Dataset Information

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

ABSTRACT: We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90°C could be achieved, with a constant 5°C offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in Bacillus subtilis up to 66°C. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of >100 μm above the surface. Suspended non-motile cells showed Brownian motion that scaled consistently with temperature and viscosity. A novel open-source automated tracking package was used to obtain 2D tracks of motile cells and quantify motility parameters, showing that swimming speed increased with temperature until ∼40°C, then plateaued. These findings are consistent with the observed heterogeneity of B. subtilis populations, and represent the highest reported temperature for swimming in this species. This technique is a simple, low-cost method for quantifying motility at high temperatures and could be useful for investigation of many different cell types, including thermophilic archaea.

SUBMITTER: Dubay MM

PROVIDER: S-EPMC9069135 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Quantification of Motility in <i>Bacillus subtilis</i> at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

Dubay Megan M MM Johnston Nikki N Wronkiewicz Mark M Lee Jake J Lindensmith Christian A CA Nadeau Jay L JL

Frontiers in microbiology 20220421

We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90°C could be achieved, with a constant 5°C offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in <i>Bacillus subtilis</i> up to 66°C. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distan ...[more]

PMID: 35531296

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Project description:Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.

Dataset Information

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

Publications

Quantification of Motility in <i>Bacillus subtilis</i> at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

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