Raw holograms frames of Bacillus Subtilis at different temperatures

<p class="MsoNormal">We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90 ºC could be achieved, with a constant 5 ºC offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in <em>Bacillus subtilis</em> up to 66 ºC. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of &gt; 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 ºC, then plateaued. These findings are consistent with the observed heterogeneity of <em>B. subtilis</em> 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.</p>