A pipeline for developing and testing staining protocols for flow cytometry, demonstrated with SYBR Green I and propidium iodide viability staining.
The increasing use of flow cytometry (FCM) for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. Viability assessment with FCM is in this context of particular interest because incorrect staining could severely affect the outcome/interpretation of the results. Here we propose a pipeline for the development and optimization of staining protocols for environmental samples, demonstrated with the common viability dye combination of SYBR Green I (SG) and propidium iodide (PI). Optimization steps included the assessment of dye solvents, determination of suitable PI concentration, and determining the optimal staining temperature and staining time. We demonstrated that dimethyl sulfoxide (DMSO) could impair membrane integrity, when used for SGPI stock solution preparation, and TRIS buffer was chosen as an alternative. Moreover we selected 6μM as optimal PI final concentration: less than 3μM resulted in incomplete staining of damaged cells, while concentrations higher that 12μM resulted in false PI-positive staining of intact cells. Low temperatures (25°C) resulted in a slow reaction and did not enable the staining of all bacteria, while high temperatures (44°C) caused damage to cells and false PI-positive results. Hence, 35°C was selected as optimal staining temperature. We further showed that a minimum of 15min were necessary to obtain stable staining results. Moreover, we showed that addition of EDTA resulted in 1-39% more PI-positive results compared to an EDTA-free sample, and argue that insufficient evidence currently exist in favor of adding EDTA to all samples in general. Altogether, the data clearly shows the need to be careful, precise and reproducible when staining cells for flow cytometric analyses, and the need to assess and optimize staining protocols with both viable and non-viable bacteria.
Riga Technical University, Ķīpsalas Str. 6A, LV-1048, Riga, Latvia; Eawag, Swiss Federal Institute for Aquatic Science and Technology, Überlandstr. 133, CH-8600 Dübendorf, Switzerland.
Alina
A
Nescerecka
Eawag, Swiss Federal Institute for Aquatic Science and Technology, Überlandstr. 133, CH-8600 Dübendorf, Switzerland. Electronic address: frederik.hammes@eawag.ch.
Frederik
F
Hammes
Riga Technical University, Ķīpsalas Str. 6A, LV-1048, Riga, Latvia.
Talis
T
Juhna
D001419:Bacteria / Q000187:drug effects; D052160:Benzothiazoles; D002463:Cell Membrane Permeability / Q000187:drug effects; D003951:Diagnostic Errors; D003959:Diamines; D004203:Disinfection; D004492:Edetic Acid / Q000494:pharmacology; D004783:Environmental Microbiology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D050296:Microbial Viability / Q000187:drug effects*; D009930:Organic Chemicals / Q000009:adverse effects / Q000737:chemistry; D011419:Propidium / Q000009:adverse effects / Q000737:chemistry; D011804:Quinolines; D045483:Rivers / Q000382:microbiology; D012997:Solvents / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods* / Q000592:standards; D013696:Temperature; D013997:Time Factors; D014871:Water Microbiology
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012997:Solvents; C098022:SYBR Green I; D011419:Propidium; D004492:Edetic Acid
Bacteria; Disinfection; Flow cytometry; Optimization; Propidium iodide; Viability