A steam-based method to investigate biofilm

Jason Tasse,Maude Saglio,Frédéric Laurent,Régis Villet,Andréa Cara

doi:10.1038/s41598-018-31437-y

Abstract

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. To be relevant, investigation of biofilm behavior requires effective and reliable techniques. We present herein a simple and robust method, adapted from the microplate technique, in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. The kinetics of steam washing indicated that the method is adapted to remove both planktonic bacteria and excess crystal violet (CV) staining for S. aureus, S. epidermidis, S. carnosus, P. aeruginosa, and E. coli biofilm. Confocal laser scanning microscopy confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing. We also investigated the measurement of the turbidity of biofilm resuspended in phosphate-buffered saline (PBS) as an alternative to staining with CV. This approach allows the discrimination of biofilm producer strains from non-biofilm producer strains in a way similar to CV staining, and subsequently permits quantification of viable bacteria present in biofilm by culture enumeration from the same well. Biofilm quantification using steam washing and PBS turbidity reduced the technical time needed, and data were highly reproducible.

Highlights

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology
We propose a new method in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification
To remove planktonic bacteria without altering biofilm integrity, the support on which biofilm has formed needs to be placed upside down above a source of steam and the bottom to be in contact with a cooling system to favor condensation and preserve viable bacteria (Fig. 1a,b)

Summary

Introduction

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. Biofilm is defined as a community of microorganisms attached to a solid surface and embedded in a matrix facilitating the survival of bacteria in hostile environments They are involved in a wide variety of biogeochemical cycling processes in water, soil, sediment and subsurface environments, and have biotechnological applications such as filtration of drinking water, degradation of wastewater and solid waste, and may be used as biocatalysis in the production of bulk and fine chemicals as well as biofuels. Dyes are toxic, mutagenic, and/or irritant for the experimenter, environmentally hazardous, and require protective equipment[17] In this context, we propose a new method in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. We investigated the measurement of turbidity to quantify biofilm matrix and bacteria as an alternative to staining

Methods

Results

Conclusion