such as "Introduction", "Conclusion"..etc
The dynamics of cell divisions was evaluated
cytometrically with a light microscope. The initial cell
density of all the cultures was set to 52x104 cells per
milliliter. The dry algal biomass of the cultures was
determined after 10 days of development of the algal
populations, when the control cultures were at the end
of exponential growth phase. Net photosynthetic
oxygen production of the algal cultures was measured
with an Oxy-Lab oxymeter at a constant light intensity
of 110 ìM photons m-2 s-1 and 20 °C [1, 12].
For determination of lipid peroxidation after 10
days of exposure to herbicides, algal suspensions were
centrifuged at 2500g for 10 minutes and pellets were
weighed. To each pellet 0.1% (w/v) trichloroacetic acid
(TCA) was added in 1:3 (g/ml) ratio. Algae were
disrupted in a Constant Systems cell disrupter, than the
extracts were centrifuged at 6300g for 10 minutes at 10
°C. 0.5% (w/v) 2-thiobarbituric acid (TBA) solution in
20% (w/v) TCA was added to the harvested
supernatants in the ratio 1:4 (v/v), in 10 ml
thermoresistant glass tubes. The extracts were heated
for 25 minutes at 96 °C and, after lowering temperature
on ice, they were centrifuged at 6300g for 10 minutes
at 10 °C. Determination of thiobarbituric acid-reactive
substances (TBARS), consisting mostly in
malondialdehyde, was performed photometrically on
the supernatants, at A532 – A600 nm, using at extinction
coefficient of 159.2 mM-1 cm-1 .
Catalase activity was evaluated spectrophotometrically
by determining the consumption of H2O2
associated with a change in absorbance at 240 nm.
Algal cultures were centrifuged at 2500g for 10
minutes. The testing medium contained 750 ìl of
sodium phosphate buffer (50 mM, pH 7.5), 100 ìl
H2O2 (200 mM), and 150 ìl of algal extract with
enzyme (5 ìg of protein) in a final volume of 1 ml.
Proteins were extracted with 1.5 ml of 0.1 M sodium
phosphate buffer (pH 7) and the extract was
centrifuged at 2300g for 20 minutes at 5 °C .
Ascorbic acid content of the algal cells was
determined titrimetrically. 25 ml algal suspension was
centrifuged at 2500g for 10 minutes, the pellet was
resuspended in 10 ml of 5% (v/v) metaphosphoric acid,
filtered and completed with 20 ml of 5%
metaphosphoric acid. This extract was titrated with
0.025% (w/v) 2,6-dichlorophenol indophenol until a
persistent light pink color appeared. Ascorbic acid
content was determined with the help of a standard
curve obtained with titration of known concentrations
of ascorbic acid .
Every measurement was repeated 5 times. The
experimental data were evaluated statistically, the
significance of the differences among the control and
the treated cultures was tested using one-way ANOVA
(after verification of variance homogeneity with the
Levene test), followed by the multiple comparison
Tukey test (P < 0.05) .
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