Counting bacteria in microfluidic devices: Smartphone compatible ‘dip-and-test’ viable cell quantitation using resazurin amplified detection in microliter capillary arrays

Abstract

Viable bacterial cell counting is fundamental to analytical microbiology and agar plate colony counting remains common yet laborious and slow. Here, we demonstrate two methods for counting bacteria using commercially available microfluidic devices. We show that accurate viable cell counting is possible using simple and easy ‘dip and test’ arrays of microcapillaries. Colorimetric and fluorescent growth detection both permit viable cell counting in microcapillaries either by limiting dilution into multiple microfluidic compartments using a single endpoint measurement, or alternatively by quantifying growth kinetics. The microcapillary devices are compatible with conventional 96 well plates and multichannel pipettes, expanding each microplate row into 120 individual 1 or 2 μL samples. At limiting dilution, counting the proportion of positive compartments permitted accurate calculation of gram-negative and gram-positive bacteria (E. coli and S. saprophyticus) at concentrations down to as low as 10 CFU/mL with almost 1:1 agreement with agar plate colony counts over four orders of magnitude. A smartphone camera was sufficient to record endpoint images of resazurin growth detection both colorimetrically and fluorescently. Viable cell counting of E. coli and S. saprophyticus was also possible through recording growth kinetics and determining the time taken to detect resazurin conversion. However, only the limiting dilution method remained consistent in the presence of urine matrix, as some interference in growth rate was observed when bacteria were spiked into higher concentrations of normal urine to simulate urinary tract infection patient samples. However, with the limiting dilution counting method endpoint growth was always detected even in the presence of 90% urine matrix, suggesting that this method might permit bacterial pathogen counting directly in clinical samples without agar plating.

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ORCID for Alexander Edwards: orcid.org/0000-0003-2369-989X

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