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

Water quality may vary greatly with time and
location in lakes, impoundments and streams. If
meaningful data are to be obtained, therefore,
the sampling program must take these variations
into account.
2.3 Apparatus and Test Conditions
2.3.1 Glassware
Use good-quality borosilicate glassware. When
studing trace nutrients, use special glassware
such as Vycor or polycarbonate containers.
Although container size is not critical, the surface
to volume ratios are critical because of
possible carbon limitation. The recommended
sample volumes for use in Erlenmeyer flasks are:
40 ml in a 125 ml flask; 60 ml in a 250 ml flask;
and 100 ml in a 500 ml flask. Use culture
closures such as loose-fitting aluminum foil or
inverted beakers to permit good gas exchange
and prevent contamination.
2.3.2 Illuflnination
After inoculation, incubate the flasks at 24 ±
2°C under cool-white fluorescent lighting: 200
ft-c (2152 lux) ± 10 percent for blue-green algae
and diatom test species, and 400 ft-c (4304 lux)
± 10 percent for green algae test species. Measure
the light intensity adjacent to the flask at
the liquid level.
2.3.3 pH
To ensure the availability of carbon dioxide,
maintain the pH of the incubating cultures
below 8.5 by using the sample volumes mentioned
above and shaking the cultures at 100
oscillations per minute. In samples containing
high concentrations of nutrients, such as highlyproductive
surface waters or domestic waste
effluents, it may be necessary to bubble air or an
air/carbon dioxide mixture through the culture
to maintain the pH below 8.5.
2.4 Sample Preparation
Two alternate methods of sample preparation
are recommended, depending upon the type of
information to be obtained from the sample:
• membrane filtration (0.45 pore diameter) ­
remove the indigenous algae by filtration if
3
Prepare test flasks in triplicate.
ALGAL ASSAY
you wish to determine the growth response
to growth-limiting nutrients which have not
been taken up by filterable organisms, or if
you wish to predict the effect of adding
nutrients to a test water at a specific time.
• autoclaving -- autoclave samples if you wish
to determine the amount of algal biomass
that can be grown from all nutrients in the
water, including those in the plankton.
Autoclaving solubilizes the nutrients in the
indigenous filterable organisms and releases
them for use by the test organisms.
2.5 Inoculum
The algal test species may be one of those
recommended in the Bottle Test or another that
has been obtained in unialgal culture. Grow the
test species in a culture medium that minimizes
the intracellular carryover of nutrients in the
test species when transferred from the stock
culture to the test water (Table I.) When taken
from the stock culture, centrifuge the test cells
and discard the supernatant. Resuspend the
sedimented cells in an appropriate volume of
glass-distilled water containing 15 mg sodium
bicarbonate per liter and recentrifuge. Decant
the supernatant, resuspend the algae in fresh
bicarbonate solution, and use as the inoculum.
The amount of inoculum depends upon the algal
test species used. The following initial cell concentrations
are recommended:
Test organism
Selenastrum capricornutum
Anabaena [los-aquae
Microcystis aeruginosa
lnitial cell count!ml
IOOO/rol
SOOOO/ml
SOOOO/rol
2.6 Growth Response Measurements
The method used to determine growth response
during incubation depends on the
equipment available. Cells may be counted with
a microscope, using a hemacytometer or a
Palmer-Maloney or Sedgwick-Rafter plankton
counting chamber. The amount of algal biomass
may be determined by measuring the optical
density of the culture at 600 -750 nm with a
colorimeter or spectrophotometer. The amount
of chlorophyll contained in the algae may be