Growth and Cell Death Kinetics
Cell growth was estimated by determining chlorophyll concentration in algal cultures over time, as previously described (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). The maximal specific growth rate (μmax) is the highest value of μ calculated according to the equation:
μ = ln(Δ[chl])/Δthours
where Δ[chl] is the ratio of the chlorophyll between the two sampling times, and Δthours is the time elapsed.
The kinetics of cell death were determined in two ways. First, the loss of chlorophyll due to exposure to heat was measured as described above. Second, culture viability was assayed by resuspending pelleted algal cells in 0.5% (w/v) Evans Blue solution. Cells were incubated for 30 min and the unbound dye was removed by extensive washing with BBM medium. The 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) to remove insoluble particles, 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. Images of algal cells were taken for observing cell morphology and chlorophyll loss due to heat stress. All images were processed using Image-Pro Premier 9.1 (Media Cybernetics, USA).