SILVER, MARY WILCOX, AND PETER J. DAVOLL. Loss of ... - ASLO

362 Notes
BIENFANG, P. 1975. Steady state analysis of nitrate-
ammonium assimilation by phytoplankton.
Limnol. Oceanogr. 20: 405411.
CAPERON, J. 1968. Population growth response of
Isochrysis galbana to nitrate variation at lim-
iting concentrations. Ecology 49: 866-872.
-> AND J. MEYER. 1972. Nitrogen-limited
growth of marine phytoplankton. 1. Changes in
population characteristics with steady-state
growth rate. Deep-Sea Res. 19: 601-618.
-, AND D. A. ZIEMANN. 1976. Synergistic ef-
fects of nitrate and ammonium ion on the
growth and uptake characteristics of Mono-
chrysis Zutheri in continuous culture. Mar. Biol.
36: 75-84.
COLLOS, Y., AND J. LEWIN. 1976. Blooms of surf
zone diatoms along the coast of the Olympic
Peninsula, Washington. Variations of the car-
bon to nitrogen ratio in field samples and lab-
oratory cultures of Chaetoceros armatum. Lim-
nol. Oceanogr. 21: 219-228.
DOTY, M. S., AND M. OGURI. 1957. Evidence for
photosynthetic daily periodicity. Limnol.
Oceanogr. 2: 2740.
DROOP, M. R. 1968. Vitamin BIz and marine ecol-
ogy. 4. The kinetics of uptake, growth and in-
hibition in Monochrysis Zutheri. J. Mar. Biol.
Assoc. U.K. 48: 689-733.
EPPLEY, R. W., AND E. H. RENGER. 1974. Nitrogen
assimilation of an oceanic diatom in nitrogen-
limited continuous culture. J. Phycol. 10: 15-
23.
-, J. N. ROGERS, J. J. MCCARTHY, AND A.
SOURNIA. 1971. Light/dark periodicity in ni-
trogen assimilation of the marine phytoplank-
ters Skeletonema costatum and Coccolithus
huxleyi in N-limited chemostat culture. J. Phy-
col. 7: 150-154.
GOLDMAN, J. C., AND J. H. RYTHER. 1976. Tem-
perature-influenced species competition in
mass cultures of marine phytoplankton. Bio-
technol. Bioeng. 18: 1125-l 144.
LAWS, E., AND J. CAPERON. 1976. Carbon and ni-
trogen metabolism by Monochrysis lutheri:
Measurement of growth-rate-dependent respi-
ration rates. Mar. Biol. 36: 84-97.
MEYER, J., AND M. CRAMER. 1948. Metabolic con-
ditions in Chlorella. J. Gen. Physiol. 32: 103-
110.
SMALL, L. I?., P. L. DONAGHAY, AND R. M. PYT-
KOWICZ. 1978. Effects of enhanced COZ levels
on the growth of two marine phytoplankton
species, p. 183-204. Zn N. R. Anderson and A.
Melhoff [eds.], The fate of fossil fuel CO, in
the oceans. Plenum.
THOMAS, !4'. J., AND A. N. DODSON. 1972. On ni-
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Oceanogr. 17: 515-523.
Submitted: 4 June 1976
Accepted: 8 June 1977
Loss of 14C activity after chemical fixation of phytoplankton: Error
source for autoradiography and other productivity measurements1
Abstract-Loss of fixed, filter-retainable 14C
due to treatment with preservatives is report-
ed in productivity studies of natural phyto-
plankton populations. Treatment with form-
aldehyde consistently results in large decreases
in activity, whereas iodine and gluteraldehyde
treatments cause losses with some populations
but not with others. The extent of loss with all
treatments varies over time in natural popu-
lations, suggesting that no simple correction
factor can be applied for all populations and
that leakage may not be uniform for different
species. Should activity losses vary by species,
then quantitative autoadiographic methods for
estimating individual species production rates
may require individual correction factors for
each species when preservative-caused losses
occur.
In developing methods measuring sin-
gle species production rates, we have re-
’ This work was supported by contract agreement
M-12 and M-25 from the Marine Research Com-
mittee, State of California.
peatedly observed the loss of 14C-labeled
material from cells after addition of
chemical preservatives. This source of
error has not been recognized widely,
and preservation of samples from produc-
tivity studies is practiced occasionally
and appears to be a regular part of most
autoradiographic methods.
Fixatives in common use (e.g. the al-
dehydes, osmium, permanganates) are
known to bind only particular classes of
cell compounds. For instance, the alde-
hydes bind proteins and partially fix nu-
cleic acids, but generally do not react
with polysaccharides and lipids (Hayat
1970; Dawes 1971). The compounds
commonly used in phytoplankton stud-
ies-formaldehyde, gluteraldehyde, and
iodine-are successful preservaties (i.e.
minimize visible alterations due to os-
motic effects, do not dissolve or distort

conspicuous cell organelles, prevent mi-
crobial activity, and prevent cell autoly-
sis), and most of these compounds are also
useful fixatives (i.e. aid retention of struc-
tural features through subsequent treat-
ment-as in embedding, etc.). The com-
pounds most widely used are successful
because of their ability to bind and sta-
bilize structural proteins (Baker 1966;
Pearse 1968; Hayat 1970), thus rendering
the cells lifelike for microscopy. How-
ever, we are unaware of any assurances
or experiments in the literature that in-
dicate general retention of most cellular
compounds after treatment with preser-
vatives. On the contrary, evidence indi-
cates that no preservative retains all com-
pounds (Baker 1966; Hayat 1970), and a
number of workers have documented
loss of particular families of compounds
due to preservatives (reviews in Pearse
1968; Stoward 1973; Rogers 1973).
Occasionally preservatives have been
used in productivity measurements by
radioactive isotopic methods. Strickland
and Parsons (1968) and Marshall et al.
(1973) suggest the use of formaldehyde
when photosynthesis must be terminated
but cell collection has to be delayed, al-
though these investigators warn that such
a process can result in a loss of some (un-
specified amount) of the label. Preserva-
tives are commonly used for autoradiog-
raphy, and the introduction of quantitative
autoradiographic methods into phyto-
plankton productivity studies (Brock and
Brock 1968) has led to widespread use of
various compounds: formaldehyde (Brock
and Brock 1969; Maguire and Neil1 1971;
Stross and Pemrick 1974), gluteralde-
hyde (Coulon and Alexander 1972), and
Lugol’s iodine (Knoechel and Kalff
1976b). The danger of the fixative react-
ing with the emulsion, or “chemograph-
ic” error, has been considered recently
(Knoechel and Kalff 1976u), but the dan-
ger of losing labeled cell components ap-
parently has not received much attention.
References to such losses have been not-
ed briefly (Cadee and Hegeman 1974;
Wood et al. 1973), and we further docu-
ment these losses.
We acknowledge the help and reviews
Notes 363
of D. L. Garrison, D. B. Seielstad, E. A.
Silver, R. B. Wood, and an anonymous
reviewer.
We examined the loss of radioactivity
from labeled phytoplankton by compar-
ing counts from populations that had
been preserved with counts from popu-
lations not treated with preservatives.
Our experiments were designed to iden-
tify a suitable fixative-one that we
hoped would cause minimal, or at least
constant leakage of labeled material. Nat-
ural populations of phytoplankton were
collected from northern Monterey Bay,
California, from the end of the Santa Cruz
municipal pier (water depth about 5 m)
or from one of three routinely studied sta-
tions between about l-9 km offshore
(depths 15-35 m). Unfiltered water sam-
ples from the surface (upper 1 m) provid-
ed our natural populations, unless oth-
erwise stated. Populations were either
incubated in situ, incubated in the labo-
ratory with natural daylight in an aquar-
ium set to temperatures within 3°C of
field temperatures, or placed in a tem-
perature-controlled chamber over “cool-
white” fluorescent bulbs (0.09 ly min-‘)
with temperatures within 3°C of field
temperatures. Productivity experiments
were conducted in flint glass bottles with
volumes of 125 to 500 ml and water sam-
ples were generally incubated for be-
tween 4 and 7 h (range 2-11 h) with 14C,
following the methods of Strickland and
Parsons (1968). At the end of the incu-
bations, portions of control samples were
filtered with low vacuum pressure (< l/5
atm) or by simple gravity filtration, the
filters rinsed repeatedly, and the moist
filters placed in vials with premeasured
Aquasol fluor. We used a variety of filters
to trap the labeled cells: glass-fiber filters
with effective pore size of 0.3 pm; 5-pm
metricel filters; and lo- and 20-pm Nytex
filters. Occasionally we split the popula-
tion into two fractions in the filtration
process; for convenience we refer to the
small (10 or 20 pm) and large (2 10 or 20
pm) fractions as ultraplankton and net
plankton. In the formaldehyde and io-
dine treatments, preservatives were
added directly to the whole water sam-

364 Notes
Table 1. Loss of filter-retainable 14C after treatment of populations with chemical preservatives. Rep-
licates of treated samples are compared with replicates of untreated samples from the same phytoplankton
populations using a t-test (one-tailed). (Glut-gluteraldehyde; SW-seawater.)
Date Preservative
21 Apr 75
II
6 May 75
12 May 75
23 May 75
9 Jun 75
12 Jun 75
10 Jul 75
11
14 Jul 75
I,
II
16 Jul 75
11
11
I,
4 Aug 75
11 Aug 75
19 Sep 75
11
II
II
II
19 Feb 76
II
26 Mar 76
II
30 Mar 76
II
1 May 76
11
II
II
II
11
7 May 76
II
,I
II
II
1 Jun 76
II
Formaldehyde
Formaldehyde
Formaldehyde
Formaldehyde
Formaldehyde
Formaldehyde
Formaldehyde
IKI
Gluteraldehyde>k
IKI
Gluteraldehyde$c
Formaldehyde
IKI
IKI
IKI
IKI
IKI
Gluteraldehyde
IKI
IKI
IKI
IKI
IKI
IKI
IKI
IKI
IKI
IKI
IKI
Glut (SW)
Gluti-rinse (SW)
Glut-0s04+rinse (SW)
Glut (sugar-buffer)
Glut+rinse (sugarbuffer
Gluti-Os04i-rinse
(sugar-buffer)
IKI
IKI
Os04 buffered
OsO unbuffered (SW)
KMN 8 buffered
KMNO: unbuffered
IKI
Gluteraldehyde
* Gluteraldehyde in 100% seawater.
ples. In the gluteraldehyde, osmium, and
permanganate treatments, samples were
filtered and rinsed immediately, as for
the controls, but the filters with labeled
cells were placed immediately into 10 to
20 ml of the preservative solution. Be-
tween 2 and 72 h after exposure to the
preservative, the experimental samples
were filtered, rinsed repeatedly, and
placed in fluor for subsequent counting.
Activity retained
after preservation (%> df prob.
35 (0 m sample) 7.25
38 (10 m sample) 6.26
14 20.6
43 10.67
29 7.79
34 24.8
51 9.29
125 -1.14
101 -0.12
107 -0.56
93 0.66
61 3.79
100 (0 m, offshore
station) 0.01
101 (10 m, offshore
station) -0.77
101 (0 m, inshore station) -0.03
88 (10 m, " II
1.53
79 5.13
68 6.15
87 (0 m netplankton) 1.99
34 (0 m ultraplankton) 7.93
52 (5 m netplankton) 9.41
51 (5 m ultraplankton) 4.98
53 (10 m netplankton) 7.64
73 (netplankton) 2.42
68 (ultraplankton) 3.74
56 (netplankton) 2.58
60 (ultraplankton) 2.27
45 (netplankton) 4.16
14 1.68
26 3.69
19 8.24
21 8.08
21 8.11
14 8.80
4
5
9
14
13
9
15
6
6
7
7
12
4
3
4
4
14
9
12
8
12
12
6
28
29
26
28
15
15
6
5
6
6
19 8.30 6
57 4.30 6
71 3.29 5
34 7.59 5
43 6.06 5
23 8.83 4
28 8.25 5
60 5.55 10
55 7.04 10
6
co.01
co.01
co.01
co.01
0.90
yo.50
,0..50

ated, unless otherwise noted. Cells
were left in this solution until filtration.
On 23 May 1975, we tested for differ-
ences in the amount of activity retained
when natural populations were pre-
served with different concentrations of
formaldehyde: 1.48%, 0.72%, 0.37%,
0.12%, 0.06%) 0.03% solutions. An anal-
ysis of variance indicated that no signif-
icant differences in activity of the phy-
toplankton were related to the different
preservative concentrations (F5,r2 = 2.75,
P > 0.10).
Average retention of activity of phyto-
plankton populations treated with form-
aldehyde was 38% (SD 14%, n = 8).
Formaldehyde always caused a signifi-
cant loss of activity in the treated popu-
lations when compared with the controls,
as indicated by t-values with probabili-
ties CO.01 on all tests.
Iodine-An IKI solution (10 g I, 20 g
KI, in 200 ml distilled water-Lugol’s
without the acetic acid) was added to the
seawater in which the cells were incu-
bated in the proportion 1 ml of IKI so-
lution per 100 ml of seawater, unless oth-
erwise noted. Cells were left in this
solution until filtration. In an experiment
on 4 August 1975 we compared the re-
tention of activity after treatment with 1
vs. 2 ml of IKI solution per 100 ml of sea-
water. A t-test showed some difference in
retention between the two concentra-
tions (79% retention with the lower con-
centration, 69% with the higher, t22
= 3.53, P < 0.01). We also tested for
differences in retention of activity when
the treatment time varied from a few
hours to a few days. Experiments on a
number of dates failed to show signifi-
cant differences in activity as a function
of the length of exposure to IKI solutions:
on 19 February 1976 an analysis of vari-
ance gave F 430 = 0.48, P > 0.10 for the
net plankton fraction, F4,20 = 0.49, P >
0.10 for the ultraplankton fraction; 26
March 1976, F,,,, = 0.38, P > 0.10 for the
net plankton, F,,,, = 1.03, P > 0.10 for
the ultraplankton; 4 August 1975, F3,8 =
1.35, P > 0.10 for the lower concentration
of preservative (1 ml per 100 ml of sea-
water), F,,, = 2.41, P > 0.10 for the high-
Notes 365
er concentration (2 ml per 100 ml of sea-
water). Only in one case did activity
decrease with time: on 30 March 1976
the ultraplankton showed 20% retention
after 3 h, 9% after 16 h (t!, = 2.95, P <
O.Ol), but the net plankton showed no
significant decline in activity over this
period (to = 1.94, P > 0.05).
Average retention of activity for IKI-
treated samples was 71% of controls (SD
18%, n = 20). IKI-treated populations did
not always show loss of materials, with
no significant decreases (i.e. P > 0.05 for
t-test) noted in 6 of the 20 tests.
Gluteraldehyde-EM grade gluteral-
dehyde (refrigerated and held in sealed
glass ampoules until a few hours before
use) was used at final concentrations of
1.5-2.0%. In most experiments, gluteral-
dehyde was added to 70% seawater (30%
distilled water), because precipitates
formed at higher concentrations of sea-
water (exceptions are noted in Table 1).
In an experiment on 1 May 1976, we
studied cell leakage with various gluter-
aldehyde treatments: gluteraldehyde-
seawater (70%) for 24 h; gluteraldehyde-
seawater for 1.5 h, then in 70% seawater
rinse for 22.5 h; gluteraldehyde-seawater
1.5 h, osmium tetroxide postfix (1% in
50% seawater) for 2 h, and then in 70%
seawater for 21.5 h; a second series with
treatments the same as the three previous
solutions, except that the 70% seawater
solution was replaced by a buffered sugar
solution (0.2 M sodium cacodylate buff-
ered to pH 7.3 and with 0.25 M sucrose).
In all of the treatments, cells were trans-
ferred to the appropriate solutions after
collection by gravity filtration (no vac-
uum applied) onto filters, and the filters
gently rinsed with the solution into
which they were subsequently placed.
There were highly significant, although
relatively small, differences in r4C reten-
tion between the treatments, with 14-
26% retention of the label (F5,r8 = 11.6 for
an analysis of variance, P < 0.01; see Tu-
ble 1 for details).
Average retention of activity for gluter-
aldehyde treatment was 44% (SD 33%,
n= 10). As in the case of the IKI treat-
ment, gluteraldehyde treatment did not

366 Notes
always result in loss of activity; in 2 out
of 10 tests, no significant losses (i.e. P >
0.05 for t-test) occurred with treatment.
Osmium-In an experiment on 7 May
1976 we tested retention of cell label af-
ter exposure to a 1% osmium solution.
Cells were placed into Dalton’s chrome-
osmium fix, buffered to pH 7.3 (Dawes
1971) for 2.5 h at room temperature, and
then placed in seawater for an additional
22.5 h. A second series of cells was
placed in osmium in seawater (99%) for
2.5 h and then transferred into seawater
for an additional 22.5 h. The difference
in retention of label between the two os-
mium treatments was not significant (t,,
= 2.44, P > 0.05). Both osmium treat-
ments caused large, significant losses of
label (Table 1).
Osmium was also used as a postfix with
gluteraldehyde initial preservation (see
section on gluteruldehyde and Table 1).
We tried the double fixation because of
its very common use in EM work and the
claim that among various fixatives cur-
rently in use, a double fixation by gluter-
aldehyde and osmium tetroxide seems to
be the most effective in reducing the loss
of cell constituents (Hayat 1970). This
postfix resulted in a small but significant
decrease in activity, as compared with
the cells that remained in gluteraldehyde
for the entire period (to = 2.98 for sea-
water solution, to = 2.57 for sugar solu-
tion, P < 0.05 for both tests). Osmium
postfix, as well as gluteraldehyde treat-
ment alone, pr;oduced large and signifi-
cant losses of label in these tests (Table
0 Permanganate-On 7 May 1976 we
tested for leakage with 0.6% KMn04 so-
lutions (KMn04 solutions made within 3
h of use) with 1.5-h treatments at 0°C.
Cells were placed in Dawes and Rham-
stine, veronal-acetate-salt buffered (pH
7.3) permanganate (Dawes 1971) or in fil-
tered seawater with unbuffered perman-
ganate. After treatment with the fixative,
cells were left in seawater for an addi-
tional 22.5 h before final filtration. The
retention of activity did not differ signif-
icantly between the permanganate treat-
ments (ts = 1.87, P > O.lO), and signifi-
cant losses were associated with both
(Table 1).
Overall treatment effects-The results
presented above demonstrate losses of
bound 14C with all preservatives tested.
Losses were highly variable for the various
study dates, and the differences presumably
reflect the variations in species
composition or state of the cells on those
particular days. Losses ranged from 0%
to 86% (i.e. retention of activity was 100%
to 14%) of the label in these natural populations.
Wood et al. (1973) mention losses
with formaldehyde fixation of around
50% of the radioactivity for natural populations
in a Scottish sea loch; we found
average losses of 62% for formaldehyde,
with a range of 86% to 39% on various
dates. Cad&e and Hegeman (1974) present
data on losses of radioactivity of the
marine microflora of tidal flats after IKI
fixation: their experiments on three dates
gave rather similar losses of about 15%.
We observed an average loss of 29%, with
ranges of 86% to 0%. Of the three preservatives
we tested most extensively,
formaldehyde generally averaged the
greatest leakage, gluteraldehyde the
next, and iodine the least; however, these
differences were not statistically significant
(analysis of variance, F2,36 = 5.95,
P > 0.10). Formaldehyde always caused
a loss of activity, whereas both gluteraldhd
e y e and iodine caused decreases in
some populations but not in others. Other
than preservative effects, we found leakage
patterns to be unrelated to testing
procedure: for example, losses were not
a function of filter mesh size or incubation
method (as indicated by analyses of
variance, with P > 0.05). Moreover, we
found that the loss of activity also could
vary for populations at different depths in
the same location (Table 1: 19 September
1975 experiment); a difference in the percentage
lost at the various depths could
be a consequence of differences in species
composition or physiological condition
of the population.
The proportion of activity lost in the
net and ultraplankton fractions usually
differed considerably, but a t-test for
paired data (net and ultraplankton sub-

samples of a given population) indicated
that these differences were not statisti-
cally significant (t4 = 1.60, P > 0.05).
True differences in retention of activity
in different sized cells may exist and not
be shown by our data, due either to the
large variations in the extent of loss on
the dates tested or to the fact that some
cells in the “net” fraction are actually
“ultra” sized cells that are taken in the
larger size fraction because of their
chain-forming habits (e.g. small Skeleto-
nemu). Chain-forming diatoms are usu-
ally the dominants in our samples during
the spring and summer upwelling pe-
riods when most of these studies were
done.
In conclusion, our results show sub-
stantial loss of filter-retainable radioac-
tive label in productivity experiments
when preservatives are used. The mag-
nitude of the losses differs considerably
throughout our experiments, and we in-
terpret these differences as due to some
property of the natural populations in the
water on the various dates. Such results
indicate that no constant correction factor
can be applied to predict the loss that
will occur with the use of a given preser-
vative; conversely, the “true” activity of
unfixed samples cannot be estimated
from a preserved sample. If, as we be-
lieve, the observed variability in loss rate
is a function of cell condition or species
composition, it is unlikely that leakage is
uniform for all cells in any one natural
population. Should leakage not be uni-
form, the true activity of a given cell can-
not be predicted from its measured activ-
ity after preservation, even if the extent
of loss for the population as a whole is
known. Thus, the value of productivity
measurements by autoradiography-i.e.
the measurement of single-species pro-
duction rates-may be lost when preser-
vatives are used, because leakage of the
label is probably nonuniform for differ-
ent species populations. Moreover, the
losses of activity are usually so great that
such errors must be considered when-
ever preservatives are used. Because
there appears to be no simple correction
factor for calculating the loss of activity
Notes 367
in natural populations after preservation,
we recommend either a search for new
fixation techniques or the substitution of
other methods which circumvent the
need for preservatives when natural pop-
ulations are investigated.
Mary Wilcox Silver
Peter J. Davoll
Center for Coastal Marine Studies
University of California
Santa Cruz 95064
References
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-, AND -. 1969. The fate in nature of
photosynthetically assimilated 14C in a blue-
green alga. Limnol. Oceanogr. 14: 604-607.
CADI~E, G. C., AND J. HEGEMAN. 1974. Primary
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COULON, C., AND V. ALEXANDER. 1972. A sliding
chamber phytoplankton settling technique for
making permanent quantitative slides with ap-
plications in fluorescent microscopy and auto-
radiography. Limnol. Oceanogr. 17: 149-151.
DAWES, C. J. 1971. Biological techniques in elec-
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KNOECHEL, R., AND J. KALFF. 1976u. The appli-
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A method for the determination of phytoplank-
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MAGUIRE, B., JR., AND W. E. NEILL. 1971. Species
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MARSHALL, N., D. M. SKAUEN, H. C. LAMPE, AND
C. A. OVIATT. 1973. Primary production of
benthic microflora. Monogr. Oceanogr. Meth-
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368 Notes
STRICKLAND, J. D., AND T. R. PARSONS. 1968. A WOOD, B. J., P. B. TETT, AND A. EDWARDS. 1973.
practical handbook of seawater analysis. Bull. An introduction to the phytoplankton primary
Fish. Res. Bd. Can. 167. production and relevant hydrography of Loch
STROSS, R.G., AND S. M. PEMRICK. 1974. Nutrient Etive. J. Ecol. 61: 569-585.
uptake kinetics in phytoplankton: A basis for
niche separation. J. Phycol. 10: 164-169. Submitted: 23 December 1976
Accepted: 26 April 1977
A modified chamber for use with an oxygen electrode to
allow measurement of incident illumination
within the reaction suspension
Abstract-A method for precise measure-
ment of incident illumination at the center of
an algal suspension within the reaction cham-
ber of an oxygen electrode allows reliable
comparisons to be made of the photosynthetic
characteristics of cultures of differing popu-
lation densities and provides estimations of
the degree of shade adaptation in cells of
Chlorella comparable with those obtained by
other methods. The reliability of the method
is further confirmed by comparing the photo-
synthetic characteristics of synchronous cul-
tures of Chlorella at different stages in the life
cycle.
Much of our knowledge of the ability
of some algal species to adapt their pho-
tosynthetic characteristics to suit the con-
ditions of light availability is based on
examination of curves relating the rate of
photosynthesis to incident illumination
(Steemann Neilsen and Jorgensen 1968).
Extrapolation from such curves (P vs. I
curves) gives a particular light intensity,
designated as II, by Talling (1957), at
which photosynthesis, for that sample,
ceases to be light limited. Comparison of
the Ik value for different natural algal
samples or for cultures grown under var-
ious conditions has been widely used as
an indication of the degree of photosyn-
thetic adaptation (reviewed by Steemann
Nielsen 1975). Thus, a low value for Ik
indicates shade adaptation and a high Ik
value is evidence of adaptation to high
light intensities.
Clearly, the reliability of such compar-
isons depends on the accuracy with
which the curves can be constructed and
on a precise measurement of the light in-
tensity at which photosynthesis is being
assessed. We have used a Rank oxygen
electrode (Delieu and Walker 1972),
based on a principle first described by
Clark (1956) for such studies because its
use avoids some of the problems inherent
in the 14C method. Since, however, with
the oxygen electrode, the beam of light
reaches the algal suspension through a
water jacket and since the suspension is
itself contained in a chamber of 15-mm
diameter, there are obvious practical dif-
ficulties in arriving at a precise measure
of the light intensity incident within the
body of the culture. We have attempted
to overcome these difficulties by the use
of a half-cell (Fig. 1) which matches the
reaction chamber in all respects but
which allows for the insertion of the sen-
sor of a light meter (Ll-185 meter, Lamb-
da Instruments Corp.) in such a way that
the light-sensitive surface of the sensor
is in a position corresponding to the cen-
ter of the algal suspension in the oxygen
electrode chamber.
The half-cell (Fig. l), constructed of
Perspex, can be mounted on the stirrer
plate of the oxygen electrode. It consists
of an inner chamber corresponding to
half of the reaction chamber of the oxy-
gen electrode and an outer sleeve corre-
sponding to the water jacket. The sensor
of the light meter is held against a cir-
cular thin glass window set in the wall
which forms the diameter of the half-
chamber. For estimation of light intensi-
ty, the half-cell replaces the oxygen elec-
trode cell on the stirrer plate and the sen-
sor is placed in position. The outer jacket
is filled with distilled water and the inner
half-chamber with the algal suspension.
The position of the half-cell is adjusted