Abstract
M.Tech
Atomic 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.