Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria)

Abstract

J. Li and L. Song. 2007. Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria). Phycologia 46: 593–599. DOI: 10.2216/07-11.1

Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions.

Key Words::

• Algae
• Oxidoreductase
• Microcystis
• MTT
• Viability assessment

ACKNOWLEDGMENTS

This research was supported by the Chinese Academy of Sciences Project (KSCX2-YW-426) and National Natural Science Foundation of China(39870091) and National Key Project for Basic Research (2002CB412306). We thank Ms Jing Zhang for useful help on FDA assay using flow cytometry. We also thank Dr Philip Orr and two anonymous referees for critical comments and suggestions.