Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Rachna Pandey,Erik M M Manders,Norbert O E Vischer,Stanley Brul,Jan P P M Smelt,Alex Ter Beek,Adam Driks

doi:10.1371/journal.pone.0058972

Rachna Pandey, Erik M M Manders + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0058972

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Journal: PLoS ONE	Publication Date: Mar 25, 2013
Citations: 137	License type: CC BY 4.0

Affiliation: University of Amsterdam, Ghent University

Abstract

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.

Highlights

Spore-forming bacteria are an aggravating problem for the food industry and public health
We propose a novel phase-contrast microscopy chamber that allows us to study at the individual cell level the real time dynamics of spore germination, outgrowth, and vegetative growth of aerobically growing B. subtilis
We demonstrate that our closed air-containing chamber and image analysis tool is suitable for the quantification of heterogeneous spore germination and outgrowth of aerobic B. subtilis

Summary

Introduction

Spore-forming bacteria are an aggravating problem for the food industry and public health. It would be advantageous to deliberately drive spores in food into their vegetative form in order to facilitate their inactivation with relatively mild food preservation treatments. In practice this should be done by triggering spores with germinants or physical treatments that allow their ‘rapid return to life’ through the process of germination and outgrowth [6]. The timing of this process is difficult to predict accurately as spores are generally seen to germinate at different times and at different rates This heterogeneity makes predictions of microbial stability of food products exceedingly challenging [2,7]

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