Membrane depolarization and ion fluxes are events that have been studied extensively in
biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including
electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored
radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using
carbocyanine or oxonol
fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM)
ester versions of ion-sensitive fluorescent
indicator dyes. We therefore established and optimized protocols for measuring membrane potential,
rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the
bis-oxonol dye DiBAC4(3) and the cell-impermeant
dye propidium iodide to measure membrane depolarization and
rupture, respectively, as well as methods to optimally load the pneumococci with the AM
esters of the ratiometric
dyes Fura-2,
PBFI, and
BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these
dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.