Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria. Barr, D. A, Omollo, C., Mason, M., Koch, A., Wilkinson, R. J, Lalloo, D. G, Meintjes, G., Mizrahi, V., Warner, D. F, & Davies, G. bioRxiv, Cold Spring Harbor Laboratory, may, 2021. ![Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology – including the tendency to clump, and “differential” culturability – and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis BCG time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique. ### Competing Interest Statement The authors have declared no competing interest.
@article{Barr2021,
abstract = {Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology – including the tendency to clump, and “differential” culturability – and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis BCG time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique. {\#}{\#}{\#} Competing Interest Statement The authors have declared no competing interest.},
author = {Barr, David A and Omollo, Charles and Mason, Mandy and Koch, Anastasia and Wilkinson, Robert J and Lalloo, David G and Meintjes, Graeme and Mizrahi, Valerie and Warner, Digby F and Davies, Gerry},
doi = {10.1101/2021.05.01.442251},
file = {:C$\backslash$:/Users/01462563/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Barr et al. - 2021 - Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria.pdf:pdf},
isbn = {10.1101/2021.05.0},
journal = {bioRxiv},
keywords = {OA,fund{\_}ack,original},
mendeley-tags = {OA,fund{\_}ack,original},
month = {may},
pages = {2021.05.01.442251},
publisher = {Cold Spring Harbor Laboratory},
title = {{Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria}},
url = {https://doi.org/10.1101/2021.05.01.442251},
year = {2021}
}
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