Application of VP‐SEM for studying biofilms

The importance of microbial existence as attached communities, embedded in an exopolymeric matrix (EPS), has profound medical, industrial and environmental implications. A major concern is their recalcitrance to antimicrobial agents, as well as to host defenses. Biofilm EPS form a hydrogel, which renders this microbial phenomenon complex to visualize using normal EM preparative techniques. However, our recent application of VP‐SEM in various biofilm studies provided supporting evidence to data which is of major concern in the 21 st century: wetland stability in wastewater treatment, the fate and potential impact of genetically modified organisms (GMOs) on the environment, and biodegradation of biomass for alternative energy sources. Biofilms were viewed unfixed and hydrated with a LEO 1430 VP‐SEM after gold sputter‐coating and application of conductive carbon paste. Operation at 7kV allowed high magnification imaging for periods of 30 min, with little evidence of structural distortion. Results obtained by molecular fingerprinting using tRFLP (Terminal Restriction Fragment Length Polymorphism) of wetland microbial communities were elucidated with SEM images of multi‐species biofilm development over time. GMO retention in soil, in comparison to unmodified organisms, was confirmed with SEM of yeast biofilms. Biodegradation by rumen bacteria, as well as by microbial consortia from compost, was demonstrated to be enhanced by attachment to lignocellulose surfaces. In conclusion, VP‐SEM of biofilms has proved to successfully support and enhance data obtained by using other experimental procedures.