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

High-throughput, multiplex microfluidic test strip for the determination of antibiotic susceptibility in uropathogenic E. coli with smartphone detection

“…Previously we established that even at low cell densities, E. coli growth can be detected within 7 h and at the inoculum recommended for AST within approximately 3-5 h (Needs et al, 2021) (Fig S1). MIC were measured and susceptibility/resistance scored for microcapillary BMD after 6 h incubation (Fig S2-7), and results compared to overnight microplate BMD results (Table 3).…”

“…This confirms that the inclusion of resazurin does not interfere with MIC determination for E. coli when mixed with the sample at 0.25 mg/mL and incubated overnight. Resazurin can be used with fluorescence detection (Needs et al, 2021) with the potential for earlier growth detection compared to colorimetric detection, however, in previous studies, some isolates showed a decay in fluorescence after the initial increase (Needs et al, 2019). This has been observed previously in assays in which fluorescent resorufin is further reduced to the colorless, non -fluorescence hydroresorufin.…”

“…Although small, the individual capillaries are large enough to be scored by the naked eye if digital camera is not available to record result. A smartphone camera is ideal for digitally recording these test results, and wide range of different quality digital cameras have been shown to be equally effective for recording microcapillary bioassay results (Needs et al, 2021, Jégouic et al, 2021).…”

“…The ability to detect growth of very low viable cell numbers – ideally down to a single colony forming unit – may be critical for some analytical applications. We previously identified the limit of detection of a 1 μL microcapillary test strip to be in the order of 5×10 3 CFU/mL; at this concentration 99% of capillaries are predicted to contain at least 1 bacteria every test, allowing MIC measurement (Needs et al, 2021). The initial inoculum density correlates to the speed of detecting resazurin conversion, as early growth at low density does not convert enough dye to influence the capillary color, thus lower densities take longer before growth can be detected (Needs et al, 2021).…”

“…Using this method, samples can be loaded easily by dipping the test strips into the well of a 96-well plate with the sample drawn up into the 10 capillaries by capillary action. Previously, we showed that these “Lab-on-a-stick” devices can perform a range of classical microbiology tests to identify and count viable cells (Reis et al, 2016, Needs et al, 2019, Dönmez et al, 2020, Needs et al, 2021). Here we present the first full demonstration of MIC measurement and antibiotic susceptibility testing for a range of antibiotics in clinically relevant UPEC isolates.…”