Composition and Biomass of Phytoplankton
143
without entrapping any air bubbles. In order to ensure complete sedimentation of
all organisms, sedimentation time in hours must be at least three times the height of
the sedimentation chamber in centimeters. Place the chamber in a vibration-free
area during the sedimentation process. Where wide fluctuations in room temperature
occur, the chambers should be covered (e.g., with an inverted styrofoam box) during
sedimentation in order to avoid convection currents.
Inverted Microscope. The basic difference between an erect and an inverted
microscope is that, in the latter, the positions of the objective and condenser are
reversed in relation to the stage (Fig. 10.1). The inverted design allows for observation
through the bottom of containers with bright-field, dark-field, phase-contrast,
and fluorescent procedures. Use of this microscope is limited by the thickness of
the bottom cover glass and by the working distance of the condenser, which must be
long enough to allow the chamber to rest between the stage and the condenser across
the optical axis.
Because of these limitations in resolution, it is difficult to use the sedimentation chamber-inverted microscope at magnifications much greater than 500 to 600 x .
With special chambers, discussed below, and ultrathin coverslips, the resolution of
the inverted microscope can be extended to include organisms smaller than 10 ,um.
The sedimentation chambers are placed carefully on the stage of the microscope;
great care is taken not to disturb the sedimented organisms. Identification, counting,
and size measurements then are made according to procedures outlined below.
OTHER MODIFIED SEDIMENTATION CHAMBERS FOR USE WITH
A COMPOUND MICROSCOPE
A number of sedimentation chambers have been designed that permit removal of the
vertical cylinder after setting of the algae. The resulting base containing the sedimented
algae in a very small, flat depression chamber is covered with a coverslip. The algae
in this chamber can be viewed on either a conventional microscope or an inverted
microscope.
The simplest compound chamber [after Lund (1951) and Utermohl (1958)] has a
base unit that contains a shallow cylindrical chamber of a volume of about 2 cm 3
and a height of about 4 mm (Fig. 10.2). The top and bottom sides of the base unit
are ground flat. The lower surface of the base plate is coated lightly with
water-insoluble grease (e.g., Apiezon M stopcock grease) and accepts a large, thin
coverslip. The upper tubular tubular chamber is open below and is glued permanently
to a square base of the same size as the base chamber. The lower surface of the base
plate of the tubular chamber is ground flat.
During use, the upper chamber, lightly coated with grease at the base, is centered
precisely over the hole of the lower chamber. After the algae have sedimented, the
upper chamber is slid laterally onto a square of plastic of identical thickness as the
base chamber, while the exposed upper surface of the base chamber simultaneously
is covered with a coverglass. In this manner, the water of the upper chamber is
removed and that of the lower chamber is intact and covered. The sedimented algae
can be counted with an inverted microscope or a compound microscope at low to
moderate magnification.
An excellent modification of this compound chamber has been devised to permit
permanent mounting of the slide (Coulon and Alexander, 1972). These slides then
143
without entrapping any air bubbles. In order to ensure complete sedimentation of
all organisms, sedimentation time in hours must be at least three times the height of
the sedimentation chamber in centimeters. Place the chamber in a vibration-free
area during the sedimentation process. Where wide fluctuations in room temperature
occur, the chambers should be covered (e.g., with an inverted styrofoam box) during
sedimentation in order to avoid convection currents.
Inverted Microscope. The basic difference between an erect and an inverted
microscope is that, in the latter, the positions of the objective and condenser are
reversed in relation to the stage (Fig. 10.1). The inverted design allows for observation
through the bottom of containers with bright-field, dark-field, phase-contrast,
and fluorescent procedures. Use of this microscope is limited by the thickness of
the bottom cover glass and by the working distance of the condenser, which must be
long enough to allow the chamber to rest between the stage and the condenser across
the optical axis.
Because of these limitations in resolution, it is difficult to use the sedimentation chamber-inverted microscope at magnifications much greater than 500 to 600 x .
With special chambers, discussed below, and ultrathin coverslips, the resolution of
the inverted microscope can be extended to include organisms smaller than 10 ,um.
The sedimentation chambers are placed carefully on the stage of the microscope;
great care is taken not to disturb the sedimented organisms. Identification, counting,
and size measurements then are made according to procedures outlined below.
OTHER MODIFIED SEDIMENTATION CHAMBERS FOR USE WITH
A COMPOUND MICROSCOPE
A number of sedimentation chambers have been designed that permit removal of the
vertical cylinder after setting of the algae. The resulting base containing the sedimented
algae in a very small, flat depression chamber is covered with a coverslip. The algae
in this chamber can be viewed on either a conventional microscope or an inverted
microscope.
The simplest compound chamber [after Lund (1951) and Utermohl (1958)] has a
base unit that contains a shallow cylindrical chamber of a volume of about 2 cm 3
and a height of about 4 mm (Fig. 10.2). The top and bottom sides of the base unit
are ground flat. The lower surface of the base plate is coated lightly with
water-insoluble grease (e.g., Apiezon M stopcock grease) and accepts a large, thin
coverslip. The upper tubular tubular chamber is open below and is glued permanently
to a square base of the same size as the base chamber. The lower surface of the base
plate of the tubular chamber is ground flat.
During use, the upper chamber, lightly coated with grease at the base, is centered
precisely over the hole of the lower chamber. After the algae have sedimented, the
upper chamber is slid laterally onto a square of plastic of identical thickness as the
base chamber, while the exposed upper surface of the base chamber simultaneously
is covered with a coverglass. In this manner, the water of the upper chamber is
removed and that of the lower chamber is intact and covered. The sedimented algae
can be counted with an inverted microscope or a compound microscope at low to
moderate magnification.
An excellent modification of this compound chamber has been devised to permit
permanent mounting of the slide (Coulon and Alexander, 1972). These slides then