Marine Micropaleontology 44 (2002) 163^185
www.elsevier.com/locate/marmicro
Techniques for quantitative analyses of calcareous marine
phytoplankton
Jo«rg Bollmann a; , Mara Y. Corte¤s b , Ali T. Haidar c , Bernhard Brabec d ,
Anne Close e , Robert Hofmann a , So¢a Palma f , Luis Tupas g ,
Hans R. Thierstein a
a
Geological Institute, ETH Zurich, Sonneggstr. 5, 8092 Zurich, Switzerland
Departemento de Geolog|¤a Marina, UABCS, Apdo. Postal 19-B, C.P. 23080 La Paz, B.C.S., Mexico
c
Department of Geology, American University of Beirut, P.O. Box 11-0236/26, Beirut, Lebanon
d
Swiss Federal Institute for Snow and Avalanche Research, Flu«elastrasse 11, 7260 Davos Dorf, Switzerland
e
Wrigley Institute for Environmental Studies, USC, AHF 232, Los Angeles, CA 900089-03171, USA
f
IPIMAR, Lab. Fitopla“ncton, Dep. Ambiente Aqua¤tico, Avenida de Bras|¤lia, 1449-006 Lisboa, Portugal
Biological Oceanography Division of SOEST, University of Hawaii at Manoa, 1000 Pope Road, Honolulu, HI 96822, USA
b
g
Received 7 February 2001; received in revised form 19 July 2001; accepted 10 August 2001
Abstract
This paper discusses the techniques used to sample and analyse living marine calcareous phytoplankton. The
various methods are described and tested within several research projects aimed at the determination of
coccolithophore cell densities in seawater. In addition, the potential advantages and drawbacks associated with the
application of light and scanning electron microscopic techniques to the quantitative analysis of coccolithophores are
discussed. Several tests have been carried out in order to quantify potential errors related to: (1) homogeneity of
material distribution on filter membranes; (2) use of different microscopes (scanning electron microscope versus light
microscope) ; (3) use of different filter membranes (cellulose mixed-ester membranes versus polycarbonate
membranes); and (4) Utermo«hl settling versus filtration method. These tests revealed that major errors in cell
density calculations could result from the uneven distribution of coccolithophore specimens on a filter membrane. The
error resulting from the use of a light microscope arises from its low resolution, which restricts the identification of
species, especially of small coccospheres. The use of different filter membranes does not show a statistically significant
difference in cell density calculations, although polycarbonate membranes can be examined much more efficiently with
the scanning electron microscopy than cellulose mixed-ester membranes. The Utermo«hl method, however, gives lower
cell densities consistently (several times) than the filtration method. : 2002 Elsevier Science B.V. All rights reserved.
Keywords: microscopy; plankton surveys; plankton collection devices; water ¢ltration; coccolithophores
1. Introduction
* Corresponding author. Tel.: +41-1-632-3684;
Fax: +41-1-632-1080.
E-mail address: [email protected] (J. Bollmann).
In recent years, there has been an increasing
interest in living calcareous marine phytoplankton
0377-8398 / 02 / $ ^ see front matter : 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 3 9 8 ( 0 1 ) 0 0 0 4 0 - 8
MARMIC 854 3-5-02
164
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
because of their role within the global carbon
cycle. In order to assess their contribution to the
global carbon cycle, absolute abundance determinations (cells/l) are needed. There are two basic
methods for quantifying the abundance of calcareous marine phytoplankton: (a) the Utermo«hl
settling method (Utermo«hl, 1931, 1958); and
(b) the ¢ltration method (McIntyre and Be¤,
1967; Okada and Honjo, 1973). Samples prepared
using the Utermo«hl method can only be analysed
by light microscopy (LM), whereas ¢lter preparations are suitable for both LM and scanning electron microscopy (SEM). The application of these
two methods depends on the purpose of the
study. The Utermo«hl method has been widely
used to quantify total phytoplankton composition
in water samples, whereas ¢lter preparations are
mainly used to analyse calcareous marine phytoplankton. A general description of how to sample
and analyse phytoplankton can be found in Sournia (1978).
The use of di¡erent methods, ¢lter funnels, microscopes or ¢lter membranes may result in di¡erent cell density estimates. In this study, we have
conducted several investigations to address this
potential error by focussing on: (a) the homogeneity of material on ¢lter membranes; (b) the application of di¡erent microscopes (SEM versus
LM); (c) the use of di¡erent ¢lter membranes
(cellulose mixed-ester membranes versus polycarbonate membranes) ; and (d) the application of
the Utermo«hl method versus the ¢ltration method. We also give recommendations on how to
collect and analyse samples for optimal reproducibility, as well as discussing the major sources
of error involved in the calculation of cell density.
In addition, we provide detailed instructions on
how to build a device for water sample ¢ltration.
2. Materials and methods
The samples used in this study were collected at
the JGOFS time series stations in Hawaii (HOT),
in Bermuda (BAT) and during the EC-MAST III
project CANIGO o¡ the Canary Islands. Details
of all samples are given in Table 1.
2.1. Settling method (after Utermo«hl, 1931, 1958)
Thirty millilitres of 20% hexamethyltetraminebu¡ered formalin (pH 7.5) were mixed in dark
plastic bottles with 250 ml of seawater transferred
from Niskin bottles (after Throndsen, 1978).
These bottles were stored in the dark prior to
analysis. For analysis, 100 ml sub-samples were
introduced into a settling chamber and the phytoplankton was allowed to settle for 72 h (Margalef, 1969). Phytoplankton cells were then identi¢ed and counted by the Utermo«hl method using
a Zeiss0 IM35 inverted microscope equipped with
phase contrast and bright¢eld illumination (Hasle,
1978).
2.1.1. Counting
Relatively large coccolithophore cells were
identi¢ed and counted in alternate ¢elds of the
settling chamber, at a magni¢cation of 160U.
This corresponds to 50 ml of seawater analysed
with a detection limit of 60 cells/l at a 95% con¢dence level. Where necessary, species identi¢cation was con¢rmed at a magni¢cation of 400U or
1000U. Relatively small species, such as Gephyrocapsa oceanica and Emiliania huxleyi, were
counted and identi¢ed at a magni¢cation of
400U in 64 squares. This corresponds to 1 ml
of seawater analysed with a detection limit of
3000 cells/l at a 95% con¢dence level.
2.2. Filtration
Up to 10 l of seawater was ¢ltered on a 47-mmdiameter cellulose mixed-ester membrane ¢lter
(Millipore0 ) or polycarbonate membrane ¢lter
(Nucleopore0 ) using Nalgene0 or Gelman0
1119 inline ¢lter gaskets (for details of samples
see Table 1). Each ¢lter membrane was rinsed
with NH4 -bu¡ered distilled water (pH 8.5) immediately after ¢ltration in order to remove all traces
of sea salt. All membranes were stored in plastic
Petri dishes and dried in an oven at 40^60‡C for
several hours. The seawater was ¢ltered with an
accuracy of P 0.1 l. A sketch of the ¢ltration apparatus used for these studies is shown in Fig. 1.
For light microscope analyses a piece of ¢lter
membrane cut along its radius was mounted onto
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
165
Table 1
Details of all samples used in this study
Sample
Filter
type
Filtration
device
Test material distribution
HS715-25-1
M
1
HS715-25-2
M
1
HS715-25-3
M
1
HS715-25-4
M
1
HOT52-2-15-17
M
2
M
2
M
2
HOT53-2-19-6
M
2
M
2
M
2
HOT52-2-15-12
M
2
M
2
M
2
HOT53-2-19-9
M
2
M
2
M
2
Test SEM versus LM
HS717
M
1
M
1
HS745
M
1
M
1
HOT57-75
M
2
M
2
HOT52
M
2
M
2
Test MF versus PC
HOT61-50
N
1
M
1
HOT61-75
N
1
M
1
HOT61-100
M
1
N
1
Test ¢ltration versus Utermo«hl
M37-31-31-0m
N
3
M37-31-31-10m
N
3
M37-31-31-25m
N
3
M37-31-31-50m
N
3
M37-31-31-75m
N
3
M37-31-31-100m
N
3
M37-31-31-150m
N
3
Method
Amount of
water ¢ltered (l)
Amount of water
analysed (ml)
Number of
specimens
Cells/l
Number
of species
Worker
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
LM
4
4
4
4
8.6
8.6
8.6
8.7
8.7
8.7
8.7
8.7
8.7
8.7
8.7
8.7
3.1
3.1
3.1
3.1
15.9
15.9
15.9
16.1
16.1
16.1
16.1
16.1
16.1
16.1
16.1
16.1
320
376
343
448
241
238
290
299
309
248
81
66
81
35
48
44
101 780
119 592
109 095
142 492
151 73
149 85
182 58
18 522
19 142
15 363
5 018
4 089
5 018
2 168
2 973
2 726
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
LM
SEM
LM
SEM
LM
SEM
LM
SEM
3.5
3.5
4
4
8.5
8.5
8.6
8.6
3.1
6.7
11.8
11.4
7.7
12.4
15.6
13.5
306
369
302
366
303
629
305
306
99 504
55 312
25 615
32 113
39 357
50 537
19 519
22 689
5
3
16
36
13
27
18
20
A
B
A
B
A
B
A
B
SEM
SEM
SEM
SEM
SEM
SEM
8.8
8.8
8.8
8.8
8.8
8.8
25.8
25.8
25.8
25.8
12.9
12.9
1062
954
946
1008
535
522
41 185
36 997
36 687
39 091
41 602
40 591
^
^
^
^
^
^
B
B
B
B
B
B
SEM
Utermo«hl
SEM
Utermo«hl
SEM
Utermo«hl
SEM
Utermo«hl
SEM
Utermo«hl
SEM
Utermo«hl
SEM
Utermo«hl
4
6.8
51.0
6.2
51.0
6.2
51.0
3.4
51.0
10.2
51.0
6.8
51.0
8.0
51.0
399
33
777
3
933
35
428
24
1147
34
467
18
91
6
58 676
14 380
125 323
3 000
150 484
10 500
125 882
6 360
112 451
7 540
68 676
5 260
11 375
2 070
15
12
26
2
33
9
21
10
30
6
17
5
7
3
C
D
C
D
C
D
C
D
C
D
C
D
C
D
4
4
4
4
4
4
HS samples were collected at the JGOFS time station Bermuda, 32‡10PN, 64‡30PW and HOT samples were collected at the
JGOFS time station ALOHA, 22‡45PN, 158‡0PW. M37 samples were collected on R/V Meteor at the location 28‡51PN, 13‡56PW.
M = Millipore0 MF cellulose mixed ester membrane; N = Nucleopore0 polycarbonate membrane. Filtration device: 1 = Gelman0
1119, 2 = Nalgene0 330^4000, 3 = Gelman0 1119 modi¢ed as described in Appendix A.2.1; LM = light microscope; SEM = scanning electron microscope. Worker: A, B, C, D indicate the scientist who has analysed the sample.
MARMIC 854 3-5-02
166
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Fig. 1. Filtration apparatus used at JGOFS time series station Bermuda, Hawaii and during the CANIGO project. (A) 10-l carboys. (B) 47-mm-diameter inline ¢lter holders. (C) Stopcock valve. (D) Vacuum pump. (E) Large water trapping steel tank.
a glass slide using Canada Balsam and ¢xed beneath a cover slip. A similar sized piece of ¢lter
membrane was mounted onto an aluminium stub
using carbon tape and coated with 15 nm of gold
for subsequent analysis in the SEM.
2.2.1. Counting
Cell counts were carried out with a Zeiss0 polarising light microscope using 63U or 100U objectives and a 1.25U Optovar lens. The area represented by one ¢eld of view is 0.0389 mm2 and
0.0154 mm2 , respectively, and the total observed
area (52 ¢elds of view along the radial cut) is
2 mm2 and 0.8 mm2 (see also Haidar and Thierstein, 2001). This corresponds to 7.86 and 3.13 ml
of seawater analysed, if 4 l were ¢ltered using the
Gelman0 1119 inline ¢lter gasket.
SEM counts of the HOT and the BAT samples
were performed with a Hitachi0 S2300 SEM
equipped with a computer controlled stage at a
magni¢cation of 1500U and 3000U. Calculation
of the observed ¢ltration area relies on the positioning accuracy of the stage and the accuracy
with which the area of one ¢eld of view can be
calculated. The area of observation is the sum of
the area of all single ¢elds of view. This is easily
estimated and controlled with LM, but it is more
di⁄cult to calculate and control in the SEM. In
both cases, it is important that single ¢elds of
view do not overlap to prevent double counting
of specimens. Using a computer-driven stage
makes it possible to control the size of the total
observed area, to prevent overlapping and, therefore, avoiding the double counting of an area.
The following set-up was used for the HOT
samples (Fig. 2). A total area of 3.88 mm2 was
analysed along a transect from the centre to the
edge of each ¢lter membrane. This corresponds to
26 ml of seawater analysed. Forty equidistant
¢elds of observation, each 0.097 Wm2 in size,
were analysed manually along this transect.
Each ¢eld of observation consisted of 81 single
monitor screens, each 1200 Wm2 in size (as calibrated with a standard metal grid, 10 P 0.2 Wm),
at a magni¢cation of 3000U or 20 single monitor
screens, each 4800 Wm2 in size (total observed area
of 7.7 mm2 ), captured at a magni¢cation of
1500U (Fig. 2, see also Corte¤s et al., 2001). This
corresponds to 52 ml of the seawater sample. One
¢eld of observation at 3000U magni¢cation is
equivalent to approximately two ¢elds of view
using a light microscope with a 63U objective
and an 8U ocular.
Coccolithophore counting of the CANIGO
samples was carried out with a computer-controlled stage and a fully automated image-capturing system. A remotely controlled Philips0 XL 30
scanning electron microscope in combination with
the imaging software analySIS0 3.0 was used to
capture and store all images collected along a prede¢ned transect (Fig. 2). The system, which captures about 1000 images/h, was used to acquire
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
167
A 95% con¢dence interval was calculated by
assuming a Poisson distribution and applying
the equation :
AC L;U
CDL;U ¼
ð2Þ
av
where : CDL;U = lower, upper con¢dence limit of
the cell density based on the Poisson distribution
and CL;U = lower, upper con¢dence limit for a
Poisson distribution.
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
CL ¼
ðN þ 0:982 Þ30:98
ð3Þ
Fig. 2. Schematic representation of the counting method
used in this study (modi¢ed after Corte¤s et al., 2001). (A) A
sector of the ¢lter is mounted on an aluminium stub. The
count direction is from U1 to U2. (B) Forty ¢elds of observation were analysed. Each ¢eld consisted of 9U9 monitor
screens at a magni¢cation of U3000 or 5U4 screens at
U1500. (C) The area of a single screen is 30U40 Wm or
60U80 Wm, respectively.
images from 135 ¢lter samples and enables nearly
optimal utilisation of the SEM. Approximately
2100 images (total area of 2.5 mm2 ) were captured
from each sample at a magni¢cation of 2000U
and all images were stored on CD-ROM. Coccolithophore cell densities were subsequently determined by manual examination of single images
using a PC. The amount of analysed seawater
varied between 5 and 20 ml.
CU ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðN þ 0:982 Þ þ 0:98Þ
ð4Þ
The cell density and the lower and upper con¢dence limit of each species was calculated by
replacing N by the number of individuals of
each species in Eqs. 1^4.
2.2.3. Detection limits
The detection limit of a species is ¢nding at
least one cell of a species in a de¢ned volume of
water, and depends on both the total amount of
seawater analysed and the total cell density. It can
be calculated from a Poisson distribution as follows:
P6X ¼ 0s ¼ expð3 M Þ
ð5Þ
where
2.2.2. Cell density calculations
A detailed description of the number of cells to
count and the type of distribution which can be
expected (e.g. Poisson or Normal distribution),
was presented by Venrick (1978a, 1978b and
references therein). The number of coccolithophore cells in 1 l of water was calculated using
the following equation:
AN
ð1Þ
CD ¼
av
where CD = cell density (cells/l water) ; A = ¢ltration area ; N = total number of cells counted ;
a = analysed area ; and v = volume of water ¢ltered
(l).
M ¼ xy
where x = number of cells/l and y = amount of
sample analysed.
Thus, the probability at any given M of ¢nding
one or more cells is:
13P6 M s0
ð6Þ
For example, if in a given amount of sample
one or more cells should be detected at 95% probability level.
13expð3 M Þs0:95
MARMIC 854 3-5-02
ð7Þ
168
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
3. Results
3.1. Material distribution on a ¢lter membrane
Fig. 3. Detection limits (DTL) for samples with 10^10 000
cells/l and an analysed volume of 1^100 ml. The detection
limit is about 3000 cells/l at a 95% probability level if 0.001 l
was analysed or 30 cells if 0.1 l was analysed.
From this follows for M (see Fig. 3):
M s3lnð0:05Þ
ð8Þ
and similarly for other probabilities
99% : M s3lnð0:01Þ
99:9% : M s3lnð00:01Þ
ð9Þ
ð10Þ
The detection limit is about 3000 cells/l at a
95% probability level if a volume of 0.001 l is
analysed, or 30 cells if 0.1 l is analysed.
Most ¢ltration devices produce an uneven distribution of material. Although these patterns are
sometimes obvious to naked eye, they are often
overlooked because of the thin ¢lm of material on
the membrane. In order to assess the distribution
of material on membranes, we put membranes
collected with di¡erent ¢ltration devices on a light
table and applied digital contrast enhancement to
the captured images (Table 2, Fig. 4). The observed pattern of uneven material distribution
can be attributed to: (a) the structure of the support sieve (Fig. 4AI, AII, BI, BII); (b) air locking
of parts of the ¢lter membrane (Fig. 4AIII, BII,
BIII) ; (c) improper ¢lter handling (Fig. 4BIII);
and (d) di¡erent £ow velocities of the ¢ltered
water in a ¢lter funnel or gasket (Fig. 5).
Air locking occurs in every low vacuum ¢ltration system as the attached vacuum ( 6 200 mm
Hg) is too weak to remove air bubbles trapped
above the ¢lter membrane and below or inside the
¢lter support (Fig. 4BII) and membrane. The former occurs especially in ¢lter gaskets without an
evacuation valve (Fig. 4AIII), while the latter occurs in every type of ¢ltration device and especially if the ¢lter membrane was not properly
moisturised. These air bubbles clog up parts of
Fig. 4. Support sieves and related material distribution of several ¢ltration devices tested. (AI) Nalgene0 330^4000 inline ¢lter
support (same type as Millipore Swinnex0 47-mm-diameter inline ¢lter support. (AII) Millipore0 cellulose mixed-ester membrane
¢ltered with Nalgene0 330^4000 47-mm-diameter inline ¢lter gasket. The membrane shows dark dots with an increased amount
of material. In addition, the lower part of the membrane was clogged by a large air bubble above the membrane. (AIII) Millipore0 cellulose mixed-ester membrane ¢ltered with Nalgene0 330^4000 47-mm-diameter inline ¢lter. The binarised image shows
that large areas (white) were clogged up by air bubbles above the membrane. (BI) Gelman0 1119 inline ¢lter plastic support.
(BII) Nucleopore0 polycarbonate membrane used with Gelman0 1119 inline ¢lter gasket and a plastic support. The membrane
re£ects the gutter structure of the plastic ¢lter support and small areas of the membrane were clogged up by air bubbles inside
the support sieve. (BIII) Nucleopore0 polycarbonate membrane used in a Gelman0 1119 inline ¢lter gasket and a plastic ¢lter
support. The material distribution was disturbed because the gasket was accidentally ¢lled with water while opening it. (CI)
Gelman0 1119 inline ¢lter metal grid support. (CII) Nucleopore0 polycarbonate membrane used in a modi¢ed Gelman0 1119 inline ¢lter gasket and metal grid support combined with a Millipore0 cellulose mixed-ester membrane for improved drainage. The
membrane shows a homogenous material distribution; however, the distribution was disturbed partly by a large gelatinous object.
(CIII) Nucleopore0 polycarbonate membrane used in a modi¢ed Gelman0 1119 inline ¢lter gasket equipped with metal grid support combined with a Millipore0 cellulose mixed-ester membrane for improved drainage. The membrane shows homogenous material distribution. Note: for all images, the contrast was enhanced by using a light table and digital contrast enhancement.
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
the ¢ltration area and therefore, the ¢ltration area
is reduced, leading to potentially erroneous cell
density determinations (Fig. 4AIII,BII).
A second cause of uneven distribution on ¢lter
membranes is the texture/structure of the support
sieve (Fig. 4AI,BI,CI). Rings, dots (Fig. 4AII) or
gutter structures (Fig. 4BII) are occasionally observed patterns in the resulting ¢lter materials
(Table 2).
169
The last cause of uneven material distribution is
related to variation in the £ow velocity of ¢ltered
water within a ¢lter funnel or inline ¢lter gasket
(Fig. 5). The number of cells per ¢eld along radial
transects of membranes used in two di¡erent inline ¢lter gaskets (47 mm diameter, Gelman0
1119 and Nalgene0 330^4000), occasionally
show a signi¢cant decrease from the edge to the
centre (Fig. 5). This is an indication that the £ow
MARMIC 854 3-5-02
170
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Fig. 5. Variation of cell counts per ¢eld of view in three radial transects of one ¢lter for di¡erent cell densities. In the top row, a
sketch shows how the analyses were carried out. Cell densities decrease from top to bottom of the graph. The solid line smoothes
the individual analyses and represents the locally weighted regression scatter plot smoothing with a 20% span. All analyses were
done with a light microscope. Data are from Corte¤s (1998) and Haidar (1997).
velocity of ¢ltered water varies from the inner to
the outer part of the ¢lter gasket. This pattern
seems to become more evident with increasing
total cell density (Fig. 5). The calculations of the
cell densities with a 95% con¢dence interval for
each analysed transect show no statistically significant di¡erence between transects of one ¢lter for
the samples with low and middle cell densities, if a
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
171
Table 2
List of all ¢ltration devices tested
Brand
Filter
Type
Support sieve
Order number
Nalgene0
Gelman0
Gelman0
Schleicher and Schuell0
Millipore0
Millipore0
Millipore0
Millipore0
Bioblock0
47 mm
47 mm
Modi¢ed 47 mm
FP 050/1
Swinnex 25 mm
Swinnex 47 mm
Pyrex 47 mm
Pyrex 47 mm
47 mm
Inline
Inline
Inline
Inline
Inline
Inline
Funnel
Funnel
Funnel
Radial plastic
Plastic grid
Metal grid
Radial plastic
Radial plastic
Radial plastic
Fritted glass
Steel grid
Fritted steel
330^4000
1119
1119
461 300
SX0002500
SX0004700
XX1004700
XX1004730
C17303
complete transect was counted. The mean values
vary from P 11 to 16% of each sample with respect to the mean (Fig. 6). Cell density calculations from the ¢lter with high cell density, however, exhibit statistically signi¢cant di¡erences
between transects 715-25-1 and 715-25-4 (Fig. 6)
which is probably the result of a clogged membrane.
3.2. SEM versus light microscope counts
The use of di¡erent microscopes may result in
di¡erent cell density values and species compositions for the same sample. This hypothesis was
tested with four di¡erent samples, one with high
cell densities (HS717), two with high species richness (HOT57-75, HS745), and one with a high
proportion of small cells ( 6 2.5 Wm) (HOT52).
Each sample was analysed with both the light
microscope and a SEM. Species richness and assemblage composition determined with both microscopes are similar for samples with high cell
densities or small cells (HS717 and HOT52, Table
1, Appendix A, Section A.1). However, the number of species obtained in the high diversity samples HS745 and HOT57-75 was much higher with
the SEM than with the light microscope (Table 1,
Appendix A, Section A.1). This is due to the occurrence of syracosphaerids, small reticulofenestrids, small gephyrocapsids and holococcolithophores, which could be identi¢ed with greater
certainty using the SEM rather than with the light
microscope.
However, the occasionally large di¡erences in
total cell density (Fig. 7) result from a combination of two e¡ects: (a) di¡erent de¢nitions of
Fig. 6. Coccolithophore cell densities determined from at least three radial counting transects for the four samples shown in
Fig. 5. Error bars show the 95% con¢dence interval for a Poisson distribution.
MARMIC 854 3-5-02
172
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Fig. 7. Mean cell densities for four di¡erent ¢lters each analysed with the light microscope and SEM, respectively. Error bars
show the 95% con¢dence interval for a Poisson distribution.
what constitutes a coccosphere ; and (b) the lower
recognition rate of small cells with the light
microscope. The higher cell density obtained
with the SEM from sample HS717 can be explained by the ¢rst point. A re-examination of
the same sample in light microscope and SEM
showed that there are many single placoliths of
Emiliania huxleyi lying relatively close together.
But this impression varies from light microscope
to SEM and it turned out that this sample was
analysed using two di¡erent concepts of what
constitutes a coccosphere. This may also be the
reason for the di¡erent cell densities recorded for
Umbellosphaera tenuis and Umbellosphaera irregularis in sample HOT57-75.
The limited recognition rate of small cells with
the light microscope is demonstrated by samples
HS745, HOT57-75 and HOT52. A di¡erence in
cell density of up to 23% (e.g. HS745) between
the light microscope and the SEM counts can be
explained by larger proportions of the small taxa
Emiliania huxleyi, Gephyrocapsa ericsonii and Gephyrocapsa protohuxleyi which were determined
with the SEM (for details see Appendix A, Section A.1).
3.3. Counts on di¡erent ¢lter membranes
The most commonly used ¢lters are cellulose
mixed-ester membranes (Millipore0 MF HAWP)
and polycarbonate membranes (Nucleopore0
PC111109 or Millipore0 ISOPORE ATTP). Cellulose mixed-ester membranes are widely used for
light microscope studies because they appear
transparent in smear slides; however, they are
not optimal for SEM analysis because of their
irregular surface. In this respect, polycarbonate
membranes are much more suited to the analysis
of coccolithophores with the SEM because a £at
surface is advantageous for the identi¢cation of
small species and therefore much less tiring for
the SEM operator. They are, however, not opti-
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Fig. 8. Comparison between cell density determinations using
Millipore0 cellulose mixed-ester membranes and Nucleopore0 polycarbonate membranes for the same sample. In
total, three di¡erent samples are shown. N = Nucleopore0
polycarbonate membranes; M = Millipore0 cellulose mixedester. Error bars show the 95% con¢dence interval for a
Poisson distribution. Data from Corte¤s (1998).
mal for light microscope analysis due to a slight
darkening in transmitted light. The sti¡er cellulose-mixed ester membranes are much easier to
handle than the polycarbonate membranes that
are prone to cling and wrinkle.
The identi¢cation of small cells and of holococcolithophores on the irregular-structured cellulose
mixed-ester membranes is clearly more di⁄cult
than on the £at polycarbonate membranes, which
may result in slightly di¡erent recognition rates
and cell densities in SEM studies. In our examples, however, SEM counts on both kinds of ¢lters show no statistically signi¢cant di¡erence
(Fig. 8).
3.4. Filtration (SEM) versus Utermo«hl settling
method
Reid (1980) reported that cell density calculations of small coccospheres obtained with the inverted light microscope technique are probably
not reliable. The comparison of seven sample
pairs that were analysed both with the ¢ltration
(SEM) and the Utermo«hl technique during the
EC-MAST III project CANIGO, support this assumption (Table 1, Appendix A, Section A.1).
Coccolithophore cell densities obtained with the
173
¢ltration technique are consistently several times
higher than those obtained with the Utermo«hl
technique (Table 1), although the sub-samples
for the di¡erent analyses were always taken
from the same Niskin0 bottle. In most samples,
the overall number of species determined with the
Utermo«hl technique is lower than with the ¢ltration (SEM) technique. However, in those samples
in which the overall number of species is comparable between the two methods, the assemblage
composition is quite di¡erent (Appendix A, Section A.1, sample M37-31-31-0m). With the Utermo«hl technique relatively large species with low
cell densities are still encountered because the
probability of detecting these species is higher
(50 ml of analysed water and therefore a detection
limit of 60 cells/l, see Fig. 3) than in the SEM (6.8
ml of analysed water and therefore a detection
limit of 430 cells/l, see Fig. 3). However, the recognition rate of small and delicate species is much
better with the SEM than with the inverted light
microscope. Therefore, the large di¡erences in
cell density and species composition are likely to
result from a combination of: (a) the restricted
recognition rate for small species in the light
microscope at a magni¢cation of 400U, as most
samples analysed were dominated by small
G. ericsonii and Emiliania huxleyi cells ; and (b)
higher probability of encountering large species
using the Utermo«hl technique because of larger
volume investigated. However, it remains unexplained why some species such as Florisphaera
profunda and Umbellosphaera tenuis were never
encountered in the light microscope although
they were found frequently in ¢lter preparations.
Filtration and subsequent analyses of some Utermo«hl preparations with the SEM suggest that
carbonate dissolution might take place during
the extended particle settling times, however,
this has to be tested in future studies.
4. Conclusion
Cell densities obtained with the Utermo«hl
method are consistently lower than those obtained
with the ¢ltration method because of the restricted recognition rate of species in the light
MARMIC 854 3-5-02
174
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
microscope. The major error in cell density calculations using the ¢ltration technique results from
the uneven distribution of plankton on ¢lter membranes. This can be attributed to: (a) air locking
of parts of the ¢lter membrane; (b) the structure
of the support sieve; (c) improper ¢lter handling;
and (d) uneven £ow velocities of the ¢ltered water
across a ¢lter funnel or gasket. The bias resulting
from the use of a light microscope or scanning
electron microscope arises because of the restricted recognition rate of species in the light
microscope, especially of small coccospheres. In
contrast, the use of di¡erent ¢lter membranes
does not show a statistically signi¢cant di¡erence
in cell density calculations, although polycarbonate membranes can be examined much more e⁄ciently with the SEM than cellulose mixed-ester
membranes.
5. Recommendation
For optimal reproducibility of coccolithophore
analyses, some recommendations are given below.
We strongly recommend testing the material distribution of any ¢ltration device before routine
application.
We suggest using inline ¢lter gaskets, as onboard a research vessel funnel systems are very
di⁄cult to use due to the limited amount of seawater that can be ¢ltered (0.5^1 l) before re¢lling
the funnel. Furthermore, each re¢ll may disturb
the material distribution on the membrane. In
addition, funnel systems are relatively large compared to inline ¢lter gaskets. This makes it di⁄cult to build a ¢ltration device that simultaneously ¢lters several samples for high resolution
plankton studies (Fig. 1).
Fig. 9. Construction plan of a multi-depth level ¢ltration device. All materials used are listed in Appendix A, Section A.2.6.
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
We suggest using a modi¢ed Gelman0 inline
¢lter gasket or a similar product that has a special
valve for releasing the air from the gasket. We
recommend replacing the plastic support sieve
that has a gutter structure, with a stainless steel
support combined with a £eece (e.g. Millipore0
membrane SCWP 04700) to increase the drainage
(Fig. 9). Some recommendations concerning the
volume of water to be ¢ltered are given in
Table 3. In the case of the clogging of a ¢lter
membrane by large objects or air bubbles, we recommend calculating the e¡ective area of ¢ltration
with help of a light table and digital image processing.
If possible, it is advisable to count random
¢elds of view on a given membrane in order to
reduce the error in cell density determinations
caused by the observed trends in the inline ¢lter
gaskets and the potential clogging of the membrane by large objects or air bubbles. However,
it is not, as yet, possible to mount a complete 47mm-diameter membrane on a SEM stub or a
glass slide. Therefore, we recommend counting a
large area on a piece of ¢lter (e.g. a full transect
from the centre to the edge of a ¢lter).
Light microscope analyses are suitable, if e⁄cient estimates of total coccolithophore cell densities of only the most common species and species groups are required. Most common species
are easily recognised with the LM. However, it
should be clearly stated for all taxa what constitutes a coccosphere. If there are signi¢cant numbers of dubious cells, additional SEM analysis
should be made to calculate the potential error.
SEM counts are required for species diversity
Table 3
Recommended ¢ltration volumes for 47-mm-diameter ¢lter
Season
Eutrophic areas
Above seasonal thermocline
Spring
1^2l
Summer
1^4l
Fall
1^4l
Winter
1^2l
Below seasonal thermocline
Spring
1^2l
Summer
1^4l
Fall
1^4l
Winter
1^2l
Oligotrophic areas
2^4l
6^8l
6^8l
4^6l
175
analysis and for the determination of cell densities
of certain taxa, especially small and delicately
structured coccolithophores. If a computer-controlled stage is available, the time needed to analyse a sample with the SEM is comparable to that
in a light microscope.
We recommend the use of polycarbonate membranes (e.g. Nucleopore0 ) rather than the cellulose mixed ester membranes for SEM analysis
because of their £at surface. Furthermore, polycarbonate membranes with a pore size of 0.8 Wm
instead of 0.45 Wm should be used because their
improved drainage. Cellulose mixed-ester membranes, however, are recommended for light
microscope analysis because of their transparency.
In addition, a pore size of 0.45 Wm instead of
0.8 Wm should be used as small coccospheres
may be hidden in large pores if the membrane
has to be analysed with the SEM.
Acknowledgements
We are grateful to Uwe Koy (IFM Kiel) and
Andrea Spiedt for their very useful advice and
their help during several cruises. Furthermore, we
would like to thank Prof. Gerold Siedler, IFM
Kiel, for his support during Poseidon cruise 212.
We wish to thank L. Fujieki, D. Hebel, R.
Johnson, D. Karl, A. Knap, A. Michaels and the
technical staff of the BATS group at Bermuda
Biological Station for Research and the Hawaiian
time-series group for the collection of water
samples. The manuscript profited from comments
by Hilary Paul, Patrick Quinn and the reviews of
Jeremy Young, Luc Beaufort and Dave Lazarus.
This work is a contribution to the EC-MAST
program CANIGO, EC contract MAS3-CT960060, and the EC-TMR program CODENET, EC
contract ERB-FRMX-CT97-0113. It was funded
by the Swiss Federal Office for Education (BBW
95.0355).
10l
10l
10l
10l
MARMIC 854 3-5-02
176
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Appendix A
A.1. Comparison between cell counts obtained with the SEM, the light microscope and the Utermo«hl settling
method
Method
SEM
Sample
HS717
N
No. of species
3
Total
369
Acantoica quattrospina
Acantoica sp.
Algirosphaera oryza
Algirosphaera quadricornu
Algirosphaera robusta
Algirosphaera sp.
Alisphaera spatula
Alisphaera unicornis
Alisphaera sp.
Alveosphaera bimurata
Anaplosolenia brasiliensis
Braarudosphaera bigelowi
Calcidiscus leptoporus
Calciopappus caudatus
Calciopappus rigidus
Calciosolenia murrayi
Ceratolithus cristatus
Coronosphaera sp.
Cyrtosphaera aculeata
Discosphaera tubifera
Emiliania huxleyi
346
Florisphaera profunda
Gaarderia corolla
Gephyrocapsa ericsonii
22
Gephyrocapsa protohuxleyi
Gephyrocapsa muellerae
Gephyrocapsa oceanica
Helicosphaera carteri
Helicosphaera pavimentum
Michaelsarsia adriaticus
Michaelsarsia elegans
Neosphaera coccolithomorpha
Oolithotus fragilis
Ophiaster hydroideus
Ophiaster reductus
Ophiaster sp.
Polycrater galapagensis
Reticulofenestra punctata
Reticulofenestra sessilis
Reticulofenestra sp.
Rhabdosphaera clavigera
Rhabdosphaera stylifera
Rhadbosphaera xiphos
Scyphosphaera apsteinii
Syracosphaera sp.
Syracosphaera anthos
Syracosphaera corrugis
Syracosphaera epigrosa
LM
%
SEM
SEM
HS745
c/l
N
55 312
5
306
%
c/l
N
99 504
36
368
3
2
4
1
1
93.8 51 864
6.0
LM
298
0.3
0.3
%
0.8
0.5
1.1
HOT57-75
c/l
32 113
262
N
%
c/l
2
0.7
27
25 615 629
6
170
1
0.3
85
2
0.7
170
16
302
%
1.0
c/l
50 537
482
N
%
13
303
c/l
39 357
8
2.6
1 039
4
0.6
321
19
3.0
1 527
1
5
0.3
1.7
130
651
2
13
22
0.3
2.1
3.5
161
1 044
1 768
8
21
2.6
6.9
1 039
2 728
24
3.8
1 928
11
11
1.7
1.7
884
884
8
2.6
1 039
3
26
0.5
4.1
241
2 089
1
0.3
130
325
325
97.4 96 903
0.3
N
175
349
2
3
0.5
0.8
175
262
7
44
1.9 611
12.0 3 840
2
0.5
80
21.7 6 981
2
4
6
2
0.5
1.1
1.6
0.5
175
349
524
175
2
2
0.5
0.5
8
2.2
1
0.3
85
3
95
1.0 254
31.5 8 058
175
3 298
1
LM
325
1
6
0.3
2.0
85
509
175
175
7
2.3
594
698
1
0.3
85
3
10
5
0.8
2.7
1.4
262
873
436
1
0.3
87
MARMIC 854 3-5-02
14
1
4.6
0.3
1 187
85
9
14
1.4
2.2
723
1 125
1
9
0.3
3.0
130
1 169
16
5.3
1 357
2
0.3
161
6
2.0
779
6
1.0
482
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Method
SEM
Sample
HS717
N
Syracosphaera haldalli
Syracosphaera histrica
Syracosphaera molischii
1
Syracosphaera nana
Syracosphaera nodosa
Syracosphaera orbiculus type
Syracosphaera ossa
Syracosphaera pirus type
Syracosphaera pulchra
Syracosphaera rotula
Syracosphaera type ‘I’
Syracosphaera type ‘K’
Gladiolithus £abellata
Umbellosphaera irregularis
Umbellosphaera tenuis
Umbilicosphaera hulburtiana
Umbilicosphaera sibogae
Other heterococcoliths
Calyptrolithophora papillifera
Calyptrosphaera oblonga
Corisphaera gracilis
Daktylethra pirus
Helladosphaera sp.
Homozygosphaera arethusae
Periphyllophora mirabilis
Syracolithus schilleri
Zygosphaera hellenica
Other holococcolithophores
LM
LM
N
%
c/l
N
%
c/l
N
%
c/l
0.3
150
1
0.3
325
1
5
0.3
1.4
87
436
14
4.6
1 187
5
1.4
436
2
0.5
175
4
1.1
349
1
5
0.3
1.4
87
436
2
131
0.5 175
35.6 11 432
1
109
0.3 85
36.1 9 245
1
3
1
2
5
0.3
0.8
0.3
0.5
1.4
87
262
87
175
436
17
5.6
5
2
1.4
0.5
436
175
1
0.3
87
2
2
SEM
HOT 52
No. of species
Total
Acantoica quattrospina
Acantoica sp.
Algirosphaera oryza
Algirosphaera quadricornu
Algirosphaera robusta
Algirosphaera sp.
Alisphaera spatula
Alisphaera unicornis
Alisphaera sp.
Alveosphaera bimurata
Anaplosolenia brasiliensis
Braarudosphaera bigelowi
Calcidiscus leptoporus
Calciopappus caudatus
Calciopappus rigidus
Calciosolenia murrayi
Ceratolithus cristatus
Coronosphaera sp.
Cyrtosphaera aculeata
Discosphaera tubifera
Emiliania huxleyi
20
306
0.7
0.7
650
650
LM
%
11
SEM
N
%
17
22 689 305
c/l
N
19 519
15
400
%
c/l
N
58 707
12
33
%
6.1
3.0
%
c/l
1
0.2
80
3
4
8
7
8
6
0.5
0.6
1.3
1.1
1.3
1.0
241
321
643
562
643
482
7
1.1
562
248
163
39.4 19 926
25.9 13 096
1
0.2
80
1
0.2
80
933
Utermo«hl
2
1
0.3
3.6
N
1 442
N
%
c/l
1
0.3
130
185
11
61.1 24 030
3.6 1 429
30
9.9
3 897
8
2.6
1 039
SEM
Utermo«hl
M37-31-31-10m
M3-31-31-10m
c/l
N
N
14 380
26
777
2
125 925 3
2
324
M37-31-31-0m
c/l
LM
HOT57-75
c/l
Sample
1
SEM
HS745
%
Method
N
SEM
177
%
c/l
%
c/l
3 000
40
20
74
1
0.3
74
1
6
0.3
2.0
74
445
3
1.0
222
1
95
0.3 74
31.0 7 044
3
3
9
173
1.0
1.0
192
192
3.0 576
56.7 1 1071
3
0.8
440
4
106
1.0 587
26.5 15 557
MARMIC 854 3-5-02
4
12.1 80
1
3.0
4
10
12.1 80
30.3 10 000
4
1.0
648
1
253
162
33.0 41 003
20
2
66.7 2 000
178
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Method
SEM
Sample
HOT 52
N
Florisphaera profunda
3
Gaarderia corolla
Gephyrocapsa ericsonii
101
Gephyrocapsa protohuxleyi
Gephyrocapsa muellerae
Gephyrocapsa oceanica
34
Helicosphaera carteri
1
Helicosphaera pavimentum
Michaelsarsia adriaticus
21
Michaelsarsia elegans
Neosphaera coccolithomorpha
Oolithotus fragilis
Ophiaster hydroideus
Ophiaster reductus
Ophiaster sp.
Polycrater galapagensis
Reticulofenestra punctata
Reticulofenestra sessilis
Reticulofenestra sp.
Rhabdosphaera clavigera
4
Rhabdosphaera stylifera
Rhadbosphaera xiphos
Scyphosphaera apsteinii
Syracosphaera sp.
1
Syracosphaera anthos
Syracosphaera corrugis
Syracosphaera epigrosa
Syracosphaera haldalli
Syracosphaera histrica
Syracosphaera molischii
Syracosphaera nana
Syracosphaera nodosa
Syracosphaera orbiculus type
Syracosphaera ossa
Syracosphaera pirus type
Syracosphaera pulchra
1
Syracosphaera rotula
2
Syracosphaera type ‘I’
Syracosphaera type ‘K’
Gladiolithus £abellata
Umbellosphaera irregularis 18
Umbellosphaera tenuis
10
Umbilicosphaera hulburtiana 1
Umbilicosphaera sibogae
1
Other heterococcoliths
Calyptrolithophora papillifera
Calyptrosphaera oblonga
Corisphaera gracilis
Daktylethra pirus
Helladosphaera sp.
Homozygosphaera arethusae
Periphyllophora mirabilis
Syracolithus schilleri
Zygosphaera hellenica
Other holococcolithophores
LM
SEM
Utermo«hl
M37-31-31-0m
%
c/l
N
%
c/l
N
%
c/l
1.0
222
1
0.3
64
2
0.5
294
33.0 7 489
1
0.3
64
243
60.8 35 665
11.1 2 521
0.3 74
45
1
14.8 2 880
0.3 64
5
5
1.3
1.3
6.9
4
2
1
1.3
0.7
0.3
256
128
64
1
0.3
64
1.3
1 557
297
9
3.0
576
11
3.6
704
734
734
2
2
0.5
0.5
294
294
3
0.8
440
N
74
3
0.3
0.7
5.9
3.3
0.3
0.3
74
148
1 335
741
74
74
1
1
3
12
0.3
1.0
3.9
64
192
768
23
7.5
1 472
0.8
0.3
c/l
4
1
12.1 4 000
3.0 20
1
3.0
20
Utermo«hl
M3-31-31-10m
N
%
c/l
N
%
5
1.0
810
360
46.0 58 343
1
33.3 1 000
5
6
1.0
1.0
1
3.0
3.0
20
324
2
324
4.0
1.0
5 348
162
810
1
11
1.0
162
17 83
14
2.0
2269
10
1
1.0
1621
162
6
4
1.0
1.0
972
648
9
1.0
1 459
9
1.0
1459
4
2
1.0
648
324
20
440
147
1
0.3
147
8
1
2.0
0.3
1 174
147
11
2.8
1 614
1
2
3.0
6.1
20
40
486
3
2
0.7
810
972
2
33
1
5
3
1
0.3
%
SEM
M37-31-31-10m
128
MARMIC 854 3-5-02
11
486
1.0
1 783
c/l
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Method
SEM
Sample
M37-31-31-25m
No. of species
Total
Acantoica quattrospina
Acantoica sp.
Algirosphaera oryza
Algirosphaera quadricornu
Algirosphaera robusta
Algirosphaera sp.
Alisphaera spatula
Alisphaera unicornis
Alisphaera sp.
Alveosphaera bimurata
Anaplosolenia brasiliensis
Braarudosphaera bigelowi
Calcidiscus leptoporus
Calciopappus caudatus
Calciopappus rigidus
Calciosolenia murrayi
Ceratolithus cristatus
Coronosphaera sp.
Cyrtosphaera aculeata
Discosphaera tubifera
Emiliania huxleyi
Florisphaera profunda
Gaarderia corolla
Gephyrocapsa ericsonii
Gephyrocapsa protohuxleyi
Gephyrocapsa muellerae
Gephyrocapsa oceanica
Helicosphaera carteri
Helicosphaera pavimentum
Michaelsarsia adriaticus
Michaelsarsia elegans
Neosphaera coccolithomorpha
Oolithotus fragilis
Ophiaster hydroideus
Ophiaster reductus
Ophiaster sp.
Polycrater galapagensis
Reticulofenestra punctata
Reticulofenestra sessilis
Reticulofenestra sp.
Rhabdosphaera clavigera
Rhabdosphaera stylifera
Rhadbosphaera xiphos
Scyphosphaera apsteinii
Syracosphaera sp.
Syracosphaera anthos
Syracosphaera corrugis
Syracosphaera epigrosa
Syracosphaera haldalli
Syracosphaera histrica
Syracosphaera molischii
Syracosphaera nana
Syracosphaera nodosa
Syracosphaera orbiculus type
Syracosphaera ossa
Syracosphaera pirus type
Syracosphaera pulchra
33
933
N
5
Utermo«hl
%
c/l
Utermo«hl
SEM
Utermo«hl
M37-31-31-75m
M3-31-31-75m
c/l
N
N
6 360
30
1 147
M37-31-31-50m
N
%
9
151 207 35
1.0
SEM
c/l
N
10 500
21
428
810
5
14.3 100
4
4
%
c/l
N
%
10
125 634 24
1.0
1.0
1 174
1 174
1
4.2
179
%
c/l
162
7
1
162
1
98
196
3
486
8
249
1
1.0 1 297
27.0 40 354
162
477
51.0 77 305
2
17
1
2.0
3
6
10
2
29
2
10
9
4.0
2.0
3.0
1.0
1.0
4
4
10
1
1
1
1
881
2
105
2
587
25.0 30 821
587
239
56.0 70 155
2.9
20
11
3.0
3 229
10
2.0
2 935
7 540
1
4.2
7
29.2 140
2
2
8.3
40
17
1
4.2
20
5
3
4
12.5 60
16.7 4 000
2
1
8.3
4.2
1.0
688
20
2000
20
1.0
14
41.2 280
9
6
26.5 180
17.6 6 000
1
1
2.9
2.9
1 000
20
3
8.8
60
1 670
491
10
381
5
9
615
1.0 982
33.0 37 422
491
1.0 884
54.0 60 406
3
7
1.0
2
295
688
196
324
1
3
4
14
2
17.1 120
28.6 10 000
1.0
324
2 755
162
1
36
22.9 160
c/l
20
1
8
%
6
112 660 34
5 834
16
162
5
491
2
4
8
196
393
786
648
2 269
324
1
2.9
20
1
2.9
20
4 700
324
1 621
1 459
648
648
1.0
1 621
162
162
162
162
3
1.0
881
12
3.0
3 522
2
5.7
40
1.0
1761
8
1.0
587
5
1
2
587
294
1
294
2
587
2
1.0
1.0
9
2
6
2
2
1
6
1.0
MARMIC 854 3-5-02
2
8.3
40
1.0
1 572
884
196
589
196
786
491
98
196
180
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Method
SEM
Sample
M37-31-31-25m
Syracosphaera rotula
Syracosphaera type ‘I’
Syracosphaera type ‘K’
Gladiolithus £abellata
Umbellosphaera irregularis
Umbellosphaera tenuis
Umbilicosphaera hulburtiana
Umbilicosphaera sibogae
Other heterococcoliths
Calyptrolithophora papillifera
Calyptrosphaera oblonga
Corisphaera gracilis
Daktylethra pirus
Helladosphaera sp.
Homozygosphaera arethusae
Periphyllophora mirabilis
Syracolithus schilleri
Zygosphaera hellenica
Other holococcolithophores
Utermo«hl
N
%
c/l
14
2.0
2 269
1
5
1
SEM
Utermo«hl
M37-31-31-50m
N
%
c/l
N
%
c/l
6
1.0
1761
3
1
1.0
881
294
3
1.0
881
6
1.0
1761
N
%
c/l
SEM
Utermo«hl
M37-31-31-75m
M3-31-31-75m
N
N
%
c/l
%
c/l
162
1.0
810
162
1
2.9
20
486
Method
SEM
Sample
M37-31-31-100m
c/l
N
No. of species
Total
Acantoica quattrospina
Acantoica sp.
Algirosphaera oryza
Algirosphaera quadricornu
Algirosphaera robusta
Algirosphaera sp.
Alisphaera spatula
Alisphaera unicornis
Alisphaera sp.
Alveosphaera bimurata
Anaplosolenia brasiliensis
Braarudosphaera bigelowi
Calcidiscus leptoporus
Calciopappus caudatus
Calciopappus rigidus
Calciosolenia murrayi
Ceratolithus cristatus
Coronosphaera sp.
Cyrtosphaera aculeata
Discosphaera tubifera
Emiliania huxleyi
Florisphaera profunda
Gaarderia corolla
Gephyrocapsa ericsonii
Gephyrocapsa protohuxleyi
Gephyrocapsa muellerae
Gephyrocapsa oceanica
Helicosphaera carteri
Helicosphaera pavimentum
Michaelsarsia adriaticus
Michaelsarsia elegans
17
467
68 541
5
18
1
1
147
147
10
55.6
200
2
294
1
3
5.6
16.7
20
3 000
N
Utermo«hl
%
1
2
1
98
196
98
2
196
2
196
SEM
Utermo«hl
M37-31-31-150m
5
145
10
1.0
31.0
2.0
734
21 281
1 468
246
52.0
36 105
7
7
1
1.0
1.0
1 027
1 027
147
2
%
11.1
c/l
N
5 260
7
91
2 000
MARMIC 854 3-5-02
%
c/l
N
11 448
3
6
4
62
4.0
68.0
503
7 800
12
13.0
1 510
%
c/l
2080
3
50.0
60
2
33.3
2 000
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
Method
SEM
Sample
M37-31-31-100m
Neosphaera coccolithomorpha
Oolithotus fragilis
Ophiaster hydroideus
Ophiaster reductus
Ophiaster sp.
Polycrater galapagensis
Reticulofenestra punctata
Reticulofenestra sessilis
Reticulofenestra sp.
Rhabdosphaera clavigera
Rhabdosphaera stylifera
Rhadbosphaera xiphos
Scyphosphaera apsteinii
Syracosphaera sp.
Syracosphaera anthos
Syracosphaera corrugis
Syracosphaera epigrosa
Syracosphaera haldalli
Syracosphaera histrica
Syracosphaera molischii
Syracosphaera nana
Syracosphaera nodosa
Syracosphaera orbiculus type
Syracosphaera ossa
Syracosphaera pirus type
Syracosphaera pulchra
Syracosphaera rotula
Syracosphaera type ‘I’
Syracosphaera type ‘K’
Gladiolithus £abellata
Umbellosphaera irregularis
Umbellosphaera tenuis
Umbilicosphaera hulburtiana
Umbilicosphaera sibogae
Other heterococcoliths
Calyptrolithophora papillifera
Calyptrosphaera oblonga
Corisphaera gracilis
Daktylethra pirus
Helladosphaera sp.
Homozygosphaera arethusae
Periphyllophora mirabilis
Syracolithus schilleri
Zygosphaera hellenica
Other holococcolithophores
Utermo«hl
%
c/l
16
3.0
2 348
4
1
1.0
587
147
1.0
2
Utermo«hl
M37-31-31-150m
N
3
SEM
181
N
%
c/l
2
11.1
40
N
%
c/l
3
3.0
377
1
1.0
126
2
2.0
252
N
%
c/l
1
16.7
20
440
294
5
1.0
734
1
1.0
126
1
10
2.0
147
1 468
6
7.0
755
N = number of specimens counted; % = relative proportion of species; c/l = cells/l; SEM = scanning electron microscope;
LM = light microscope; Utermo«hl = Utermo«hl settling method.
MARMIC 854 3-5-02
182
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
A.2. Description of ¢ltration equipment and
procedure
The following descriptions provide assistance
in: (a) the construction of a ¢ltration device (Appendix A, Section A.2.6 Fig. 10) and (b) the sampling of coccolithophores. The ¢ltration system
and the sampling strategy described below, were
developed within the sampling campaigns at the
JGOFS stations near Bermuda and Hawaii and
during several cruises within the EC MAST-III
project CANIGO.
A.2.1. Modi¢cation of a Gelman0 ¢lter holder
(catalogue number 1119, see Fig. 10)
We have modi¢ed the inline ¢lter holder in order to: (a) obtain a more even distribution of
material ; (b) increase the ¢ltration e⁄ciency;
and (c) improve the handling of Nucleopore0
PC111109 membrane ¢lters.
(1) Replace the plastic support sieves (K),
which produce a cross on the membrane ¢lter,
with the stainless steel support sieve (J), which
allows for a homogeneous distribution.
(2) Screw the two hose connectors (A) into the
lower (L) and the upper ¢lter holder (E).
(3) Replace the closing cap (B) from the evacuation valve with a 5-cm piece of silicon tube (D).
(4) Mount one part of the PVC quick connector
at the end of the 5-cm piece of the silicon tube
(D).
(5) Fasten a tube clip (C) at the 5-cm piece of
the silicon tube and the hose connector.
A.2.2. Modi¢cation of carboys
(SEMADENI0 order number 1531, see Fig. 11)
We have modi¢ed the ‘normal’ carboys because
they are more robust onboard than for example
Nalgene0 carboys, which have a protruding spigot/outlet at the bottom that can be easily broken
o¡.
(1) Remove the evacuation valve/lid (B) and
drill a hole into the plastic lid.
(2) Press the female part of a quick connector
from the inner side of the lid into the hole.
(3) Connect 47 cm of a hard PVC tube (R) of
6 mm diameter to the barbed part of the tubing
connector and mount a check valve (Q) at the end
of the tube.
(4) Put the tube into the carboy and fasten the
lid. If the opening of the evacuation valve is too
small for the check valve, introduce the tube with-
Fig. 10. Modi¢cation of a Gelman0 inline ¢lter gasket. A, hose connector; B, evacuation valve and closing cap; C, tube clip; D,
silicon tube; E, ¢lter holder top; F, red sealing ring; G, white Te£on ring; H, Isopore0 /Nucleopore0 polycarbonate membrane;
I, millipore0 cellulose mixed-ester membrane; J, support sieve stainless steel; K, plastic support sieve; L, ¢lter holder base.
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
183
Fig. 11. Schematic of a single ¢ltration line. A, carboy; B, modi¢ed outlet with female quick connector (former evacuation
valve); C, male quick connector; D, PVC tube, 7 mm diameter; E, female quick connector; F, silicon tube; G, tube clip; H,
evacuation valve; I, ¢lter holder; J, PVC tube, 5 cm long, 7 mm diameter; K, male quick connector; L, PVC tube, 8 mm diameter; M, vacuum pump; N, stopcock valve; O, metal sheets for gasket holding; P, outlet; Q, check valve; R, PUR tube; S, ¢lling
inlet.
out the check valve and get the tube with the help
of a wire loop through the large ¢lling inlet (S)
and connect the check valve to the tube.
Note: one disadvantage of this set-up is that it
is necessary to ¢ll the inner tube and the connection tube between carboy and ¢lter holder with
water before ¢ltration can start. This is necessary
because air trapped inside the tubes and the ¢lter
gasket can prevent the gravity £ow of water.
A.2.3. Mounting of a membrane ¢lter
(see Fig. 10)
(1) Put a Millipore0 cellulose mixed-ester membrane (I), pore size of 8 Wm, directly on the stain-
less support sieve (J) and moisten this membrane
with distilled water to get a £at surface. Make
sure that the whole membrane is wet and that
there are no white areas with air bubbles. This
membrane prevents formation of pressure rings
on the ¢lter as well as air bubbles and it supports
the drainage.
(2) Place a 0.8-Wm Isopore0 /Nucleopore0 polycarbonate membrane (H) on top of membrane (I).
Make sure that there are no air bubbles between
these two membranes (this membrane will ¢lter
the coccolithophores). In order to hold the membrane in position we recommend attaching a low
vacuum under the membrane ¢lter (connection to
lower hose adapter A).
MARMIC 854 3-5-02
184
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
(3) Put the white Te£on ring (G) on top of the
0.8-Wm Isopore0 /Nucleopore0 polycarbonate
membrane (H). This prevents wrinkling of the
membrane during closing of the ¢lter holder.
(4) Put the red sealing ring (F) on the top of the
white Te£on ring (G).
(5) Close the ¢lter holder (E+L) carefully and
check for membrane wrinkling.
The ¢lter holder is now ready for use.
A.2.4. Filtration procedure (see also Fig. 11)
The sampling and ¢ltration procedures can be
summarised as follows:
(1) Draw o¡ the desired amount of water from
the Niskin0 bottles, from di¡erent depth levels,
into a carboy (A).
(2) Connect tube (L) to the ¢lter holder and
close valve (N). Be sure to close valve (N) after
connecting the ¢lter holder because the membrane
might be damaged by air pressure.
(3) Connect tube (D) to the ¢lter holder and ¢ll
both with distilled water. Note: this step is not
necessary if carboys with a spigot/outlet at the
bottom of a carboy are used.
(4) Connect this tube (D) to the carboy (A, B).
(5) Connect a silicon tube to the silicon tube (F)
that is attached to the evacuation valve (H) (use
the quick connector E) and put the end of the
tube in a beaker.
(6) Open the tube clip (G). The water will now
start £owing (gravity £ow). Make sure that large
air bubbles migrate out of the ¢lter gasket
through the evacuation valve. Then close the
tube clip. It is best to tilt the ¢lter holder a little
so that the evacuation valve is slightly elevated.
(7) Make sure that the vacuum is not higher
than 200 mm Hg (otherwise cells may be deformed) and then open valve (N).
(8) If large air bubbles are observed in the ¢lter
holders during the ¢ltration, close valve (N) (disconnect the vacuum) and repeat the procedure as
described in steps 5 and 6.
(9) After all water has been ¢ltered, close the
valve (N), disconnect tubes (D) at the carboy. Fill
tube (D) with bu¡ered distilled water (NH4 , pH
8.5) until the gasket is £ooded. If needed, open
tube clip (G) carefully to release the air, and open
valve (N) again.
(10) After rinsing the ¢lter with distilled water
under the vacuum, remove tube (D) and open the
¢lter holder carefully. Make sure that there is no
water left in the ¢lter holder, otherwise some material will bloat and change the distribution.
Drops of water on the white Te£on ring should
be carefully removed with a tissue before removing the membrane.
(11) Close the valve (N) and remove the ¢lter
membrane and store it in a labelled petri dish on
top of the absorber pads. You may have to exchange absorber pads several times because the
polycarbonate membrane tends to roll up.
(12) Dry the membrane immediately in an oven
at 40^60‡C for several hours. It is very important
to remove the membrane as soon as possible after
¢ltration has ¢nished, otherwise any coccolithophores may begin to dissolve.
A.2.5. Sample preparation
We strongly recommend using Canada Balsam
for light microscope preparations because of the
improved durability of the slides. Canada Balsam
preparations of the Ehrenberg collection are after
150 yr still usable (Dave Lazarus, personal communication, 2001).
The examination of old SEM stubs (older than
1 yr) that were prepared using conductive carbon
tape revealed decomposition of the gold coating.
These samples could not be recovered. We suspect
that a chemical reaction takes place between the
conductive glue, the ¢lter membrane and the gold.
Therefore, we currently mount ¢lter samples with
the help of alcohol and liquid conductive silver
directly onto the aluminium stub (or glass). We
put a drop of alcohol on the stub and, onto the
drop, place the piece of ¢lter. The ¢lter membrane
clings tightly to the aluminium stub as a result of
the strong capillary adhesion. In order to mount
the ¢lter permanently, we add a drop of conductive silver to the corners of the ¢lter and wait for
the alcohol to evaporate. Subsequently, the stub is
sputtered with gold. Ongoing tests have shown
that these preparations last at least 3 yr.
MARMIC 854 3-5-02
J. Bollmann et al. / Marine Micropaleontology 44 (2002) 163^185
185
A.2.6. List of materials used to build a ¢ltration device (including supplier information)
Item
Quantity
Brand
Order number
Te£on ring 47U37U1 mm
Dual head membrane vacuum pump
Inline ¢lter gasket
Photo etched steel support sieve
Tube clip 60 mm long
Carboy 9 l
Aluminium sheets, 1.5 and 2 mm
Stopcock valve, 7 mm diameter
Carboy 10 l
PVC tubes, 7 mm diameter
T-tube connector, 7 mm diameterU10 mm
PVC Tubes, 8 mm diameterU12 mm
PUR tubes, 8 mm diameterU10 mm
Check or no return valve, 6 mm diameter
Quick disconnectors, 6 mm diameter
Quick disconnectors, 7 mm diameter
Vacuum carboy 10 l
Filling/venting closure
Press nuts, m 6
Stainless steel tank
MF-membrane ¢lter, 8 Wm pore size
ISOPORE membrane ¢lter, 47 mm diameter, 0.8 Wm
Polycarbonate membrane, 47 mm diameter, 0.8 Wm
12
1
12
12
12
12
2 m2
14
12
12 m
12
10 m
3m
12
12
12
1
1
20
1
ADIPAC0
Cole-Parmer0
Gelman0
Gelman0
Merck0
Nalgene0
NN
Semadeni0
Semadeni0
Semadeni0
Semadeni0
Semadeni0
Semadeni0
Semadeni0 /Nalgene0
Semadeni0 /Nalgene0
Semadeni0 /Nalgene0
Semadeni0 /Nalgene0
Semadeni0 /Nalgene0
Tubtara0
NN
Millipore0
Millipore0
Nucleopore0
Special order
E-07061-00
1119
72970
3802002
2320-0020
Special order
03097
1531
1345
0485
1348
2591
0523/6120-0010
00517/6150-0010
00518/6150-0010
00970/2210
01409/2162
References
Corte¤s, M.Y., 1998. Coccolithophores at the time-series station
ALOHA, Hawaii: population dynamics and ecology. Ph.D.
Thesis, University of Zurich, Zurich, unpublished, 176 pp.
Corte¤s, M.Y., Bollmann, J., Thierstein, H.R., 2001. Coccolithophore ecology at the HOT station ALOHA, Hawaii.
Deep-Sea Res. II 48, 1957^1981.
Haidar, T.A., 1997. Calcareous phytoplankton dynamics at
Bermuda (N. Atantic). Ph.D. Thesis, No. 12084, ETH
Zurich, unpublished, 168 pp.
Haidar, A.T., Thierstein, H.R., 2001. Coccolithophore dynamics o¡ Bermuda (N. Atlantic). Deep-Sea Res. II 48, 1925^
1956.
Hasle, G.R., 1978. The inverted microscope method. In: Sournia, A. (Ed.), Phytoplankton Manual. Monographs on Oceanic Methodology, UNESCO, Vol. 6. Paris, pp. 88^96.
Margalef, R., 1969. Counting. In: Vollenweider, R.A., Talling,
J.F., Westlake, D.F. (Eds.), A Manual on Methods for Measuring Primary Production in Aquatic Environments including a Chapter on Bacteria. International Biological Programme (IBP Handbook 12). Blackwell Scient¢c, Oxford,
pp. 7^14.
McIntyre, A., Be¤, A.W.H., 1967. Modern coccolithophoridae
of the Atlantic Ocean-I. placoliths and cyrtoliths. Deep-Sea
Res. 14, 561^597.
special order
SCWP04700
ATTP 04700
111109
Okada, H., Honjo, S., 1973. The distribution of oceanic cocolithophorids in the Paci¢c. Deep-Sea Res. 20, 55^374.
Reid, F.M.H., 1980. Coccolithophorids of the North Paci¢c
Central Gyre with notes on their vertical and seasonal distribution. Micropaleontology 26, 151^176.
Sournia, A., 1978. Phytoplankton Manual. Monographs on
Oceanographic Methodology, UNESCO, Vol. 6. Paris, 337
pp.
Throndsen, J., 1978. Preservation and storage. In: Sournia, A.
(Ed.), Phytoplankton Manual. Monographs on Oceanographic Methodology, UNESCO, Vol. 6. Paris, pp. 69^
74.
Utermo«hl, H., 1931. Neue Wege der quantitativen Erfassung
des Planktons (mit besonderer Beru«cksichtigung des Ultraplanktons). Verh. Int. Ver. Theor. Angew. Limnol. 5, 567^
596.
Utermo«hl, H., 1958. Zur Vervollkommnung der quantitativen
Phytoplankton-Methodik. Mitt. Int. Ver. Theor. Angew.
Limnol. 9, 1^38.
Venrick, E.L., 1978a. The implication of sub-sampling. In:
Sournia, A. (Ed.), Phytoplankton Manual. Monographs on
Oceanographic Methodology, UNESCO, Vol. 6. Paris, pp.
75^87.
Venrick, E.L., 1978b. How many cells to count? In: Sournia,
A. (Ed.), Phytoplankton Manual. Monographs on Oceanographic Methodology, UNESCO, Vol. 6. Paris, pp. 167^180.
MARMIC 854 3-5-02