Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The development of an electron spin resonance biosensor, with both biomedical and biophysical implications

There is a need for new and innovative biosensing technologies. Biosensors are extremely useful in: relieving the diagnostics burden of healthcare professionals, environmental safety screening, drug and food testing, etc. Here, we have utilized the inherent advantages of continuous wave electron spin resonance (CW-ESR) over other biosensing platforms to develop ESR biosensing metrologies. Initially, we extrapolate the work of the ESR biosensing company Syva, established in the 1970’s. They previously performed various small molecule competition immunoassays, through spin labeling of small antigens e.g. morphine, but not large biomolecules. We attempted to strategically develop an assay for large biomolecule sensing - using similar assay principles to Syva enabled by epitope tagging. However, competition immunoassays suffer from a range of pitfalls, so a CWESR direct immunoassay was developed for the first time. Consequently, a proof-of-concept ESR direct immunoassay for a cystine stabilized single chain variable fragment (sscFv) for the carcinoembryonic antigen (CEA) was developed using disulfide bond labeling. The direct immunoassay was performed successfully in human plasma and whole human blood, both without the need for any sample pretreatment. Furthermore, using the direct immunoassay method, a thermal stability shift assay (TSSA) was developed. To truly evaluate the potential of the direct immunoassay, the disulfide bridging nitroxide spin labeling methodology was utilized to modify full-length antibodies. An anti-FLAG antibody, two human IgG1 antibodies varying in light chains only and anti-C-reactive protein (CRP) antibodies were modified and show interesting binding effects. Finally, a competition glucose sensing assay utilizing the displacement of concanavalin-A bound nitroxide spin labeled mannan complexes by glucose and a direct sensing assay utilizing chromium ions were developed. Overall, the biosensing capabilities of ESR are explored, and the respective advantages and disadvantages are discussed. Furthermore, the commercialization potential of the work is considered and critically evaluated