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.