Biological field and laboratory methods for measuring the quality of ...

of counts to numbers per ml is quite simple:
. ex 1000 mm 3
No. per ml (stnp count) = L X W X D X S
where:
C = number of organisms counted (tally)
L = length of a strip, mm
W= width of a strip (Whipple grid image
width), mm
D = depth of chamber, mm
S = number of strips counted
ex 1000 mm 3
No. per ml (field count) = A X D X F
where:
C = number of organisms counted (tally)
A = area of a field (Whipple grid image area),
mm 2
D = depth of chamber, mm
F = number of fields counted
Diatom Analysis
Study objectives often require specific identification
of diatoms and information about the
relative abundance of each species. Since the
taxonomy of this group is based on frustule
characteristics, low-power magnification is
seldom sufficient, and permanent diatom
mounts are prepared and examined under oil
immersion.
To concentrate the diatoms, centrifuge 100
ml of thoroughly mixed sample for 20 minutes
at 1000 X g and decant the supernatant with a
suction tube. Pour the concentrated sample into
a disposable vial, and allow to stand at least 24
hours before further processing. Remove the
supernatant water from the vial with a suction
tube. If the water contains more than I gm of
dissolved solids per liter (as in the case of
brackish water or marine samples), salt crystals
form when the sample dries and obscure the
diatoms on the finished slides. In this case,
reduce the concentration of salts by refilling the
vial with distilled water, resuspending the plankton,
and allowing the vial to stand 24 hours
before removing the supernatant liquid. Repeat
the dilution several times if necessary.
If the plankton counts are less than 1000 per
ml, concentrate the diatoms from a larger
volume of sample (1 to 5 liters) by allowing
them to settle out. Exercise caution in using this
11
PLANKTON COUNTING
method, however, to ensure quantitative
removal of cells smaller than 10 microns in
diameter.
Thoroughly mix the plankton concentrate in a
vial with a disposable pipet, and deliver several
drops to a No. I, circular 18-mm coverglass. Dry
the samples on a hotplate at 95°C. (Caution:
overheating may cause splattering and crosscontamination
of samples.) When dry. examine
the coverglasses to determine if there is su fficient
material for a diatom count. if not, repeat
the previous steps one or two more times,
depending upon the density of the sedimented
sample. Then heat the samples on a heavy-duty
hotplate 30 minutes at approximately 570°C to
drive off all organic matter. Remove grains of
sand or other large objects on the cover glass
with a dissection needle. The oil immersion
objective has a very small working distance, and
the slide may be unusable if this is not done.
Label the frosted end of a 25- X 75-mm
microscope slide with the sample identification.
Place the labelled slide on a moderately warm
hotplate (l57°C), put a drop of Hyrax or
Aroc1or 5442 (melt and use at about 138°C)
mounting medium (Index of Refraction
1.66-1.82) at the center, and heat the slide until
the solvent (xylene or toluene) has evaporated
(the solvent is gone when the Hyrax becomes
hard and brittle upon cooling).
While the coverglass and slide are still hot,
grasp the coverglass with a tweezer, invert. and
place on the drop of Hyrax on a slide. It may be
necessary to add Hyrax at the margin of the
coverglass. Some additional bubbles of solvent
vapor may appear under the coverglass when it is
placed on the slide. When the bubbling ceases,
remove the slide from the hotplate and place on
a firm, flat surface. Immediately apply slight
pressure to the coverglass with a pencil eraser (or
similar object), and maintain until the Hyrax
cools and hardens (about 5 seconds). Spray a
protective coating of clear lacquer on the frosted
end of the slide, and scrape the excess Hyrax
from around the coverglass.
Identify and count the diatoms at high
magnification under oil. Examine random lateral
strips the width of the Whipple grid, and identify
and count all diatoms within the borders of
the grid until 250 cells (500 halves) are tallied.