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

BIOLOGICAL METHODS
for the Petroff-Hausser bacterial counting cell is
based on the volume over the entire grid. The
dimensions are 1 mm X 1 mm Xl/50 mm.
which gives a volume of 1/50 mm 3 and a factor
of 50,000.
Carefully follow the manufacturer's instructions
that come with the chamber when
purchased. Do not attempt routine counts until
experienced in its use and the statistical validity
of the results are satisfactory. The primary
disadvantage of this type of counting cell is the
extremely limited capacity, which results in a
large multiplication factor. Densities as high as
50,000 cells/ml are seldom found in natural
waters except during blooms. Such populations
may be found in sewage stabilization ponds or in
laboratory cultures.
For statistical purposes, a normal sample must
be either concentrated or a large number of
mounts per sample should be examined.
Membrane Filter
A special filtration apparatus and vacuum
source are required, and a 1-inch, 0.45tL membrane
filter is used.
Pass a known volume of the water sample
through the membrane filter under a vacuum of
0.5 atmospheres. (Note: in coastal and marine
waters, rinse with distilled water to remove salt.)
Allow the filter to dry at room temperature for
5 minutes, and place it on top of two drops of
immersion oil on a microscope slide. Place two
drops of oil on top of the filter and allow it to
dry clear (approximately 48 hours) at room
temperature, cover with a cover slip, and
enumerate the organisms. The occurrence of
each species in 30 random fields is recorded.
Experience is required to determine the
proper amount of water to be filtered. Significant
amounts of suspended matter may obscure
or crush the organisms.
Calculate the original concentration in the
sample as a function of a conversion factor
obtained from a prepared table, the number of
quadrates or fields per filter, the amount of
sample filtered, and the dilution factor. (See
Standard Methods, 13th Edition.)
10
Inverted Microscope
This instrument differs from the conventional
microscope in that the objectives are mounted
below the stage and the illumination comes from
above. This design allows cylindrical counting
chambers (which may also be sedimentation
tubes) with thin clear glass bottoms to be placed
on the stage and sedimented plankton to be
examined from below, and it permits the use of
short focus, high-magnification objectives
including oil immersion. A wide range of concentrations
is automatically obtained by merely
altering the height of the chamber. Chambers
can be easily and inexpensively made: use tubular
Plexiglas for large capacity chambers, and
flat, plastic plates of various thick.'1esses, which
have been carefully bored out to the desired
dimension, for smaller chambers; then cement a
No. 1 or No. 1-1/2 cover slip to form the cell
bottom. Precision-made, all-glass counting
chambers in a wide variety of dimensions are
also available. The counting technique differs
little from the S-R procedure, and either the
strip or separate field counts can be used. The
Whipple eyepiece micrometer is also used.
Transfer a sample into the desired counting
chamber (pour with the large chambers, or pipet
with 2-ml or smaller chambers), fill to the point
of overflow, and apply a glass cover slip. Set the
chamber aside and keep at room temperature
until sedimentation is complete. On the average,
allow 4 hours per 10 mm of height. After a suitable
period of settling, place the chamber on the
microscope stage and examine with the use of
the 20X, 45X, or 100X oil immersion lens.
Count at least two strips perpendicular to each
other over the bottom of the chamber and average
the values. Alternatively, random field
counts can be made; the number depends on the
density of organisms found. As a general rule,
count a minimum of 100 of the most abundant
species. At higher magnification, count more
fields than under lower power.
When a 25.2 mm diameter counting chamber
is used (the most convenient size), the conversion