Immobilization of microorganisms for AFM studies in liquids

In this paper a new preparation method is described that allows the in-vivo AFM imaging of wide range of different microorganisms. The work was mainly focused on the immobilization of fixed and living cells of various microorganisms of our interest on substrates. Tested organisms are Gram-negative and Gram-positive bacteria, yeast and algae. For AFM of biological samples their reliable immobilization on a sample holder is crucial. Lateral forces of the probe tip can alter or remove sample material during scanning. This effect occurs especially on soft biological samples, causing artifacts in imaging and leads to a loss in quality and structural information. For the immobilization, organisms were deposited on polyelectrolyte coated surfaces by centrifugation. Microorganisms were imaged without drying steps either living or with glutaraldehyde fixation. Glutaraldehyde fixation enables long time scans that cover wide areas or the investigation of organisms in special growth stages such as cell division or budding. Skipping fixation steps allows in vivo imaging to investigate living organisms and cellular processes under physiological conditions. The reliable immobilization is demonstrated by imaging the proteinaceous surface layer (S-layer) of living Lysinibacillus sphaericus and Viridibacilli arvi cells. In additional experiments, cell division of E. coli was successfully imaged. During repeated scans of wide areas fixed sample material was not removed by the AFM tip, proving the suitability of the methods for AFM analyses. In conclusion, the method can be easily applied for a wide range of microorganisms and enables the in vivo imaging of whole cells and cell ultrastructure.