A rapid and efficient method for visualisation of microbial biofilms on natural, and industrially- and...

A rapid and efficient sample preparation method for visualising surface-associated microbial biofilms using Field Emission-Scanning Electron Microscopy (FE-SEM) was developed by optimising the fixative concentration and dehydration process. First, different concentrations of the fixative, glutaraldehyde (5-50 %), were tested on Escherichia coli biofilms formed on smooth glass surface for a fixation period of 30 min, followed by a 10-min dehydration each in increasing grades (10-90 %, with 10 % increment) of alcohol. The highest (50 %) glutaraldehyde concentration resulted in the sharpest biofilm micrographs. Further, the incubation period in each alcohol grade was reduced to 2 min to lower the sample preparation time. Improved imaging using the developed protocol was quantified using a newly developed metric, 'Cellular Integrity Index' (CII), which evaluates the morphological integrity of biofilm-cells. The protocol preserved the cellular integrity of individual biofilm-associated cells of Aeromonas hydrophila, A. salmonicida, Pseudomonas fluorescens, and Bacillus mycoides. The optimised method generated distinctive high-resolution micrographs of E. coli biofilms pre-formed on different medically-, industrially-, and environmentally-relevant surfaces, such as polypropylene plastic, catheter, and paper, all with high CII values (95-97 %) with least deformation. Further, the method was used for visualising naturally-formed biofilms on poultry ceca, plant roots, and rock surfaces with impeccable clarity, even effectively resolving different microorganisms, like fungus, algae and bacteria Thus, the developed method will be a valuable asset for any research dealing with the visualisation of naturally-formed or laboratory-developed biofilms on any sort-of surface, be it of a single individual or mixed species, thus enriching environmental, industrial, and medical research.