Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality

Abstract

UV] irradiation, heat, and cavitation) treatment technologies (Shannon et al. 2008, Werschkun et al. 2012, 2014). Traditionally, water analysis used to assess most treatment technologies has depended on cultivation, such as the plate count or the most probable number (MPN) technique. Cultivation methods measure viability of organisms present, i.e. the ability of a cell to reproduce. Vital (live) cells can be either viable or non-viable, whereas non-vital (dead)

Effects on inactivation of Tetraselmis suecica following treatment by KBAL: a UV-based ballast water treatment system with an in-line vacuum drop

“…a Analysis of the lab-culture before added to the KBAL test rig, 10,000 cells were analyzed. b Analysis of 1 ml seawater sample (dual stained) shows that most other organisms and/or particles were excluded from the gate The FCM signals for sample 3 (test water treated once, incubated in the lab) followed a pattern as previously described [26]. The cells were in Q1 (live) at day 0 with some cells in Q2 (severely damaged), and the population in Q2 increased during days 1 and 2 (Fig.…”

“…3), displaying membrane permeability and esterase activity, respectively. These dot plots were separated into 4 quadrants (Q) by a vertical and a horizontal line based on the green and blue fluorescence intensity from the cells, as described previously [26]. The quadrants reflect the physiological cellular characteristics of esterase activity and membrane permeability, and thus vitality.…”

“…To get an indication on effects caused by the P/V technology, the results of T. suecica cells treated at the KBAL test rig were compared with previous results from T. suecica cells treated with UV irradiation only. Dot plot analyses from days 0, 1, 2 and 7 of seawater (sample 1) and test water treated once at the KBAL test rig (sample 3) were compared to a sample irradiated with an MP UV lamp at 100 mJ cm −1 [26] (Fig. 5).…”

“…352360, BD, USA) to prevent large organisms and/or particles from clogging the tubing of the FCM instrument. All samples were dual stained with the esterase substrate CFDA-AM (C1354, Thermo Fisher Scientific, USA) and SYTOX Blue Dead Cell Stain (S34857, Thermo Fisher Scientific, USA), as described previously [26]. 4 ml of each sample was analyzed at a flow rate of 1000 µl min −1 and the sample dispensed options were set at standard collection mode.…”

“…Several studies have previously argued that Tetraselmis is well suited as a standard test organism for validation of UVbased systems, since they are more or at least as resistant to LP and MP UV treatments as other naturally occurring seawater organisms, and do not generate a positive or negative bias to any technology [24,25]. We used a dual staining flow cytometry (FCM) protocol with 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue [26], plate count analysis, and a most probable number (MPN) analysis to analyze the effect of the treatments. We aimed at answering the following questions:…”