A steam-based method to investigate biofilm

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.

Figure 1

Steam washing process design. (a) Cross sectional view of steam washing system. After 24-h formation of the biofilm in the microplate, the liquid media is removed by inversion and the microplate is hung upside down in the system. The biofilm wash system (1) is composed of a holding device (2) configured to hold a microplate (3). A steam generating system (4), composed of a heating unit (4a) and a reservoir (4b) creating a flow of steam (F1) leading to the formation of droplets (F2) by condensation that removes non-adherent bacteria and media. To enhance condensation and protect bacteria of the biofilm from heat, a cooling system (5), composed of a cooling device (5a) and a thermal transmission device (5b) is in contact with the microplate. A semi-closed cover (6) enhances condensation. (b) Enlarged view of a microplate well in the wash system.

Figure 2

Steam washing kinetics for removal of the planktonic bacteria. A 24h-old biofilm was formed by (a) SH1000 (S. aureus), (b) ATCC 12228 (S. epidermidis), (c) TM300 (S. carnosus), (d) PA01 (P. aeruginosa), and (e) TG1 (E. coli) which were washed using the steam method. Viable cell count was measured every 10 min over 1 h. Data represent the median and interquartile range of three experiments performed in triplicate.

Figure 3

Steam washing kinetics for removal of excess stain. A 24h-old biofilm was formed by (a) SH1000 (S. aureus), (b) ATCC 12228 (S. epidermidis), (c) TM300 (S. carnosus), (d) PA01 (P. aeruginosa) and (e) TG1 (E. coli) which were washed using the steam method before staining by crystal violet. The capacity of the steam washing to remove excess stain was evaluated by measuring OD₆₂₀ every 10 min over 1 h. Data represent the median and interquartile range of three experiments performed in quadruplicate.

Figure 4

Application of steam washing process. (a) Biofilm quantification by crystal violet (CV) coloration (OD₆₂₀) or PBS turbidity (OD₄₉₀) for SH1000 (S. aureus), ATCC 12228 (S. epidermidis), TM300 (S. carnosus), PA01 (P. aeruginosa), and TG1 (E. coli). Biofilms were washed using the steam (S) or pipette-based method (P). Data are presented as box plots of three experiments performed in quadruplicate. (b) Viable cell count of bacteria assessed after PBS turbidity. Data are presented as box plots of three experiments performed in quadruplicate. ***P value < 0.001, as determined by the Mann-Whitney U test.

Figure 5

Confocal laser scanning microscopy (CLSM) of bacteria biofilm washed using the steam or pipette-based method. (a) Images are 3D visualizations of representative biofilm structures washed using the steam (top) or pipette-based method (bottom) stained with Syto-9 and PI for SH1000 (S. aureus), ATCC 12228 (S. epidermidis), TM300 (S. carnosus), PA01 (P. aeruginosa), and TG1 (E. coli). Images represent an area of approximately 320 × 320 µm. (b) Quantification of biomass and mean thickness of the biofilm measured by CLSM using Comstat2. *P value < 0.05, **P value < 0.01, as determined by the Mann-Whitney U test.

Figure 6

Reproducibility of crystal violet (CV) staining using the pipette-based method and PBS turbidity using the steam method among 3 technicians. Biofilm quantification by CV staining (OD₆₂₀) or PBS turbidity (OD₄₉₀) for SH1000 (S. aureus). Data represent three experiments in performed in quadruplicate.