Application of scanning electron microscopy and IR spectroscopy for a timely evaluation of the morphology and chemical composition of bacterial films during batch cultivation

Abstract

Bacteria most commonly exist in nature in the form of bacterial biofilms, i.e. associations of cells attached to a substrate surrounded by a polymer matrix. Research into biofilm formation and functioning is fundamental to the management of microbial associations in ecology, biotechnology and medicine. This task requires the development of analytical approaches capable of providing timely information throughout the life cycle of microbial communities at the same time as maintaining their intact structure. In this paper, we apply scanning electron microscopy and IR spectroscopy as rapid methods for analysing microbial biofilms. To this end, the growth kinetics of a Bacillus subtilis culture cultivated on a solid substrate for 24 h was comparatively studied by the methods of classical microbiology and biochemistry, electron scanning microscopy and Fourier-transform IR spectroscopy. The biofilm morphology was found to vary from a uniform settlement of planktonic cells over the substrate surface at the initial stage of growth (6 h) followed by the accumulation of the extracellular matrix and the formation of microcolonies at the exponential and stationary stage (12–18 h) and a gradual depletion of the matrix at the stage of cell death (24 h). The results of IR spectroscopy were established to agree well with those of biochemical studies, thereby demonstrating the potential of the method for a timely evaluation of the accumulation of proteins, polysaccharides and nucleic acids and for obtaining information about their structural state in the studied biofilm. It is concluded that scanning electron microscopy and Fourier-transform IR spectroscopy can be used for obtaining complementary information about the morphology and chemical composition of microbial biofilms during their cultivation.