Ocena zmian morfologii, składu chemicznego i struktury elektronowej powierzchni bakterii z wykorzystaniem skaningowego mikroskopu elektronowego oraz spektroskopu fotoelektronów

Abstract

The aim of the study was the use of modem physical techniques to study biological materials. Knowledge of the structure and elemental composition of the bacterial cells surface and the modifications occur in them is important due to the fact that the surface structures of the first contact with the environment, to protect cells from harmful substaces and are involved in interactions between other cells of the bacteria. The experiments were carried out using X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM) equipped with "Cryo-chamber”, which gives the possibility of testing biological samples in a frozen state. These methods enabled receiving images of surface topography of the bacteria and demarcated their chemical composition. This work presents the results of studies on bacteria isolated from the air indoor spaces, such as dental offices and gyms, and the external environment sewage treatment plant, located in the province of Silesia. In addition, the chemical composition of the surface layer of the bacteria and morphological changes were studied in the cell wall bacteria Bacillus subtilis and Escherichia coli that were grown at different temperatures and different pH. X-ray photoelectron spectroscopy (XPS) gives high information content and yields quantitative data on elemental composition and chemical bonding of the surface layer of the bacteria. Based on the registered inspection XPS spectra, in the energy 0 - 1400 eV, average atom concentration elements on the surface of the tested microorganisms was determined. The calculations were performed using the Physical Electronics Multipak which includes only those elements for which the characteristic lines are clearly visible in the spectrum. When interpreting the results the special attention was put on the determination of major elements (C, O, N, P) and remaining (K, Cl, Mg). An interesting observation was made in the elemental analysis of the bacterial surfaces for two examples of XPS spectra of Bacillus subtilis in the temperature 37°C and 20°C. Fitted lines of carbon (C1 s) and oxygen ( 0 1 s) of the bacterial samples tested for both temperatures (20°C and 37°C) using SimPeak. Analysis of this main line allowed the identification of various functional groups participating in the biomaterial of the bacterial surface. The XPS lines related to the optimal temperature are narrower and the functional groups are better distinguishable. This indicates the better quality of the cell wall surface. Observations of frozen the bacteria in Cryo-SEM have allowed to gain information about their surface structure, both at low and high magnifications and to determine their size. The length, thickness and smoothness of the surface of the bacteria grown in the optimal temperature are different than at a sub-optimal temperature. Also, the electronic structure is different, and the deconvolution lines C 1 s and O 1 s in bacteria grown at temperature 20°C revealed an additional peak. This is due to immaturity of the cell wall surface. Presence of the additional lines indicates that the temperature is one of environment of the main factors affecting the structure of the bacteria.