumes
72 C. BUTTERWICK, S. I. HEANEY AND J. F. TALLING
were filtered on to Whatman GF/C glass fibre filters, diameter 5.5 cm, to give absorbance
readings between 0.1 and 1.0 A. Filters were extracted by addition of 7"0 ml of 94-6 ~ methanol,
yielding a 9 0 ~ solution after allowing for 0.36 ml of water in the filter, and by subsequent
boiling for a few seconds.
After cooling and centrifuging, absorbance of the extracts were read at 655 and 750 nm in
cells of 5cm path-length against a reference of 90 ~ methanol. Absorbance values at 665 nm
were corrected (as Acorr) for residual turbidity by subtracting the absorbance at 750 nm. The
equation of Tailing (1974) was used to calculate the original concentration of chlorophyll a in
t~g 1-1 (uncorrected for possible degradation products) as Aeorr× 13"9× v x 1000, where d=cell
V d
length (5 cm), v=volume of solvent (7.36 ml) and V=volume filtered in ml.
(h) Extracted chlorophyll a by fluorometry
Extraction was again by hot 9 0 ~ methanol, after filtration of appropriate volumes of
sample on to 12-5mm GF/C filters. Fluorescence was measured with a Turner fluorometer
used and equipped as described in (e) with zero set by 90 ~ methanol.
Fluorescence measurements were calibrated against chlorophyll a concentration, determined
spectrophotometrically as in (g). For estimation of an extract by both methods (g and h), it
was prepared in a suitable concentration for use in the spectrophotometer, and then diluted
× 100 with 90 ~ methanol for estimation of fluorescence.
The readings obtained by all methods were converted to equivalent cell quantities of Asterio-
nella, using relationships obtained by direct cell counts on the same samples.
STATISTICS
The parameters calculated were defined according to Sutcliffe (1979). The LIMITOFDETECTION
(LD) is five times the standard deviation (SD) of blank determinations, with freshly prepared
growth medium used as blank. SENSIantVlTYis the increment in response per unit increment of
concentration, expressed as cell density. PRECISIONis expressed as the coefficient of variation
(CV), which is the percentage ratio of the SD of the mean (at least six samples) to the mean
(i.e. 100 SD/g). Precision was found at more than one cell density for the methods which
utilize a single fixed volume, and is also given as the equ;.valent cell density.

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

cells m|-lx IO-5

FIG. ]. Estimations of Asterionella biomass by four methods over a dilution series ( x 1,
× 112, × 1/8, × 1/16, × 1/32). Broken lines indicate extrapolation of the initial linear
relationships. (a) Spectrophotometry, (b) nephelometry, (c) fluorometry, (d) Coulter
counter.
76 C. BUTTERWICK, S. I. H E A N E Y A N D J. F. TALLING

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

Nevertheless, mainly because of the large quantities of sample required (II-

mavirta, 1979), especially for colonial forms, poor precision in our measure-
ments plus interference from debris with small cells, the technique is rarely used
in this laboratory. With some manipulative adaptation and modem instruments
these disadvantages could well be reduced.
Whatever method is adopted for determining algal biomass some degree of
compromise has to be accepted regarding specificity, manipulative ease, pre-
cision and limit of detection. The methods selected here include many in
common use, but there are obvious omissions (e.g. dry weight, nucleic acids,
protein, ATP) that deserve further systematic intercomparison. We believe
qualitative microscopical observations should supplement any chosen method
to indicate cell "condition" and to avoid' a fundamental error--the possible
change of organism involved, as through contamination.
In the light of the present results, the heavy reliance on visual counting in past
experimental work on cultures of Asterionella formosa was generally not mis-
placed. Alternative methods might be preferred for obtaining generalized
measures of biomass directly, rather than indirectly via cell numbers. For
growth studies involving low concentrations of this and other micro-algae,
there could well be greater use of extract-fluorometry, and of attenuance in
instances where its rapidity and precision are not vitiated by interferences.

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(Accepted 21 August 1981)