PNNL

Abstract

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we explored if white-light interferometry (WLI) would be a practical tool for reducing the wait time required before colonies can be identified and counted. High resolution surface profile imaging was used for non-destructive characterization and counting of bacterial colonies on agar before they became visible to the naked eye. The three-dimensional (3D) morphology of Gram-negative (Pseudomonas fluorescens) and Gram-positive (Bacillus thuringiensis) bacterial species were visualized over time by collecting surface profiles of colonies on agar plates with high vertical resolution (3-5 nanometers) and large field of view (3-5 mm). This unique combination of sensitive vertical resolution at low magnification enabled simultaneous detection of hundreds of microcolonies. The colonies were accurately counted within the first few hours after plating with results comparing favorably to counts made by traditional methods. Since the imaging is non-destructive it was used to track single cells multiplying into small colonies and the volumetric changes in the colony were used to accurately measure colony growth rates over time. Based on the results herein, bioimaging with WLI was demonstrated as a powerful bacterial culture assay. Fast non-destructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates with this method suggest that it may lead to beneficial future applications in research, clinical, and other settings.