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

Alyssa Francesca Levy,Leslie Knecht,J David Van Dyken,Anthony Labrador,David T Eddington

doi:10.1371/journal.pone.0225020

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.

Highlights

ΜPAD assay for microbial growth rate and motility on solid surfaces
Jokerst and colleagues [16] optimized a wax-printed μPAD assay protocol to determine the prevalence of foodborne pathogens via bacteria-specific enzyme, live bacteria, μPAD assay for microbial growth rate and motility on solid surfaces and food sample analyses in 2012
Wax-printed μPADs take advantage of the wax’s hydrophobic μPAD assay for microbial growth rate and motility on solid surfaces properties to direct the spatial distribution of growth medium, and microorganisms, in a desired shape and direction

Summary

Introduction

It has been shown that artificial selection of microbes during lab domestication to a regime of serial liquid culture propagation has led to the loss of many ecologically significant traits, those associated with sociality, biofilm formation and motility [5, 6], highlighting how cryptic phenotypic variation may be revealed only when grown on solid agar surfaces. It has been demonstrated that growth relying on exoenzyme production is qualitatively different in shaken liquid cultures (such as those in a plate reader) than on solid surfaces [7, 8]. Motility assays are typically performed in agar plates, in part because many motility phenotypes, such as twitching and social swarming motility, are not expressed in liquid culture, and because motility is easier to measure in macroscopic colonies than at the single-cell level. Traditionally-used agar plates are expensive, bulky, contribute to lab waste, and generally lead to low-throughput assays

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