Modified osmium tracer technique enables precise microscopic delineation of hydrocarbon-degrading bacteria in clay aggregates

Abstract Clay minerals can support bacterial proliferation, induce the formation of clay–bacterial aggregates, and finally a clay-based biofilm. However, how these abiotic and biotic entities interact in a microhabitat is not fully understood. Visualization of the clay–bacterial micro-aggregate under scanning electron microscope (SEM) and profiling the associated elemental signature through energy dispersive X-ray spectroscopy (EDS) can potentially unravel the mechanisms of a complex clay–bacterial interaction. Osmium (Os) was used previously to enhance the visualization of microbial substances, but the delineation of bacterial cells from clay particles in a micro-aggregate was not tried before. In this study, bacterial cells in a clay–bacterial aggregate ( Burkholderia sartisoli with montmorillonite and kaolinite) were specifically stained with osmium (Os) which served as the EDS tracer of the biotic component of the interaction. Simultaneously silicon (Si) provided the signature of the clay minerals. X-ray elemental profiling (line and field mapping) successfully delineated the individual components of the clay–bacterial aggregate. Thus, this study presented a simple Os-based SEM-EDS technique which could facilitate the microanalysis of bacterial microhabitat within a complex environmental substrate.