Composition and Biomass of Phytoplankton
151
FILTRATION ONTO MEMBRANE FILTERS
A number of methods of similar principle have been devised to concentrate
phytoplankton onto membrane filters. The filter material then is cleared by various
means and the filter can be mounted permanently on a microscope slide. The
advantages of this method are that high magnification can be used and the samples
are permanent. Severe disadvantages exist, however. By applying pressure or a
vacuum, even at low pressure differentials of 0.3 atm, which is necessary to pass water
through membranes of a pore size of 0.5 ,um, severe rupture and deformation of small,
delicate algae often occur. Flagella are almost always lost as microflagellates strike
the filter surface. Certain algae with heavy cell wall structures, such as diatoms, are
preserved relatively well. When using this method, examine separate samples by other
techniques as well.
Procedures
Well-mixed, preserved water samples of known volume should be passed through
gridded, white membrane filters of a pore size of ca. 0.5,um (e.g., Millipore HA,
gridded, 0.45-,um pore size). Vacuum of less than 0.5 atm should be applied to the
filtration system.
Labeled microscope slides are prepared, and two or three drops of microscope
immersion oil are placed onto each slide. The gridded filters are removed from the
filtration apparatus while they are still wet and placed directly onto the small pool
of immersion oil. A small ring weight of sufficient diameter to hold down the edges
of the filter but not to come into contact with the algae is then placed carefully on
the filter. Add two drops of oil on the top of the filter. Under normal conditions of
humidity, the oil will displace the water and render the filter transparent within 12
to 24 h at room temperature. A mounting medium of good refractive index (e.g.,
Fisher Permount) then can be applied, and the cleared filter is covered with a
coverslip. A number of modifications, particularly in relation to staining techniques,
are discussed in McNabb (1960), Holmes (1962), Moore (1963), Clark and Sigler
(1963), and deNoyelles (1968).
Enumeration of membrane filters is confounded by the distribution of the algae.
The distribution of organisms on the filter is nonrandom, since there is a tendency
for algae to be concentrated near the edges of the filters (Holmes, 1962). Therefore
it is recommended that the entire filter be counted or at least on strips covering the
entire diameter of the filter. Knowing the size of the area examined, the total area
of the circle onto which the algae were filtered (A = nr2), and the volume of water
filtered, it is simple to convert to number of organisms per milliliter.
OTHER CONCENTRATION METHODS
Centrifugation
Concentration by centrifugation is not recommended as a quantitative technique to
collect algae from water. Removal is not complete at normal centrifugal speeds,
damage to many algae is great, and it is difficult to transfer algae quantitatively from
the centrifugation apparatus to counting chambers.
151
FILTRATION ONTO MEMBRANE FILTERS
A number of methods of similar principle have been devised to concentrate
phytoplankton onto membrane filters. The filter material then is cleared by various
means and the filter can be mounted permanently on a microscope slide. The
advantages of this method are that high magnification can be used and the samples
are permanent. Severe disadvantages exist, however. By applying pressure or a
vacuum, even at low pressure differentials of 0.3 atm, which is necessary to pass water
through membranes of a pore size of 0.5 ,um, severe rupture and deformation of small,
delicate algae often occur. Flagella are almost always lost as microflagellates strike
the filter surface. Certain algae with heavy cell wall structures, such as diatoms, are
preserved relatively well. When using this method, examine separate samples by other
techniques as well.
Procedures
Well-mixed, preserved water samples of known volume should be passed through
gridded, white membrane filters of a pore size of ca. 0.5,um (e.g., Millipore HA,
gridded, 0.45-,um pore size). Vacuum of less than 0.5 atm should be applied to the
filtration system.
Labeled microscope slides are prepared, and two or three drops of microscope
immersion oil are placed onto each slide. The gridded filters are removed from the
filtration apparatus while they are still wet and placed directly onto the small pool
of immersion oil. A small ring weight of sufficient diameter to hold down the edges
of the filter but not to come into contact with the algae is then placed carefully on
the filter. Add two drops of oil on the top of the filter. Under normal conditions of
humidity, the oil will displace the water and render the filter transparent within 12
to 24 h at room temperature. A mounting medium of good refractive index (e.g.,
Fisher Permount) then can be applied, and the cleared filter is covered with a
coverslip. A number of modifications, particularly in relation to staining techniques,
are discussed in McNabb (1960), Holmes (1962), Moore (1963), Clark and Sigler
(1963), and deNoyelles (1968).
Enumeration of membrane filters is confounded by the distribution of the algae.
The distribution of organisms on the filter is nonrandom, since there is a tendency
for algae to be concentrated near the edges of the filters (Holmes, 1962). Therefore
it is recommended that the entire filter be counted or at least on strips covering the
entire diameter of the filter. Knowing the size of the area examined, the total area
of the circle onto which the algae were filtered (A = nr2), and the volume of water
filtered, it is simple to convert to number of organisms per milliliter.
OTHER CONCENTRATION METHODS
Centrifugation
Concentration by centrifugation is not recommended as a quantitative technique to
collect algae from water. Removal is not complete at normal centrifugal speeds,
damage to many algae is great, and it is difficult to transfer algae quantitatively from
the centrifugation apparatus to counting chambers.