Heat-fixation Method Used in an Atomic Force Microscopy Study of Cell Morphology

Abstract

Extremely halophilic archaea are a diverse group of prokaryotes that inhabit highly saline environments. H. salinarum, a species of archaebacteria, is a photosynthetic bacteria that can survive in 4 M NaCl solutions. When the concentration of Na + is low, the cell wall will break apart and the cell will be lysed. 1 To date, the reason why H. salinarum requires a high concentration of sodium ions for growth has not been fully explained. Archaebacteria have a different morphology than other prokaryotes, such as E. coli. 2,3 As is known, the functions of the cells are related to the structure of the cell membrane. Thus, investigations of the structure of the H. salinarum surface are quite significant. Atomic force microscopy (AFM) is a nondestructive imaging technique with nanometer resolution. Since its introduction in 1986, AFM has been used more extensively in the field of life sciences. [4][5][6] AFM images can reveal the surface ultrastructure of living microbial cells with unprecedented resolution. [7][8][9] AFM can be operated in several operational modes, including contact, tapping and MAC modes. 10In the MAC mode, an alternating magnetic field, generated by a solenoid placed under the sample, is used to directly drive a magnetically coated cantilever to enable a stable operation. The advantages of the MAC mode are high resolution and easy operation in fluids for in situ studies, and little harm to soft biological samples due to intermittent contact.For AFM imaging, samples must be immobilized on a substrate. A variety of methods have been used to immobilize cells for AFM imaging, including trapping living cells in filtrate films, placing cells in agar, or immobilizing cells onto a surface such as mica or glass. 4 In microbiology, the heat-fixation method is widely used for immobilizing bacterial cells onto glass slides for observing cell morphology by optical microscopy. For low-resolution observations with an optical microscope, it seems that this method does not affect the cell morphology. However, the effect of this method on the ultrastructure of the cell morphology has not been examined.Herein, the effect of the heat-fixation method on cell morphology studied by MAC mode atomic force microscopy is reported. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were investigated, respectively, as representatives of Gram-negative bacteria and Gram-positive bacteria. The heatfixation method was then employed to prepare H. salinarum samples for AFM study.Details of the ultrastructural morphology of H. salinarum surface were obtained by MAC mode AFM. ExperimentalCulture of bacteria E. coli JM109 and B. subtilis DB104 were grown in LuriaBertani (LB). H. salinarum R1 was grown in a medium containing 0.25% lactalbumin hydrolysate, 0.2% yeast extract, 25% NaCl and 3% magnesium chloride heptahydrate at pH 7.0. The three species of bacteria were cultured at 37˚C with shaking and harvested in the late exponential phase of growth. Bacteria were centrifuged at 5000 rpm for 4 min, and the wet pellets were r...