College
College of Engineering
Mentor Information
Katherine Alfredo
Description
All living organisms share a fundamental biological drive to survive through adaptation. In drinking water systems, pathogens like E. coli utilize survival mechanisms to withstand the chemical disinfectants designed to eliminate them. While treatment with free chlorine or monochloramine successfully stops bacteria from multiplying into colonies, many cells respond to this oxidative stress by shifting into a viable but non-culturable (VBNC) state. In this state, bacteria remain physically intact and potentially virulent but fail to grow on standard agar plates, effectively “disappearing” from the conventional monitoring used to verify water safety. This investigation resolves this monitoring blind spot by utilizing flow cytometry (FCM) with SYBR Green I and propidium iodide co-staining to characterize total and intact cell counts (TCC/ICC) after disinfection contact. Dosing mirrors typical municipal concentrations (~1 mg/L), with efficacy quantified using the Ct concept and numerical integration of measured residuals to calculate “true Ct”. Bacterial cultures are harvested during the growth phase (OD600) to ensure robust populations prior to disinfection. Survival is monitored by simultaneously tracking culturability (CFU) and physical integrity (ICC) across a shared Ct axis. Initial observations revealed a distinct population shift from high nucleic acid (HNA) to low nucleic acid (LNA) intact cells following disinfectant exposure. Notably, intact cells remained detectable at Ct values where culturability approached the detection limit, providing a clear VBNC window. These results define the specific thresholds required for true inactivation, establishing a more accurate framework for water safety assessments that accounts for hidden microbial risks overlooked by standard culture-based methods.
The Invisible Threat: Traditional culture-based monitoring misses the VBNC state
All living organisms share a fundamental biological drive to survive through adaptation. In drinking water systems, pathogens like E. coli utilize survival mechanisms to withstand the chemical disinfectants designed to eliminate them. While treatment with free chlorine or monochloramine successfully stops bacteria from multiplying into colonies, many cells respond to this oxidative stress by shifting into a viable but non-culturable (VBNC) state. In this state, bacteria remain physically intact and potentially virulent but fail to grow on standard agar plates, effectively “disappearing” from the conventional monitoring used to verify water safety. This investigation resolves this monitoring blind spot by utilizing flow cytometry (FCM) with SYBR Green I and propidium iodide co-staining to characterize total and intact cell counts (TCC/ICC) after disinfection contact. Dosing mirrors typical municipal concentrations (~1 mg/L), with efficacy quantified using the Ct concept and numerical integration of measured residuals to calculate “true Ct”. Bacterial cultures are harvested during the growth phase (OD600) to ensure robust populations prior to disinfection. Survival is monitored by simultaneously tracking culturability (CFU) and physical integrity (ICC) across a shared Ct axis. Initial observations revealed a distinct population shift from high nucleic acid (HNA) to low nucleic acid (LNA) intact cells following disinfectant exposure. Notably, intact cells remained detectable at Ct values where culturability approached the detection limit, providing a clear VBNC window. These results define the specific thresholds required for true inactivation, establishing a more accurate framework for water safety assessments that accounts for hidden microbial risks overlooked by standard culture-based methods.
