An inexpensive, high-throughput µPAD assay of microbial growth rate and motility on solid surfaces using Saccharomyces cerevisiae and Escherichia coli as model organisms.

ABSTRACT

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (µPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed µPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed µPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the µPAD design for channel width (3 mm), agar volume (780 µL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed µPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the µPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and µPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, µPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.