Composition and Biomass of Phytoplankton
147
outlined in Fig. 10.6. These measurements apply only to the specific combination of
oculars and objectives on the microscope for which they were determined.
Alternatively, a series of parallel and perpendicular cross hairs, made of molten
glass drawn to very fine threads, can be affixed permanently to the internal flange
within the ocular [see Lund et al. (1958) for details] or a Porton sizing reticle [see
Brock (1983)]. Calibration procedures would be the same as discussed for the Whipple
reticule.
COUNTING AND SIZE DETERMINATIONS
Counting procedures are similar whether sedimentation chambers with an inverted
microscope or slides or counting cells such as those discussed below are used. Before
counting, a portion of the edges and central areas of the chambers or slides should
be examined to ensure that definition is good and distribution of the organisms is
relatively uniform.
By moving the mechanical stage, the entire bottom of the sedimentation chamber,
counting cell, or slide area can be examined in a systematic manner. Organisms lying
between two parallel cross hairs are counted as they pass a vertical line. Proceed
from left to right from edge to edge; move down to the next full field within the
horizontal parallel lines, and then, in the next row, proceed from the right edge to
the left. Only part of the optical field is covered by the first and the last traverses
because of its circular nature. When organisms lie across a horizontal line, those
along the upper one are counted as lying within the lined area, but those lying across
the lower are not counted. When the next lower area is counted, those along
the previous lower line will overlap with the upper one and be counted.
Often time constraints prohibit counting of all organisms of the entire area of the
tube bottom, cell, or slide when the organisms are dense. In this case, portions of the
total are counted such as several diagonal rectangular strips near the center of the
field from one edge to the other. Or, when the organisms are particularly dense,
diagonals may be counted over specific distance, e.g., 1.0 cm, as determined by stage
calibrations and mechanical stops on the stage of the microscope. Knowing the
surface area of that portion counted in relation to that of the total, a factor can be
determined to expand the average counts of several replicates to the total area of
the counting surface. This total area represents the number of organisms per given
volume of sample; this volume then can be expanded by an appropriate factor to
yield the organisms per liter of water from the given depth of the lake.
Use of phase contrast optics is recommended. Phase contrast takes advantage of
slight differences in refractive indices between the water and the cell and increases their
contrast. Definition of refractile vacuoles, nuclei, eyespots, flagella, and other cellular
components is enhanced, and thus their identification is made more certain.
Sources of Error
The main sources of error in enumerating organisms include those associated with
sampling and counting techniques. A particular problem arises as a result of counting
colonies and then multiplying them by a mean number of cells per colony to derive
an estimate of cell number (Lund et ai., 1958). Many studies are concerned with
generations of populations, or changes in abundance. In such studies, a method that
estimates abundance to an accuracy of ± 50% of the true value often is adequate, as
147
outlined in Fig. 10.6. These measurements apply only to the specific combination of
oculars and objectives on the microscope for which they were determined.
Alternatively, a series of parallel and perpendicular cross hairs, made of molten
glass drawn to very fine threads, can be affixed permanently to the internal flange
within the ocular [see Lund et al. (1958) for details] or a Porton sizing reticle [see
Brock (1983)]. Calibration procedures would be the same as discussed for the Whipple
reticule.
COUNTING AND SIZE DETERMINATIONS
Counting procedures are similar whether sedimentation chambers with an inverted
microscope or slides or counting cells such as those discussed below are used. Before
counting, a portion of the edges and central areas of the chambers or slides should
be examined to ensure that definition is good and distribution of the organisms is
relatively uniform.
By moving the mechanical stage, the entire bottom of the sedimentation chamber,
counting cell, or slide area can be examined in a systematic manner. Organisms lying
between two parallel cross hairs are counted as they pass a vertical line. Proceed
from left to right from edge to edge; move down to the next full field within the
horizontal parallel lines, and then, in the next row, proceed from the right edge to
the left. Only part of the optical field is covered by the first and the last traverses
because of its circular nature. When organisms lie across a horizontal line, those
along the upper one are counted as lying within the lined area, but those lying across
the lower are not counted. When the next lower area is counted, those along
the previous lower line will overlap with the upper one and be counted.
Often time constraints prohibit counting of all organisms of the entire area of the
tube bottom, cell, or slide when the organisms are dense. In this case, portions of the
total are counted such as several diagonal rectangular strips near the center of the
field from one edge to the other. Or, when the organisms are particularly dense,
diagonals may be counted over specific distance, e.g., 1.0 cm, as determined by stage
calibrations and mechanical stops on the stage of the microscope. Knowing the
surface area of that portion counted in relation to that of the total, a factor can be
determined to expand the average counts of several replicates to the total area of
the counting surface. This total area represents the number of organisms per given
volume of sample; this volume then can be expanded by an appropriate factor to
yield the organisms per liter of water from the given depth of the lake.
Use of phase contrast optics is recommended. Phase contrast takes advantage of
slight differences in refractive indices between the water and the cell and increases their
contrast. Definition of refractile vacuoles, nuclei, eyespots, flagella, and other cellular
components is enhanced, and thus their identification is made more certain.
Sources of Error
The main sources of error in enumerating organisms include those associated with
sampling and counting techniques. A particular problem arises as a result of counting
colonies and then multiplying them by a mean number of cells per colony to derive
an estimate of cell number (Lund et ai., 1958). Many studies are concerned with
generations of populations, or changes in abundance. In such studies, a method that
estimates abundance to an accuracy of ± 50% of the true value often is adequate, as