Effects of temperature and heat activation on

Letters in Applied Microbiology 1999, 29, 228–232

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis R.G.K. Leuschner and P.J. Lillford Unilever Research Colworth, Sharnbrook, Bedford, UK 2208/99: received 3 June 1999, revised and accepted 8 June 1999 R .G .K . LE US C HN ER A ND P. J . L IL L FO RD . 1999. Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in L-alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to L-alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.

INTRODUCTION

Bacterial spores are resistant to extremes of temperatures and pH, to desiccation, UV irradiation, enzyme action, organic chemicals and may remain dormant for long periods (Moir & Smith 1990; Gould 1997). A successful survival of a bacilli species by formation of a metabolically inactive endospore depends mainly on the ability of this dormant system to resuscitate, allow outgrowth and multiplication under favourable environmental conditions. Germination can be induced by nutrient germinants (Johnstone 1994). Germination involves a sequence of events which result in a breakdown of the spore structure. As a consequence, spores lose their resistant properties and become hydrated which can be observed as a phase change from bright to dark by phase-contrast microscopy (Moir et al. 1994). A chronology of events during germination has been summarized by Moir & Smith (1990) and Johnstone et al. (1982). Cortex hydrolysis begins and allows the core to swell and hydrate which results in a loss of refractility. A phase change from bright to grey is a late event during germination (Stewart et al. 1981; Foster 1994; Atrih et al. 1998, 1999). It is known that spore populations vary in the degree of dormancy, with some spores being more dormant than others. Correspondence to: R. Leuschner, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK (e-mail: [email protected]).

The heterogeneous distribution of dormancy in spore populations results in small numbers of spores which are reluctant to germinate (superdormant) and a general biovariability of spores to respond to a germinant. This explains the difficulties to use germination as a spore controlled procedure for scientific investigations (Gould et al. 1968; Gould 1970; Gould & Sale 1970). To gain knowledge of the germination characteristics of spore populations involves the investigation of a representative number of individual spores in a population as described for Bacillus cereus, B. megaterium and Clostridium botulinum (Coote et al. 1995; Billon et al. 1997). Hashimoto et al. (1969a,b) measured single spore germination of B. cereus and B. megaterium and observed a biphasic germination curve which was characterized by an initially fast drop in refractility followed by a slow asymptotic levelling off decrease. The authors attributed the first phase to an initial hydration and release of ions. The second phase was dependent on germination conditions and was explained by enzymatic degradation of the cortex and hydration of the core. Coote et al. (1995) used confocal scanning laser and phase contrast light microscopy to study single spore germination of B. cereus in combination with image analysis. The authors calculated germination characteristic parameters for single spores and studied their distribution within the total population. Billon et al. (1997) applied their method to study effects of tem© 1999 The Society for Applied Microbiology

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perature on the germination of individual spores of C. botulinum. The effect of five temperatures between 20 and 37 °C was investigated on the initial germination lag of spores in a population (biovariability). In the present study, we recorded the germination behaviour of individual spores of B. subtilis in dependence of temperature and heat activation. Image analysis was applied to determine greylevels in individual spores during the phase change and to measure the time prior to an onset of a phase change.

MATERIALS AND METHODS Microorganism

B. subtilis CMCC 604 was present in the Colworth Microbiology Culture Collection, Unilever Research Colworth, Bedford, UK.

Recording of germination kinetics

Preparation of a slide for germination studies was carried out as follows: A drop (4 ml) of a spore suspension in L-alanine buffer was transferred onto a glass microscope slide. The drop was covered with a 22 × 40 mm cover slip and sealed with nail varnish to avoid dehydration. The slide was imaged under phase-contrast optics of a light microscope. Groups of single spores which were attached to the underside of the cover slip were chosen for analysis. An important criteria was that all spores were in a well defined focal plane and immobilized under the cover slip surface. An objective magnification of 60× was chosen (Nikon × 60, Plan Apo 1.40 na, Kingston, Surrey, UK). Images were recorded using a video camera (Sony DXC-950P, Sony Corporation, Tokyo, Japan). The settings of the camera were optimized and standardized. As an internal standard, latex spheres of a diameter of 1.14 mm (Malvern Instruments Ltd, Worcestershire, UK) were mixed into suspensions of spores and served as constant controls for phase-brightness. This was particularly important in calibrating spore intensities during subsequent image analysis.

Preparation of spore suspensions

Fresh overnight cultures of bacilli were prepared by agitation at 30 °C in Heart Infusion broth (Difco Laboratories, Detroit, USA). Aliquots of 1 ml were plated on heart infusion agar plates. The agar plates were incubated at 30 °C until preparations showed between 90 and 99% phase-bright spores as examined by phase-contrast microscopy (Ortholux II Leitz, Leica Microsystems Ltd, Milton Keynes, UK). Spores were washed from agar plates with 10 ml aliquots of cold sterile distilled water. The combined suspensions were centrifuged at 4000 g (Rotor, JLA-10.500, Beckman, CA, USA) for 20 min at 4 °C. The spore pellet was resuspended in distilled water and centrifuged at 4000 g for 30 min at 4 °C. The washing procedure was repeated at least 4–5 times. The wet spore pellet was divided into aliquots and stored at −20 °C until use. Throughout the study, spores originating from the same spore crop were investigated.

Analysis of recorded data

Images from the video tape were captured and saved using image analysis software (Qwin, Leica Quantimet 570C, Leica Microsystems Ltd). The total magnification on the analysis monitor was 6000×. The diameters of spores on the screen were 6 mm. For the germination experiment several groups were observed over a time period of 1–24 h and at least 50 spores per batch were recorded. Intensities were quantified manually for each spore in the centre (normally the lightest area) in triplicate and averaged by the software package. Fast germinating spores were analysed every 2 min for 1–2 h. The initial greylevel in pixel of phase-bright spores was adjusted close to the maximum of 255. The applied method was validated by measuring greylevel intensities of particles with a constant intensity and on spores repetitively (n 100) whereby the software calculated the mean and statistical parameters such as standard deviation and standard error. Experiments were carried out at least in triplicate and on independent occasions.

Germination conditions and heat treatment

For germination studies, 5 ml of a spore suspension with 107– 108 spores ml−1 were added to 25 ml L-alanine buffer [10 mmol l−1 L-alanine in 50 mmol l−1 Tris HCL buffer, pH 7 (Sigma)]. The mixture was immediately used for light microscopical studies. Heat activation was carried out by heating spore water suspensions in a waterbath at 65 °C for 10 min. Germination kinetics were investigated at 20 and 37 °C. The temperature during germination was controlled by a thermometer which was integrated in the slide holding stage of the microscope.

RESULTS

Dormant spores of B. subtilis 604 displayed a high germination rate (90–100%) in the presence of L-alanine at 37 °C and were therefore suitable for the study of single spore germination. The germination characteristics of four single spores at 37 °C is shown in Fig. 1. We investigated the effect of a suboptimal germination temperature of 20 °C on the germination characteristic of this strain. In addition, spores

© 1999 The Society for Applied Microbiology, Letters in Applied Microbiology 29, 228–232

230 R .G .K . LE US C HN ER A ND P. J . L IL L FO RD

Fig. 1 Germination kinetic expressed as decrease in refractility of four individual spores of B. subtilis 604 at 37 °C in Lalanine buffer.

were heat activated for 10 min at 65 °C and exposed to Lalanine buffer at 20 °C. The effects of these three different experimental conditions on the lag time prior to a phase intensity change is shown in Fig. 2. The majority of spores started to germinate between 10 and 19 min at a temperature of 37 °C. When the temperature was decreased to 20 °C changes in phase-brightness were increasingly evident after 1 h to 1.5 h. Heat activation reduced the lag phase prior to a decrease in phase brightness of spores which were incubated at 20 °C and the majority of spores started to change their phase after around 20 min.

Fig. 2 Lag times prior to the onset of a phase change of individual spores (n 40) of B. subtilis 604 in L-alanine buffer at 37 °C (Ž), at 20 °C () and after heat activation (65 °C per 10 min) at 20 °C (*).

Spores which stayed dormant during the experimental time were observed in few cases (³10%) at 37 °C and increased up to 40% at 20 °C. Heat activation reduced this figure to 25%. The germination temperature and heat activation had also effects on the duration of the phase intensity changes of spores. Figure 3 shows that at 37 °C the majority of spores needed between 20 and 29 min to become phase dark. The time distribution required for this process was broader at 20 °C. The maximum was between 40 and 49 min but a large number of spores needed around 80–100 min for a conversion into a dark spore after an initial loss in brightness. Heat activation combined with a germination temperature of 20 °C led to a narrower distribution and almost all spores were dark within 40–49 min. It was obvious that heat activated spores displayed an increase in homogeneity and converted almost simultaneously after a short lag period from bright to dark. Our results indicated that spores which are exposed to Lalanine at an optimal temperature (37 °C) germinated earlier and faster than spores at room temperature (20 °C). Heat activation enhanced germination and increased the homogeneity of both germination phases. DISCUSSION

The study of phase changes of single spores revealed an insight in the effects of temperature and heat activation on spore resuscitation, whereby the present investigation distinguished between the lag time prior to an onset of a phase change and the duration of a phase intensity change. Germination of individual spores by phase intensity changes has

Fig. 3 Duration of the phase intensity change of individual germinating spores (n 40) of B. subtilis 604 in L-alanine buffer at 37 °C (Ž), at 20 °C () and after heat activation (65 °C per 10 min) at 20 °C (*).


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been investigated for B. cereus, B. megaterium (Hashimoto et al. 1969a,b; Coote et al. 1995) and Clostridium botulinum (Billon et al. 1997). Coote et al. (1995) used heat activated spores throughout their study and determined a mean value for the lag time of 23 min before visible germination started and an average duration of the germination process of 14 min. We obtained similar results for B. subtilis 604 at a germination temperature of 37 °C where a maximum of spores started to change their phase after 20–29 min and the time needed to complete the phase change was between 10 and 19 min. A sequence of phase contrast photographs of a germinating spore of B. cereus is shown in Hashimoto et al. (1969a) and indicates that the process was finished in 3 min, which is a very short time according to our results and those of Coote et al. (1995). An explanation for this might be the different microscope type Hashimoto et al. (1969a,b) used in addition to the number of spores which were evaluated. A temperature of 37 °C reduced the lag phase of spores prior to germination compared with 20 °C and resulted in a more homogenous and narrower distribution of the germination profile. We observed that heat activation effected both phases and led to a faster and more homogeneous germination. The lag phase prior to an onset of a phase change was reduced but the duration of the phase intensity change of the majority of spores was not effected. Hashimoto et al. (1969a,b) described a biphasic germination characteristic for individual spores of B. cereus and B. megaterium. They found that the first phase of an initial rapid decrease in phase brightness lasted for B. cereus 75 2 15 s and the second phase 3–4.5 min and for B. megaterium 2 min and 7 min, respectively. They also observed that the kinetics of the first phase were similar under all germination conditions, whereas the duration of the second phase was dependent on environmental conditions. The authors concluded that the first rapid decline was due to an initial hydration and the second phase reflected degradation of the cortex and hydration of the core. We also observed an initial fast decline of phase brightness in single B. subtilis spores which was followed by a levelling off behaviour. Coote et al. (1995) did not observe two phases during single spore germination of B. cereus. The authors used spores which were heat treated in nutrient broth prior to an exposure to a germinant. This could have effected the speed of the phase intensity change. A biphasic germination behaviour can be seen in some individual spore germination characteristics of C. botulinum (Billon et al. 1997). Our results reveal a temperature dependence of the lag time before a decrease in phase brightness was observed. This indicated a probable involvement of enzymatic activity at this stage which would be in accordance with recent reports on activity of a lytic transglycosylase and glucosamidase early during germination (Atrih et al. 1998, 1999). The time required to complete the phase change was dependent on the

germination temperature and heat activation decreased the time distribution but not the duration of a phase intensity change of the majority of spores. This finding might implicate that heat activation facilitates the access of the germinant. It can be concluded from the present study that enzymatic activity seems to be involved before a phase change becomes visible and is maintained during the phase intensity change.

ACKNOWLEDGEMENTS

The authors thank Dudley P. Ferdinando and Helen Hunt for expert support during experimental analysis. This work was funded by an European Community Marie Curie Research Training Grant in the Framework of the FAIR Program (FAIR CT 975014).

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