Differentiation of DNA or membrane damage of the cells in disinfection by flow cytometry

Abstract

Recently, the viabilities changes of fungal spores in the water supply system during different disinfection processes have been revealed. SYBR Green I (SG), a nucleic acid stain, its fluorescence intensity is correlated with the amount of double-stranded DNA. This study established a new method through successive SG-SG-PI staining (PI: Propidium Iodide) with flow cytometry (FCM). It could successfully distinguish DNA damage and membrane damage of fungal spores, clearly elucidating the intrinsic disinfection mechanism during the chemical disinfection. This method was briefly described as follows: firstly, (1) the fungal spores were stained with SG and washed by centrifugation; and then, (2) the washed spores were treated with disinfectants and terminated; after that, (3) the disinfected spores were re-stained with SG and analyzed by FCM; finally, (4) the SG re-stained spores were stained with PI and analyzed by FCM. The percentages of spores with DNA damage and membrane damage were determined by the fluorescence intensity obtained from steps (3) and (4), respectively. The repeatability and applicability of this developed method were confirmed. It was further applied to explore the inactivation mechanism during chlorine-based disinfection, and results demonstrated that chloramine attacked the DNA more seriously than the membrane, while chlorine and chlorine dioxide worked in a reverse way.

Keywords: DNA damage; Flow cytometry; Fungal spores; Inactivation mechanism; Membrane damage.