Evaluation of atomic force microscopy techniques for imaging and studying surface characteristics of bacterial systems involved in bioleaching

M.TechAtomic force microscopy (AFM) has been an integral tool in bacterial studies for resolving surface structures. Novel applications of this instrument in research require the development of sample preparation techniques and improvement of existing ones. Careful selection of the scanning parameters is particularly crucial when exploring the full potential of the AFM. The objective of this study was to design sample preparation methods for AFM imaging bioleaching bacteria and optimise the scanning parameters (deflection setpoint, feedback loop and the scan rate) for contact mode (CM) imaging in air. The method should be simple, fast and cost effective. The strategy used in this study of (i) evaluation of support substrates for bacterial attachment, (ii) investigation of the effect of pH and centrifugation on cell samples during imaging. Centrifuged and noncentrifuged cell samples suspended in either deionised water (pH 7) or acidified water (pH 1.5) were tested for imaging. Mica and glass cover slips were used as potential substrates for attachment. Cells were attached to substrates for imaging by simple adsorption (‘air-drying’ method). To optimise the scanning parameters, the effect of different values of the scan rate, deflection setpoint and the feedback gains on the quality of AFM imaging was investigated. Optimisation of these parameters was found to be instrumental when imaging weakly adsorbed samples prepared by simple adsorption and ‘soft’ samples such as bacterial cells. The results obtained from these experiments were used during preparation of iron- oxidising leaching bacteria for AFM imaging. The surface morphology of iron-grown bacterial samples was investigated with contact mode AFM in air. Reproducible results obtained in each scan shown by the stability of morphological characteristics of bacterial samples indicate that (i) mica can be used successfully as a substrate for attaching cells, (ii) centrifuged bacterial samples can be easily imaged (iii) scanning with scan rate values of <0.5Hz, deflection setpoint of between 0.2-0.5V and feedback values of < 5.000V improve the image quality and can prevent deformation of the bacterial cells by the tip. Non-centrifuged samples could not be imaged, indicating that bacterial cells need to be separated from growth residues as a prerequisite for successful AFM imaging