Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata.
Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5 μmol/l SYTOX Green for 40 min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6 h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617 mg/l 1-pentanol (95 % confidence limits 4,670-9,327) and 12,818 mg/l 1-pentanol (95 % confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.
Bioengineering Laboratory, Chemical Engineering Department, Superior Institute of Engineering of Porto Polytechnic Institute, Rua Dr António Bernardino de Almeida 431, Porto, Portugal.
Manuela D
MD
Machado
Eduardo V
EV
Soares
D002462:Cell Membrane / Q000378:metabolism; D000460:Chlorophyta / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results