Comparison of Pre-Stain Suspension Liquids in the Contrasting Ability of Neutralized Potassium Phosphotungstate for Negative Staining of Bacteria

Image contrast of whole bacteria was compared in Staphylococcus aureus and Escherichia coli depending on pre-stain suspension liquids by energy-filtering transmission electron microscopy. The two bacterial strains were suspended in three most commonly used liquids for negative staining (triple distilled water [DW], phosphate-buffered saline [PBS], and nutrient broth [NB]) and directly observed without staining or stained with neutralized potassium phosphotungstate (PTA), respectively. Even though in low contrast, unstained bacteria were observed owing to their inherent electron density and cell shape in zero-loss (elastic scattering) images. After being suspended in PBS, unstained bacteria appeared to have higher contrast and more refined periphery than DW-suspended ones, and extracellular appendage structures such as fimbriae and flagella could be discerned. The unstained bacteria appeared to be invariably surrounded with electron-lucent precipitates, possibly from PBS. As far as delineation of the structures, the combination of DW or PBS suspension with subsequent staining provided the most satisfactory results, as evidenced by the high contrast of bacterial morphology and appendage structures. However, after being suspended in NB and stained with PTA, bacteria often had too high contrast or poor staining, with electron-dense aggregates around the bacteria. These results suggest that suspension with concentrated organic aliquots including broth media before PTA staining could deteriorate image contrast, and should be used only in dilute form for visualizing bacterial morphology and appendage structures. Moreover the contrast enhancement of unstained bacteria by salt granules would be advantageous in demonstrating bacterial sorption of environmental particles like heavy metals, maintaining minimal contrast for cell imaging.