Chapter 8
Single-Cell Analysis of Mycobacteria Using Microfluidics
and Time-Lapse Microscopy
Giulia Manina and Neeraj Dhar
Abstract
Studies on cell-to-cell phenotypic variation in microbial populations, with individuals sharing the same
genetic background, provide insights not only on bacterial behavior but also on the adaptive spectrum of
the population. Phenotypic variation is an innate property of microbial populations, and this can be further
amplified under stressful conditions, providing a fitness advantage. Furthermore, phenotypic variation may
also precede a latter step of genetic-based diversification, resulting in the transmission of the most beneficial
phenotype to the progeny. While population-wide studies provide a measure of the collective average
behavior, single-cell studies, which have expanded over the last decade, delve into the behavior of smaller
subpopulations that would otherwise remain concealed. In this chapter, we describe approaches to carry out
spatiotemporal analysis of individual mycobacterial cells using time-lapse microscopy. Our method encompasses the fabrication of a microfluidic device; the assembly of a microfluidic system suitable for long-term
imaging of mycobacteria; and the quantitative analysis of single-cell behavior under varying growth
conditions. Phenotypic variation is conceivably associated to the resilience and endurance of mycobacterial
cells. Therefore, shedding light on the dynamics of this phenomenon, on the transience or stability of the
given phenotype, on its molecular bases and its functional consequences, offers new scope for intervention.
Key words Microfluidics, Time-lapse microscopy, Fluorescent reporters, Mycobacteria, Image analysis, Single-cell biology, Spatiotemporal dynamics, Gene expression, Protein localization, Growth rate
1 Introduction
Bacterial populations are heterogeneous due to the inherent stochasticity of biological processes. This heterogeneity is further
modified either by the architecture of gene networks or upon
exposure to stressful environments [1–5]. In isogenic populations,
this potentially serves as a mechanism of diversification, which
could benefit subpopulations of cells that can survive stressful and
lethal environmental fluctuations, resulting in a fitness advantage
[6–10]. As phenotypic heterogeneity is ubiquitous, occurs on short
time scales, and does not require genetic modification, it is being
increasingly studied in the context of chronic and persistent infections [11–13].
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
205
Single-Cell Analysis of Mycobacteria Using Microfluidics
and Time-Lapse Microscopy
Giulia Manina and Neeraj Dhar
Abstract
Studies on cell-to-cell phenotypic variation in microbial populations, with individuals sharing the same
genetic background, provide insights not only on bacterial behavior but also on the adaptive spectrum of
the population. Phenotypic variation is an innate property of microbial populations, and this can be further
amplified under stressful conditions, providing a fitness advantage. Furthermore, phenotypic variation may
also precede a latter step of genetic-based diversification, resulting in the transmission of the most beneficial
phenotype to the progeny. While population-wide studies provide a measure of the collective average
behavior, single-cell studies, which have expanded over the last decade, delve into the behavior of smaller
subpopulations that would otherwise remain concealed. In this chapter, we describe approaches to carry out
spatiotemporal analysis of individual mycobacterial cells using time-lapse microscopy. Our method encompasses the fabrication of a microfluidic device; the assembly of a microfluidic system suitable for long-term
imaging of mycobacteria; and the quantitative analysis of single-cell behavior under varying growth
conditions. Phenotypic variation is conceivably associated to the resilience and endurance of mycobacterial
cells. Therefore, shedding light on the dynamics of this phenomenon, on the transience or stability of the
given phenotype, on its molecular bases and its functional consequences, offers new scope for intervention.
Key words Microfluidics, Time-lapse microscopy, Fluorescent reporters, Mycobacteria, Image analysis, Single-cell biology, Spatiotemporal dynamics, Gene expression, Protein localization, Growth rate
1 Introduction
Bacterial populations are heterogeneous due to the inherent stochasticity of biological processes. This heterogeneity is further
modified either by the architecture of gene networks or upon
exposure to stressful environments [1–5]. In isogenic populations,
this potentially serves as a mechanism of diversification, which
could benefit subpopulations of cells that can survive stressful and
lethal environmental fluctuations, resulting in a fitness advantage
[6–10]. As phenotypic heterogeneity is ubiquitous, occurs on short
time scales, and does not require genetic modification, it is being
increasingly studied in the context of chronic and persistent infections [11–13].
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
205