Resazurin as an indicator of reducing capacity for analyzing the physiologic status of deep-sea bacterium Photobacterium phosphoreum ANT-2200

Abstract : Resazurin (RZ) is a weakly fluorescent blue dye and can be reduced irreversibly to highly fluorescent pink resorufin (RF) that is reduced reversibly to colorless dihydroresorufin (hRF) by photo, deoxygenation, chemical reaction and reductive organic compounds produced through cell metabolism. Because of the reliable and sensitive fluorescence-color change and noninvasive features, RZ has been used widely as a redox indicator in cell viability/proliferation assays for bacteria, yeast, and mammalian cells. However, RZ is used rarely for physiological characterization of marine microorganisms. Here, we developed a custom-made irradiation and absorption-analysis device to assess the reducing capacity and physiologic status of marine bacterial cultures. We measured the absorption spectra of RZ, RF, and hRF in the presence of the reducing compound Na2S and under visible-light irradiation. After establishing appropriate parameters, we monitored the color changes of RZ and its reduced derivatives to evaluate the coherence between reducing capacity, bioluminescence and growth of the deep-sea bacterium Photobacterium phosphoreum strain ANT-2200 under various conditions. Emission of bioluminescence is an oxidation process dependent upon cellular reducing capacity. Growth and bioluminescence of ANT-2200 cell cultures were impeded progressively with increasing concentrations of RZ, which suggested competition for reducing molecules between RZ at high concentration with reductive metabolism. Therefore, caution should be applied upon direct addition of RZ to growth media to monitor redox reactions in cell cultures. Analyses of the instantaneous reduction velocity of RZ in ANT-2200 cell cultures showed a detrimental effect of high hydrostatic pressure and high coherence between the reducing capacity and bioluminescence of cultures. These data clearly demonstrate the potential of using RZ to characterize the microbial metabolism and physiology of marine bacteria.