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.

Keywords: tracking; Bacillus subtilis; bacterial motility; heat shock; holographic microscopy; temperature effects; thermophile.

FIGURE 1

Temperature-controlled microscope setup. (A) Schematic of off-axis DHM used in these experiments. (B) The microscope was protected by a heavy-duty plastic bag and submerged in heated water to control the sample temperature. (C) Photograph of the complete setup showing the insulating materials necessary to attain the highest temperatures. The microscope is submerged into the pot with the sample chamber located several cm below the water surface. The insulation shown is required for achieving water and sample temperatures above ∼70°C. (D) Correspondence between the sous vide measured water temperature (verified with a laboratory thermometer) and thermocouple measurements of the sample chamber at top and bottom, along with the average between top and bottom. The slope of the temperature change was consistent between the bath and the chamber, with a nearly constant offset of 5°C. The lines are linear fits with slopes indicated.

FIGURE 2

Sample chamber arrangement. (A) Schematic of the 1 mm deep chamber, showing the sample (S) and reference (R) channels that permit the off-axis DHM geometry. The volume of view of each snapshot is 365 μm × 365 μm × 1 mm in x, y, and z. (B) The chambers were loaded using a 1 mL syringe and placed onto the microscope stage at a fixed focus before immersion.

FIGURE 3

General appearance and size of B. subtilis at normal and elevated temperatures. Shown are phase contrast (top row) and autofluorescence (bottom row); scale bar = 5 μm. (A) 30°C. (B) 60°C. (C) 90°C.

FIGURE 4

Cell sizes and diffusion coefficients with temperature. (A) Cell area as estimated by microscopy, shown as mean ± standard deviation. The differences in the means from one temperature to the next was not significant. (B) Measured diffusion coefficients at 3 temperatures (black dots), with measured standard deviations, compared with predicted values according to Eq. (1) for particle radii of 2.0, 2.5, and 3.0 μm.

FIGURE 5

Data reconstruction and processing for tracking. (A) A portion of the field of view of a median-subtracted hologram from the 34°C dataset. The inset shows part of the image magnified 4x to show the fringes. (B) Amplitude reconstruction of the same field of view in (A) at +50 μm. (C) Maximum Z projection of the same field of view, representing reconstructions from −200 to +200 μm in steps of 10 μm.

FIGURE 6

MHI analysis shows changes in motility patterns with increasing temperature and assists in identifying motile tracks. The tracks are time-coded, with the time index indicating frame number (15 frames/s). The scale bar applies to all panels. (A–F) Full field of view of all identified tracks in selected datasets. (A) 28°C, showing a distribution of motile and non-motile cells and distinct swimming patterns. (B) 34°C, showing increased motility and speed. (C) 44°C, showing nearly all cells motile at high speed. (D) 61°C, showing a reduction in the number of motile cells, but some swimming at high speed. (E) 66°C, showing a large amount of thermal drift with a few motile cells. (F) 84°C, with all motion due to thermal drift. (G–J) Selected examples of swimming types identified in the tracks. Magenta indicates tracks identified as motile by the software; tracks in cyan were identified as non-motile. (G) Helical swimming. (H) Long straight runs. (I) Circling or spinning. (J) Run and tumble.

FIGURE 7

Selected motility parameters. Definitions of the parameters, values and statistical significance are given in the text. Error bars shown are mean ± standard error of the mean unless noted. Numbers of analyzed tracks and their classifications are given in Table 1. When error bars do not appear, they are smaller than the symbols. (A) Mean speed. (B) Mean speed times viscosity of water at that temperature. The lines are linear fits to temperatures above or below 310 K. (C) Total displacement normalized to track length and average speed<v>. (D) Histogram of selected values of D/ <v> T, showing classification into long runs vs. run-and-tumble traces. (E) Sinuosity (no error bars given as the distributions were not Gaussian and the mean value had little significance). These values are plotted on a Log_2 scale for ease of visualization of the ranges involved. Circular tracks were identified as those with sinuosity >20. (F) Correlation matrix of the measured parameters.

FIGURE 8

Collections of non-motile cells on the chamber surface at 66°C. (A) Still image; the circle indicates a cluster of cells. (B) Schematic of surface clustered cells with relative positions of selected motile tracks. (C) Corresponding image of the slide surface with the killed cell dataset at 66°C.