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Although tedious, time-consuming, and requiring an excellence in taxonomic identifications, the
traditional counting of phytoplankton is still unsurpassed for quantifying plankton, especially at
low limits of detection. The examination and counting of preserved material also allows for
direct observation and assessment of cell condition. The fundamental issue that must be addressed
prior to choosing a counting technique is whether samples must be concentrated. The three routine
methods used to concentrate phytoplankton samples are centrifugation and filtration for live
samples and sedimentation (gravitational settling) for preserved samples. Centrifugation and sedimentation
are the most commonly used, although filtration onto membrane filters is an effective
procedure for fluorescence microscopic enumerations. If the cell density is less than 105 cells l-1,
the sample needs to be concentrated; if cell density is too high to allow direct counting,
the sample will be diluted. Dilution or concentration factors need to be taken into account for
calculating the final cell concentration. |
Species containing gas vacuoles (cyanobacteria) are unlikely to fully sediment despite the
Lugol’s fixation. Two methods are available for collapsing vacuoles to assist sedimentation.
Samples can either be exposed to brief ultrasonification (<1 min, but may vary for different
species) or alternatively, pressure can be applied to the sample by forcing it through a syringe
with a fine needle; this will collapse the vacuoles and the cells will then settle through gravity.
If the water sample contains sufficient number of algae and concentration is not required,
a direct count can be undertaken. The sample is thoroughly mixed, treated with a few drops of
Lugol’s solution, mixed again and allowed to stand for 30–60 min. The sample is mixed again
and the subsamples are used for direct counting and taxonomic identification.
When concentration is required, a simple procedure for gravitational settling is to pour a wellmixed
volume of the sample of water containing the microalgae in a measuring cylinder (100 ml),
add Lugol’s solution to it (1% by volume), and either allow the sample to stand overnight or use a
centrifuge so that the cells sink. The iodine in Lugol’s solution not only preserves and stains the
cells but also increases their density. When the column of water appears clear, the top 90 ml
will be gently siphoned off without disturbing the sediment at the base of the cylinder. This
leaves the cells concentrated in the bottom 10 ml. Subsamples of this sedimented fraction can be
used for counting procedure and examined under the microscope for identification, keeping in
mind that the cells of the original sample have been concentrated 10 times. |
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At present, there are many kinds of counting procedures available for enumerating phytoplankton,
depending on whether counting algae in mixtures, as from field sampling, or unialgal samples,
such as in growth or bioassay experiments in a laboratory. Some techniques are relatively “low-
tech,” and can be used also in remote locations, other are “high-tech,” and require an expensive
instrument.
The Sedgewick-Rafter counting chamber (Figure 6.2) is a low tech device routinely used for
counting algae in mixed assemblages. This cell limits the volume and area of the sample to
enable easier counting and calculation of phytoplankton numbers. It consists of a brass or polystyrene
rectangular frame mounted on a heavy glass slide from which a precise internal chamber
has been cut; its dimensions are 50 x 20 x 1 mm
3, with an area of 1000 mm
2, and a volume of
1.0 ml. The base is ruled in a 1 mm grid. When the liquid sample is held in the cell by its large,
rectangular glass cover slips, the grid subdivides 1 ml volume into microliters.
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| FIGURE 6.2 Schematic drawing of the Sedgewick-Rafter counting chamber. |
To fill the Sedgewick-Rafter chamber, the cover slip is placed on top of the chamber, diagonally
across the cell, so that the chamber is only partially covered. This helps to prevent the formation of
air bubbles in the cell corners. The cover slip will rotate slowly and cover the inner portion of the
cell during filling. The cover slip should not float, nor should there be any air bubbles; the former
results from over-filling, the latter from under-filling the chamber. In both cases, the depth of the
chamber will be different from 1 mm, and the calculations will be invalidate. A large-bore
pipette should be used to transfer the sample into the chamber; after filling, the cover slip is
gently pushed to cover the chamber completely. The phytoplankton sample placed into the Sedgwick-
Rafter counting chamber is allowed to stand on a flat surface for 20 min to enable the phytoplankton
to settle. It is then transferred to the stage of an upright light microscope and securely
positioned, ready for counting.
Counts are done with the 4x or (more usually) the 10x objectives of the compound microscope
(depth of field and lens length preclude the use of higher magnification objectives). A Whipple disk
is inserted into one of the ocular lenses in order to provide a sample grid. It is necessary to first
determine the area (A) of the Whipple field for each set of ocular and objective lenses used. This
is accomplished with a stage micrometer.
There are 50 fields in the length and 20 fields in the width of the chamber (comprising a total of
1000 fields). A horizontal strip corresponds to 50 fields. All cells within randomly selected fields are
counted. A convention needs to be followed for cells or units lying on a boundary line or field, such
as all cells or units overlapping the right hand and top boundary are counted, but those overlapping
the bottom and left hand boundary are not. The number of units per milliliter for each taxon is
calculated according to following formula:
where
C is the number of cells counted,
A is the area of field in mm
2,
D is the depth of a field
(Sedgewick-Rafter chamber depth) in mm, and
F is the number of fields counted.
For colonial taxa multiply the count of units by the average number of cells per unit and use the
resulting value as
C in Equation (
6.1). To adjust for sample concentration of dilution the result is
divided or multiplied by the appropriate factor. To obtain total cell density per milliliter, sum all
counts of individual taxa.
If cell density is low (<10 units per field) counting of long transects to cover a large proportion
of the chamber floor is more appropriate. Several transects with a width of a chamber field are
counted. The number of strips depends on the required precision and the phytoplankton density.
The number of cells per millimeter is calculated according to the following formula:
where
C is the number of cells counted,
L is the length of strip in mm,
D is the depth of a field
(Sedgewick-Rafter chamber depth) in mm, W is the width of strip in mm, and
S is the number
of strips counted. To adjust for sample concentration or dilution the result is divided or multiplied
by the appropriate factor.
A “high-tech” alternative to counting algae in mixed assemblages with the Sedgewick-Rafter
cell is the inverted microscope method. The expensive component here is the inverted microscope,
whose great advantage is that settling chamber depth does not preclude the use of high magnification
objective lenses. In 1931, Utermo¨hl solved the problem of concentrating and enumerating
algae in mixed populations when he described a one step settling and enumeration technique
using the inverted microscope. The procedure involved the gravitational sedimentation of preserved
phytoplankton into a counting chamber. This counting technique correctly assumed that phytoplankton
would fall randomly to the bottom of the chamber and that counts would then be made
on random fields or transects. The inverted microscope counting technique has gained broad popularity
for phytoplankton enumeration. One of the advantages of this randomized counting technique
is the capability of calculating error estimates to verify the accuracy of the enumeration. Through
the years, many modified chambers have been designed and used with the inverted microscope.
Special and expensive “Utermo¨hl” chambers can be purchased, but cheaper ones can be constructed
from large cover slips, and plastic syringe barrels. If a long focal-length lens is available,
chambers may be constructed from glass slides.
A measured volume of preserved sample is added to the settling chamber and allowed to settle
for at least an hour. Time periods as long as 24–48 h are preferred, especially if small algae are
present in the sample (these will settle only very slowly). Upon settling, the upper portion of the
chamber is removed and replaced with a glass plate. The sample is then transferred to an inverted
microscope (condenser numerical aperture 0.70; objectives 25 x and 40x; oculars 12.5x) with
phase contrast optics.
The sample is initially enumerated at 500 x using a random fields technique. A minimum of 20
random fields and 200 individual cells are enumerated. Additional fields are counted until the
minimum count is attained. When there is a large number of cells of a particular taxon in a
sample, fewer than 20 random fields are enumerated with a minimum of five random fields examined
for this taxon. Individual cells are enumerated, whether in chains, filaments, or colonies. This
allows for a more accurate estimate of biomass which is determined from the cell densities. Upon
achieving 20 random fields and 200 individual cells, a low magnification scan (25x) of 20 random
fields is used to estimate the rarer, larger forms within the sample.
In the case of unialgal samples (unicells, small colonies, or relatively short filaments) chambers
such as the haemacytometer, the Thoma chamber (Figure 6.3), the Fuchs-Rosenthal or the Burker
chambers are effective and commonly used for estimating the densities of cultures. The
hematocytometer was developed for counting cells in blood samples (now this ismostly done with electronic
particle counters). Most of these counting chamber have delicate, mirrored surfaces that must not
be scratched. Each mirrored surface has a grid etched upon the surface. In the case of Thoma chamber
each grid is composed of 16 fields of 0.2 mm side, separated by three boundary lines (Figure 6.4). These
16 fields are further subdivided into 16 smaller areas each, for a total of 256 counting fields. The
chamber is 0.1 mm deep; hence each grid holds exactly 64 x 10
-3 ml of sample. You have the choice
of counting the algae in the entire grid; counting algae in only one of the 16 squares, then multiplying by
16; or counting algae in an even smaller area and multiplying accordingly.
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| FIGURE 6.3 Schematic drawing of the Thoma counting chamber. |
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| FIGURE 6.4 Schematic drawing of the Thoma chamber ruling. |
When counting cell in the entire grid, that is, 16 fields, the cell concentration is calculated
according to the following formula:
where
C is the number of cells counted.
Counts of about 30 cells per field are desirable for accuracy. If there are more than 30 cells
per field, dilute the sample, or count algae in a lower number of fields and multiply. As for the
Sedgewick-Rafter a convention needs to be followed for cells lying on a boundary line or field,
such as all cells overlapping the right hand and top boundary are counted, but those overlapping
the bottom and left hand boundary are not (Figure 6.5). The counting process has to be repeated
at least ten times to determine an accurate mean.
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| FIGURE 6.5 Schematic drawing of the counting convention: all cells overlapping the right-hand and top boundary are counted (black cells), but those overlapping the bottom and left-hand boundary are not (gray cells). |
“High-tech” methods for counting unialgal samples are the electronic particle counter (e.g.,
Coulter counter) and the digital microscopy. In spite of relatively high cost, an electronic particle
counter is highly recommended for performing growth or bioassay studies that require many counts
and high accuracy. In addition, the instrument will provide particle size/biovolume distributions.
The principle of operation is that particles, suspended in an electrolyte solution, are sized and
counted by passing them through an aperture having a particular path of current flow for a given
length of time. As the particles displace an equal volume of electrolyte in the aperture, they
place resistance in the path of the current, resulting in current and voltage changes. The magnitude
of the change is directly proportional to the volume of the particle; the number of changes per unit
time is proportional to the number of particles in the sample. When opened, the stopcock introduces
vacuum into the system, draws sample through the aperture, and unbalances the mercury in the
manometer. The mercury flows past the “start” contact and resets the counter to zero. When the
stopcock is closed, the mercury starts to return to its balanced position and draws sample
through the aperture. Variously sized aperture tubes are available for use in counting variously
sized particles; the aperture size is chosen to match that of particles.
Digital microscopy necessitates of a microscope equipped with a digital TV camera connected
to a personal computer running dedicated application software for cell recognition and cell counting.
It represents an automatic, reliable, and very fast approach to growth determination.
GROWTH RATE AND GENERATION TIME DETERMINATIONS
Growth curves are prepared from cell density data obtained with a hemocytometer or electronic
particle counter on cultures sampled at intervals, such as once per day, depending on the growth
rate of the alga. Plots of number of cells against time (in days) can be made, and from these
curves can be calculated specific growth rate or growth constant (µ) and division or generation
time (T
g).
A typical growth curve will show a lag phase, an exponential or log phase, and a stationary or
plateau phase, where increase in density has leveled off. In the stationary phase, growth is likely
limited by resources such as light or nutrients. Growth rate (µ) is calculated with the following
equation:
where
N2 and
N1 are number of cells at times t
2 and t
1.
When
N2 is twice
N1, for example, the population has undergone a doubling, the growth rate can
be expressed as
As ln(2) is equal to 0.6931, generation time can be calculated with the following equation:
