Efficacy of a dual fluorescence method in detecting the viability of overwintering cyanobacteria

Abstract

Significance and Impact of the Study: This study established the efficacy of the dual fluorescence method in evaluating the viability of cyanobacteria under chill-light stress. The results provided the direct evidence for acquired chill-light tolerance and the viability of overwintering Microcystis colonies. Such information can be useful in prediction of cyanobacterial blooms. AbstractChill in the light is the major environmental stress that cyanobacteria encounter in winter. Cyanobacterial cells may acquire chill-light tolerance upon exposure to low temperature in autumn and early winter. We sought to establish the efficacy of the dual fluorescence method in detecting the viability of overwintering cyanobacteria and to provide further evidence for the chilllight tolerance of preconditioned cyanobacteria. Synechocystis sp. PCC 6803 and Microcystis aeruginosa PCC 7806 were exposed to chill (5°C)-light stress with or without pretreatment at 15°C and stained with SYTO 9 and propidium iodide. Live and dead cells were observed under a fluorescence microscope, and the percentage of viable cells was quantified on a microplate reader. The dual fluorescence method showed consistent results with tests of the ability to reinitiate growth. Cell viability was quantitatively correlated with ratio of SYTO 9/propidium iodide fluorescence. Previously, Microcystis colonies in Lake Taihu had been found to accumulate RNA-binding protein 1 in autumn and winter. Use of this method directly showed the viability of such Microcystis colonies throughout the winter.

Role of Spermidine in Overwintering of Cyanobacteria

“…Studies using Synechocystis sp. strain PCC 6803 (Synechocystis 6803) showed that preconditioning at a low temperature, such as 15°C, could greatly enhance its survivability under long-term chill (5°C)-light stress (7,8). This finding suggests that cyanobacteria acquire the capability to overwinter in late autumn and early winter.…”

“…In previous reports, we showed that ␣-tocopherol, which is membrane localized, is essential for the acquired chill-light tolerance (ACLT) of cyanobacteria (7) and that accumulation of RNAbinding protein 1 (Rbp1) leads to the formation of overwintering capability of cyanobacteria (8,12). Nonetheless, information about the biology of overwintering cyanobacteria is still very limited.…”

“…Studies on how cyanobacteria adapt to low temperatures can be roughly divided into two categories: (i) cold acclimation of cells exposed to a suboptimal growth temperature, such as 22 to 24°C (9,10) or 15°C (11) and (ii) survival under long-term (8 to 10 days) chill (5°C)-light stress as a simulation of overwintering (7,8,12). In previous reports, we showed that ␣-tocopherol, which is membrane localized, is essential for the acquired chill-light tolerance (ACLT) of cyanobacteria (7) and that accumulation of RNAbinding protein 1 (Rbp1) leads to the formation of overwintering capability of cyanobacteria (8,12).…”

Increasing the tolerance of filamentous cyanobacteria to next-generation biofuels via directed evolution

“…Typically, SYTO® 9 is used in conjunction with propidium iodide (PI) as a dual-stain assay in which SYTO® 9 causes viable cells to fluoresce in the green spectrum and PI causes non-viable cells to fluoresce in the red spectrum. This assay has previously been used in unicellular cyanobacteria [89], however our research group determined that PI binds to both viable and non-viable cells in filamentous cyanobacteria and cannot accurately quantitate non-viable cells in the strains used in this study [36]. We later determined that SYTO® 9 does stain viable filamentous cyanobacteria cells as expected and was used to monitor growth in this study [38].…”

Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles

“…Fluorescence microscopy was carried out using a Zeiss AxioImager Z1 microscope following staining of samples with Cyto9 and propidium iodide (Invitrogen), using FITC (490/530 Ex/Em) and TRITC (547/572 Ex/Em) filters. Samples were prepared by mixing 1 ml of the suspension with 3μl of the combined (premixed) stain in sterile Eppendorf tubes and incubating for 15 minutes (Zhu and Xu, 2013). Stained suspensions were centrifuged for 10 minutes and rinsed three times in 10 mM NaCl in order to remove the stain and hence avoid artefacts due to cell death post-staining.…”