A Novel Method for Continuous Determination of the Intracellular pH in Bacteria with the Internally Conjugated Fluorescent Probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester

Abstract

A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.

Antibacterial activity and proposed action mechanism of a new class of synthetic tricyclic flavonoids

“…We presume that the flavonoid 1 mechanism of action is rather related to the impairment of the cell membrane integrity, as our fluorescent image analysis showed. The viable cells, having an intact membrane, do not allow penetration of EB or PI and thus are not stained, whereas dead cells or cells with permeable and disrupted membranes give a fluorescent signal due to the penetration of EB/PI and intercalation into cellular DNA and RNA (Ueckert et al 1995;Breeuwer et al 1996). The high percentages of fluorescent cells (e.g.…”

Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis

“…4). The £uorescence intensity is believed to re£ect di¡erent properties of the cells, including the ability of the cells to accumulate CFDA-SE, the esterase activity of the cells and pH i [9,16]. In addition, measuring forward scatter proper-ties in combination with the green £uorescence of the CFDA-SE stained cells by £ow cytometry (results not shown) indicated a correlation between the size of the cells and the £uorescence intensity.…”

Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment

“…The principle of the FRIM technique is that a bacterial cell is loaded with a non-fluorescent dye (such as 5(6)-carboxyfluorescein diacetate-N-succinimidyl ester). The dye is cleaved inside bacterial cells by non-specific cellular esterases and becomes fluorescent (Breeuwer et al, 1996). Fluorescent emission is recorded at two wavelengths, only one of which is pH-dependent.…”