Growth and Cell Death Kinetics
Cell growth was estimated by measuring optical density at 750 nm
(OD750) and chlorophyll concentration in algal cultures
over time (Possmayer et al. 2011). The concentrations of
chlorophyll a and b were measured spectrophotometrically
at 647 and 664 nm (Cary 50 Bio; Varian, USA) and calculated as described
(Jeffrey & Humphrey 1975). Maximum growth rates were calculated using
natural log transformation of the optical density values during the
exponential phase. Culture viability was assayed by resuspending
pelleted algal cells in 0.5% (w/v) Evans Blue solution, incubating for
30 min and removing the unbound dye by washing with BBM medium. Dye
bound to dead cells was solubilized in 50% (v/v) methanol and 1% (w/v)
SDS, and extracted by incubation at 50°C for 30 min. The suspension was
centrifuged (16,000g, 3 min) and absorbance was measured
spectrophotometrically at 600 nm. The absorbance of cells treated with
1% (v/v) chloroform (100% death) were equivalent to values obtained
after prolonged exposure to heat. Light microscopy was carried out using
a Zeiss Axioimager Z1 Microscope (Carl Zeiss AG, Germany) at the
Integrated Microscopy Facility, The Biotron, Western University.