An acoustically focused flow cytometry assay to rapidly and accurately count prokaryotic cells.

Abstract

Introduction: The use of traditional culture techniques for viable counting is laborious and requires time to allow the bacteria to grow. There has been limited innovation in this field; both measurement of nucleic acid by qPCR and spectrophotometric methods rely on standard curves that are generated from traditional plate count methods. While spot plating or spread plating of serial dilutions is sufficient on a small scale, in a high throughput environment it becomes uneconomical in terms of time and cost. Problem Statement: The project aims to develop and validate acoustically focused flow cytometry (AFC) as a reliable alternative to culture methods to quantify prokaryotic cells. Procedures/Data/Observations: Bacterial cultures of four different genera representing each typical bacterial cell morphotype (Gram-negative cocci, Gram-negative bacilli, Gram-positive cocci, and Gram-positive bacilli) were each inoculated into heart infusion broth at approximately 106 CFU/mL and cultured for 24h. At both 0h and 24h, culture samples were stained using a nucleic acid dye to identify particles that contained nucleic acid (presumptive cells) in conjunction with an amine reactive dye to indicate cell viability. AFC was used to quantify stained cells. The AFC data were analysed using FlowJo and a gating strategy was employed to count presumptive cells and to exclude non-viable presumptive cells. Results: AFC and viable plate count data were highly correlated (Pearson’s correlation coefficient r = 0.97) although AFC detected approximately 2-5 times more viable cell-like events than traditional culture plate counting. Time from sampling to obtaining a cell count was approximately 2h using AFC compared to culture methods that take 18-48h. Conclusions: AFC was shown to be a reliable and high-throughput approach that should be considered as both a diagnostic and research tool.