Ethanol and lysozyme immobilize phosphatidylserine on cell surface for evaluating apoptosis-like decay in activated sludge bacteria.

Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using Annexin-V-FITC (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pre-treatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an Ochrobactrum sp. and a Micrococcus sp.. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating a higher extent of externalization of PS than cells harvested at logarithmic growth. A multi-species microbial sludge was artificially decayed by heat treatment or anoxic-oxic alternating treatment, which also induced increased AVF stain, again presumably via decay-related PS externalization. The developed method proved to be efficient for identification of bacterial decay and has potential for evaluation of multi-species bacterial samples such as soil matrix, bioaerosol, and activated sludge. Importance Since the externalization of phosphatidylserine (PS) was considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show this is possible, provided the bacteria are pretreated with ethanol+lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in a bacterial cell membrane, this approach also has potential for evaluation of cell decay of other bacterial species.