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Help us improve our products. Sign up to take part. A Nature Research Journal. 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. 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 Biofilms Investigative Methods And Applications 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.
Biofilm is defined as a community Biofilms Investigative Methods And Applications 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, but also 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.
They are also responsible for persistent contamination in the food industry, impacting the safety and quality of products 1. In addition, they are now recognized as a major virulence factor in infections of plants and animals, including humans, especially in the presence of medical devices or implants 23. Since its first description in by Costerton et al. One of the most commonly used biofilm models for high-throughput analyses is based on well microtiter plates 78.
This historical device is adaptable to most bacterial or fungal species, and remains widely used to compare the behavior of bacterial biofilms, screen for potential anti-biofilm agents, and identify genes involved in biofilm formation in liquid media 9. In the microplate-based method, cells are grown for a specific period before being washed in order to remove planktonic bacteria.
The biofilm Biofilms Investigative Methods And Applications can then be assessed using various approaches including staining with a dye Crystal Violet — CV, Safranin Red, or Congo Red for total biomass quantification 10growth ability colony forming units for viable cell enumeration 11and specific fluorescent probes FITC, SYPRO Ruby, Calcofluor White for the quantification of extracellular polymeric components For the staining protocol using CV, both living and dead bacteria Biofilms Investigative Methods And Applications well as the matrix are colored.
An alternative method has been proposed in which biofilm is resuspended in phosphate-buffered saline PBS instead of staining 1415yet, to the best of our knowledge, no comparison with the classical staining method has been performed. All these methods have several limitations. The main one is the lack of reproducibility of the results notably due to aggressive pipette-based washing 16which is likely to cause the random detachment of large amounts of biofilm.
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 also investigated the measurement of turbidity to quantify biofilm matrix and bacteria as an alternative to staining. The new washing method is based on water vapor condensation. To remove planktonic bacteria without altering biofilm integrity, the support microplate 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.
Water droplets form and accumulate in each well on the biofilm surface before falling back down along with the non-adherent bacteria.
This system can be easily adapted in any laboratory by using boiling water to produce the steam, and a cooling system a simple ice pack for transport being adequate to chill the microplate. This soft washing process was initially tested on a 24 h-old biofilm of an S. During the washing process, the spreading of bacteria was investigated by taking multiple samples from the various parts of the steam washing system steam generating system, reservoir, water, holding device and around it; no dissemination of viable bacteria was found which confirmed the safety of the system.
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Of note, water drops containing the washed planktonic bacteria fall back into the boiling water reservoir, that allow the destruction of viable bacteria. Steam washing process design. After 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 4composed 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 5composed of a cooling device 5a and a thermal transmission device 5b is in contact with the microplate. A semi-closed cover 6 enhances condensation.
Steam washing kinetics for removal of the planktonic bacteria. A 24h-old biofilm was formed by a SH S. Data represent the median and interquartile range of three experiments performed in triplicate. The homogeneity of the steam washing between wells of the same microplate was investigated. For this, the mean numbers of viable bacteria among wells of the same row and then the same column were compared using one-way analysis of variance ANOVA ; the null hypothesis was that the biofilm formed was identical between rows and between columns.
The steam washing method therefore appears adapted to Biofilms Investigative Methods And Applications removal of planktonic bacteria. In this context, we tested the possibility to combine the steam washing method with the CV assay. For this, three reference strains described as a biofilm producer S.
The biofilms were then stained using CV, and excess stain was removed by steam washing. Steam washing kinetics for removal of excess stain. Data represent the median and interquartile range of three experiments performed in quadruplicate.
Biofilms Investigative Methods And Applications CV assay using the steam washing method was then compared to the classical CV assay using pipette-based washing. Staining for all strains was greater using steam washing Fig. Interestingly, we noticed that the standard deviation for the SH strain was lower using steam washing 0. Interestingly, as control wells without bacteria were prepared and ODs were subtracted from the values for strain samples, the data suggest that ATCC and TM were not strictly non-biofilm producers, and have a residual even low ability to form weak biofilm.
Application of steam washing process. Biofilms were washed using the steam S or pipette-based method P. Data are presented as box plots of three experiments performed in quadruplicate. To explore the capacity of steam washing to be used with other species, Pseudomonas aeruginosa PA01 and Escherichia coli TG1, two isolates known to be biofilm producer strains, were tested under the same conditions as SH As for S.
Interestingly, for TG1 there was no significant difference between the washing methods Supplementary Fig. To conclude, all washing kinetics indicate that bacteria were effectively removed until a plateau was reached. This was reproducible and was observed for all bacteria tested, strongly suggesting that the final balance achieved corresponded to the removing of non-adherent bacteria, and that the greater amount of biofilm measured using the new method is not a consequence of a partial wash.
We then hypothesized that biofilm formation could be measured without a staining step by measuring the turbidity of a biofilm resuspended in PBS.Your Account Logout. Search all titles. Until now, techniques for studying biofilms- the cellular colonies that live in drinking water systems, wastewater operations, even ground and surface water- have been limited. Yet during the last decade, biofilms have become a critical element in water quality preservation systems, a key component of wastewater treatment biological reactions and t. Search all titles Search all collections.
Using PBS turbidity ODwe were able to discriminate staphylococcal biofilm producer strains from non-producer strains in a way similar to that when CV staining and OD were used Fig. It is of note that biofilm production of the ATCC strain was less important regarding the OD than regarding OD when steam washing was used.
This could be explained by the additional step needed to add CV to the well using a pipette, that can lead to detachment of the biofilm as is the case during pipette-based washing. Regarding P. This composition probably induced a high capture of CV, leading to high OD values, but does not induce a high turbidity when the biofilm was resuspended in PBS, leading to low OD values.
Interestingly, using the non-bactericidal protocol of PBS turbidity for biofilm quantification, viable cell count can be performed on the same well by subsequently plating the bacterial suspension. Results showed that, when steam washing was used, the bacterial counts were significantly higher than Biofilms Investigative Methods And Applications obtained using pipette-based washing for all strains, except P.
Again, the standard deviations for all strains were lower using steam washing, indicating that the reproducibility was better than for pipette-based washing, except for E. In addition, the better reproducibility and the ability to discriminate high from low biofilm producers using PBS turbidity for staphylococcal species was confirmed by using a set of 5 clinical S. Finally, the impact of growth media on steam wash was investigated using BHIg and BHI media and showed that they influence the time to reach the plateau Supplementary Fig.
These results indicate that any modification of the parameters used to form the biofilm may impact the time for washing. Qualitative image analysis found that bacteria were alive green irrespective of the washing method Fig. Furthermore, the morphology of the biofilm was preserved with steam washing, while holes and alterations were present within the biofilm after pipette-based washing strain SH, Fig. The biomass and mean thickness calculated using Comstat2 software were significantly greater for steam washing than for pipette-based washing for all strains except TG1, for which they were not significantly different Fig.
These data confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing that leads to detachment of thin and fragile biofilm such as that produced by ATCC and TM For E. Taken together, these data confirmed that steam washing was adapted to the study of both robust and fragile biofilm from different species. Confocal laser scanning microscopy CLSM of bacteria biofilm washed using the steam or pipette-based method.
Finally, the reproducibility of CV staining Biofilms Investigative Methods And Applications pipette-based washing and PBS turbidity using steam washing as performed by three distinct technicians was investigated using S. The standard deviations were lower using steam washing and turbidity measurement than when using pipette-based washing and CV staining for all three Biofilms Investigative Methods And Applications 0. Furthermore, the technical time required to perform each of the methods on 4 or 96 wells was measured for each technician the time during which the microplate was on the steam system was not included as this step is performed without any technician intervention.
Reproducibility of crystal violet CV staining using the pipette-based method and PBS turbidity using the steam method among 3 technicians.
Data represent three experiments in performed in quadruplicate. The antimicrobial susceptibility of biofilms using steam washing was not evaluated in the present study. This approach could theoretically be performed using this new method, but the time required to completely remove the antimicrobial agent by steam needs to be determined.
Regarding the Calgary Biofilm Device 18steam washing is probably not adapted to remove non-adherent bacteria from pegs because the cooling system would not be able to chill the extremity of the peg as much as the bottom of a microplate. Furthermore, there is probably a risk of cross-contamination from peg to peg in this setup because bacteria and biofilm are on the outer side of the pegs and not inside a well. We propose herein two ways to easily improve the study of biofilms; first a new method for microtiter plate assays to softly wash the biofilm with steam instead of pipetting, and second the quantification of the biofilm using PBS turbidity instead of staining with a dye.
These processes can be easily adapted in all laboratories and at low cost. They require less technical time, and reduce steps involved which leads to highly reproducible data compared to the methods used up to now.
To conclude, steam washing is a simple, rapid, inexpensive and robust method which has all the features needed to replace the techniques currently used to study biofilms in medical, environmental, and industrial fields. Negative control wells were prepared with medium alone.