Rapid nondestructive measurement of bacterial cultures with 3D interferometric imaging

Abstract

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline and is a staple bioanalytical method for efforts ranging from research to standard clinical diagnostic tests. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we demonstrate white-light interferometry (WLI) as a practical tool for accelerated and improved measurement of bacterial cultures. High resolution WLI surface profile imaging was used for nondestructive 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 monitored with WLI 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 and large field of view uniquely provided by WLI enables measurement of colony morphologies and nondestructive monitoring of hundreds of microcolonies. Individual bacteria were imaged within the first few hours after plating and colonies were accurately counted with results comparing favorably to counts made by traditional methods that require much longer wait times. Nondestructive imaging was used to track single cells multiplying into small colonies and the volume changes over time in these colonies were used to measure their growth rates. Based on the results herein, bioimaging with WLI was demonstrated as a novel rapid bacterial culture assay with several advantageous capabilities. Fast nondestructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates and colony morphology with this method may be beneficial in research and clinical applications where current methods are either too slow or are destructive.

Highly Sensitive and Label-free Digital Detection of Whole Cell E. coli with Interferometric Reflectance Imaging (Conference Presentation)

“…This rapid technique could replace the time-consuming culture method which is the current clinical practice for bacteria resistivity studies. It has been also shown recently that using an interferometric imaging technique with appropriate image processing and analysis allows detecting and counting of colony forming units in a more rapid way compared to traditional colony counting techniques (Larimer et al 2019). We believe the large field of view and real-time detection capability of our proposed biosensor, allows us to monitor the bacteria size growth continuously and recognize and count the colony forming units much sooner than the time it takes for the colony units to be visible to the naked eye in a traditional counting method.…”

Scanning ion conductance microscopy revealed cisplatin-induced morphological changes related to apoptosis in single adenocarcinoma cells

“…13 However, imaging the complexity of cell surfaces such as microvilli is beyond the capability of this technique, and its potential applications in microbiology measurements remain unexplored. 14,15 Although uorescence microscopy has enabled the imaging of labelled structures on the cell membrane, the uorophore used might be invasive. [16][17][18] The 3D images of cells can be captured with a confocal microscope, but the technique is used in conjunction with uorescence microcopy, which involves labelling and sometimes xation of cells.…”