Our newly made closed air-containing chamber described in this paper has proven to be very useful for the observation of growth of single cells of aerobic bacteria and for germination and outgrowth of individual spores. shows the closed chamber, which is made by using a spacer (Gene Frame®). The chamber meets four important criteria for good growth of bacteria and outgrowth of spores. One important criterion is to minimize the evaporation of water from the medium pad at 37°C, thereby maintaining the osmotic balance in the chamber. While the experiments discussed lasted on average around 5 h we have incubated in a preliminary test, the chamber with cells for up to 24 h and observed no drying of the agarose pad with cells (data not shown). Another important criterion of the closed chamber is to ensure sufficient oxygen (air) needed for the growth and outgrowth of bacteria and spores. The oxygen diffuses from the space in the chamber into the pad towards bacterial cells and spores, while these are being observed through the microscope. The third criterion is that the cells or spores are sandwiched between the agarose-medium pad and glass cover slip. This immobilizes the spores/cells on the pad so that they remain in focus throughout the experiment while recording the images and can germinate and grow(out), in monolayer form.
Experiments with vegetative cells showed that identical growth rates could be obtained under the microscope as in well-aerated shake flasks when grown in complex media (TSB, LB) (). Only in minimal defined MOPS medium a slightly longer generation time was calculated. The reason for this observed difference is until now unclear. The transition from liquid cultures to agar plates of Escherichia coli
cells grown in rich medium has been described to cause a stress 
. Perhaps the transition for the cells taken from the pre-culture (grown in shake flasks) to solid medium under the microscope is more stressful when the cells are grown on minimal medium instead of on rich media. This is also exemplified by the relative bigger standard deviation for the average generation time for cells grown on MOPS medium (). Our experiments with vegetative B. subtilis
cells showed that the cells have larger sizes when grown in complex medium compared to the defined minimal (MOPS-buffered) medium (). Noticeable too was the observation that higher amount of nutrients present in the complex media lead to fast increase in cell biomass but not a similar increase in cell division (data not shown). As a result, long chains of undivided B. subtilis
cells (filaments) were formed. This is a common phenomenon when cells are grown in complex solid medium. Therefore, a lower percentage (2.5%) of the original complex medium concentration was used to obtain clear division of cells under the microscope. Weart et al.
showed that the nutrients dissolved in the culture medium have a strong influence on cell size through their influence on glycolipid biosynthesis. Information on the nutrient concentration is sensed most in that process and stimulates cells to grow in size until the appropriate mass is reached. Weart and Levin suggested that high growth rate delays the tubulin-like FtsZ assembly, the FtsZ ring formation, and subsequent cytokinesis 
, thereby delaying the division. High nutrient levels hence result in a delayed cell division.
spores are ubiquitously present in foods, and since they may survive preservation treatments and grow out in end-products, efforts are being made to eliminate or inactivate spores of these bacteria from foods. Moist heat (85°C for 10 min) is routinely used in industry for inactivation in food products of vegetative cells and often leads to spore injury 
. Hence, before and/or during outgrowth spores must first undergo repair of damage. Both protein denaturation and enzyme inactivation have been associated with spore inactivation by wet heat 
. Our presented results of wet-heat-treated spores are in line with the observations of Li and co-workers 
for germination of different Bacillus
species, and of Stringer et al.
for germination of C. botulinum
(, and ). Time to start of germination and germination time were both affected (delayed). Outgrowth and subsequent vegetative growth from cells emerging from heat-treated spores was not significantly changed in terms of the average and frequency distribution. Inflicted damage caused by the heat treatment is likely repaired during and/or before outgrowth, in where the spore becomes metabolically active. Although Stringer et al.
observed in C. botulinum
that outgrowth is affected, the heat treatment mainly extended germination and they conclude that damage was quickly repaired and not evident by the time the outgrowing cells started to double.
Noteworthy, when compared to the other investigated processes, the largest variation in the average (standard deviation) is observed in the time to start of germination (). This might indicate that heterogeneity within the population is intrinsically most apparent in the time to start of germination, e.g.
due to differences in the amount of germination receptors per spore, rather than in the subsequent stages of germination, outgrowth, and vegetative growth. This reasoning is in line with observations, and their interpretation, reported by Zhang et al.
The newly developed program SporeTracker, with its incorporated macros for analyses, allowed for a comprehensive and efficient data analysis. The relevance of our analysis for microbiological risk assessment of foods is significant as it is likely that spores that have turned phase-dark are more likely to become metabolically active and successfully repair damage. Modeling risk of food borne bacterial spores in the context of food safety as well as spoilage is highly topical 
To calculate the growth rates of emerging cells from spores and vegetative cells growing into monolayer-microcolonies SporeTracker determines the increase in area over time. Total cell mass of the two-dimensionally growing microcolonies was assumed to be proportional to area including inner gaps, which is only correct for large colonies. However, we felt we could apply this method already after the first cell division, as the exponential curve fit remained typically better than R
0.99. A disadvantage of determining the growth rate by this method is that not the growth rate of each individual cell within the microcolony is determined, but the average growth rate of the cells from the microcolony as a whole, either started as a single vegetative cell or as a cell emerging from a single spore. Importantly, we calculated in a few randomly picked experiments also growth rates using manually determined cell-lengths of individual cells. These rates were highly similar to the ones obtained by applying SporeTracker (data not shown).
In conclusion, with the use of single-cell analysis techniques we can enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores. The above-mentioned closed air-containing chamber and image analysis method can be used for assessing the effect of different stresses, e.g.
different acids and temperature stresses on different stages of germination and outgrowth of the bacteria. Currently we are extending our analyses to include the ratiometric assessment of the dynamics of the internal pH of spores and resulting vegetative cells using pHluorin 
. In addition, we aim at developing a micro-fluidics system that should allow for the change of growth media while observing the spore population. Such developments should provide the means to study the effect and dynamics of the response of B. subtilis
spores and cells at the single cell level, upon exposure to e.g.
the common weak acid preservative sorbic acid.