Imaging of sub-cellular fluctuations provides a rapid way to observe bacterial viability and response to antibiotics

Determining the viability of bacteria in a sample is an essential microbiological technique used in healthcare, industrial bioprocesses and research. Increasingly, attention has been focussing on antimicrobial susceptibility tests (AST), allowing rapid and appropriate prescribing of antibiotics. Current AST are limited in speed as they rely on detecting growth of microorganisms. Faster AST could be enabled by the recent discovery that living bacteria manifest nano-scale fluctuations, which reduce when the bacteria die. Here, we demonstrate a direct method of visualising fluctuations within bacterial cells using Sub-Cellular Fluctuation Imaging (SCFI), which is based on Total Internal Reflection Microscopy (TIRM). We show that SCFI can measure the viability of bacterial samples within minutes, distinguishing not only between live and dead bacteria but also live bacteria in different metabolic states. Importantly, we subsequently show that SCFI can rapidly distinguish antibiotic-treated resistant and susceptible bacteria, and therefore has particular application as a rapid AST.