With respect to coefficient of linear thermal expansion, bacterial vegetative cells and spores resemble plastics and metals, respectively

Background If a fixed stress is applied to the three-dimensional z-axis of a solid material, followed by heating, the amount of thermal expansion increases according to a fixed coefficient of thermal expansion. When expansion is plotted against temperature, the transition temperature at which the physical properties of the material change is at the apex of the curve. The composition of a microbial cell depends on the species and condition of the cell; consequently, the rate of thermal expansion and the transition temperature also depend on the species and condition of the cell. We have developed a method for measuring the coefficient of thermal expansion and the transition temperature of cells using a nano thermal analysis system in order to study the physical nature of the cells. Results The tendency was seen that among vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content, overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed phenomenon similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. Conclusions We show that vegetative cells occur phenomenon of similar to plastics and spores to metals with regard to the coefficient of liner thermal expansion. Cells may be characterized by the coefficient of linear expansion as a physical index; the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds promise as a new index for furthering the understanding of the characteristics of cells. It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells.


Background
When solid materials are heated, they expand and generally exhibit a thermal creep curve. If a fixed stress is applied to the three-dimensional z-axis of a material and the material is then heated, the amount of expansion resulting from thermal stress increases according to a fixed coefficient of thermal expansion [1]. As the temperature approaches the transition temperature, at which the physical properties of the material change, the rate of expansion decreases and the material reaches its maximum expansion. If expansion is plotted against temperature, the transition temperature is at the apex of the curve. In the case of a solid, the transition temperature may be determined as the melting point of the solid [1]. Different materials, such as metal and plastic, differ in their melting points and their patterns of thermal expansion, so they provide different curves. Microbial cells are not made of a single solid material, but rather of various constituent materials, and the composition varies according to the species and the condition of the cell. The rate of expansion and the transition temperature determined from the amount of expansion thus differ depending on the species and the condition of the cell, and may therefore offer a new approach for the study of cellular structure. However, currently there are no valid methods for determining the rate of expansion and the transition temperature from the amount of expansion of a single cell, and to date there have been no studies conducted on this topic. To address this, we have developed a method for measuring the coefficient of thermal expansion and the transition temperature of microbial samples using a nano thermal analysis (nano-TA) system.

Results and discussion
Change in the coefficient of linear expansion and its relationship to the transition temperature, determined by nano-TA-SPM A scanning probe microscope (SPM) combined with a nano-TA system was used to measure the coefficient of linear expansion. This coefficient defines the percentage of thermal expansion from the axial distortion of the z-axis and the transition temperature [2,3]. The cantilever of the SPM was brought to contact the surface of the cell and was applied to force it with a fixed stress. The cell was heated and the amount of expansion monitored, from which the transition temperature was determined. Figure 1 shows the principle behind determining the coefficient of linear expansion, α (Figure 1 (A)), and a model for the change in the amount of distortion of the cell, which gives a creep curve resulting from heating the cell (Figure 1 (B)). Expansion, softening, and distortion of the material accompanying the increase in temperature are measured by changes in the vertical position (changes in height along the z-axis) of the SPM probe in contact with the material. In stage A, the cell expands at a fixed rate due to heating, and the coefficient of linear expansion of the cell can be determined from changes in the z-value. In stage A, the coefficient of linear expansion, α = ΔL / L 0 ΔT, of the cell is calculated as the rate of increase in the z-value per unit temperature (×10 -6 /°C) [1], where L 0 is the height of the microbial cell (nm) along the z-axis before heating, ΔL is the amount of expansion of the cell (amount of deflection of the cantilever) (nm) when the temperature of the probe increases from t a to t b , and ΔT is the change in temperature (°C) from t a to t b . The probe temperature at which the z-value is maximum (z max) is the transition temperature Tg. Stage B covers the change in probe temperature from t b to Tg, where the coefficient of linear expansion of the cell decreases to 0. Stage C is at Tg, where the coefficient of linear expansion is 0. Stage D is reached if the cell is heated above Tg, when the z-value decreases.

The coefficient of linear expansion and transition temperature of bacteria and yeast
The strains investigated were vegetative cells of two strains of Gram-positive bacteria, two strains of Gramnegative bacteria, and one strain of yeast. In addition, spores of five strains of bacteria of the genus Bacillus and two strains of anaerobic thermophilic bacteria were also tested. Four types of plastic with different melting points were selected as materials for comparison. The curves showing changes in the amount of expansion resulting from heating the cells and the plastics were compared (Table 1). For the purpose of comparison, the coefficients of linear expansion and the transition temperatures of various metals [4,5] (where the melting point was taken as the transition temperature) are cited in Table 2. Among the vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content [6], overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed behaviour similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. B. subtilis spores. Figure 2 (B) shows changes in the amount of expansion of four types of plastic. These results show that the degree of deformation of spores as a result of local application of heat is less than that of vegetative cells and plastics ( Figure 2).

Comparison of the coefficient of linear expansion and transition temperature between bacteria, yeast, and materials
Figure 1 (C) shows SPM images before and after heating of the deformation of a spore in contact with the probe. The transition temperature is the temperature at which some physical properties of the microbial cell changes due to heating, it is like the melting point which changes state of the material. This is the thermal death temperature, at which the structure of the microorganism undergoes irreversible physical damage. Figure 3 shows the transition temperature plotted against the coefficient of linear expansion for the vegetative cells and spores and in Table 1, it can be seen that there is a strong negative correlation between transition temperature and coefficient of linear expansion for both vegetative cells (open triangles in   furthering the understanding of the characteristics of cells.
It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells.

Conclusions
Cells may be characterized by the coefficient of linear expansion as a physical index, and the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds promise as a new index for furthering the understanding of the characteristics of cells. One of the problems in the future includes a difference in the water content of the vegetative cells and the spores.
Estimating the possibility to influence the difference that this difference is a coefficient of linear thermal expansion to be high. We would investigate whether the quantity of water contained in a cell effects on the coefficient of linear thermal expansion. It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells.

Bacterial and yeast strains
The spores used were prepared from Geobacillus stearothermophilus NBRC 13737,   Spores were collected from the culture fluid as reported previously [7].

Measurement of transition temperature and coefficient of linear thermal expansion of bacteria
A Nano Search Microscope type SFT-3500 (Shimadzu Corporation, Kyoto, Japan) was combined with a nano-TA system (nano thermal analysis) (Anasys Instruments, Santa Barbara, CA, USA) [2,3]. The cantilever was brought into contact with a single microbial cell at a constant stress of 200 nN and heated from 25°C at 10°C/s to a temperature of 100°C or 400°C, continuously. The measurement point was the highest point, determined as reported previously [7].