Phage-Biofilm Visualization Assays

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Introduction

Microbial biofilms are generally defined as sessile microbial masses built on a 3D structure, consisting of multicellular communities of prokaryotic and/or eukaryotic cells embedded in a matrix composed, at least in part, of materials synthesized by the microbial community. Therefore, understanding the effects of phages on bacterial biofilms is crucial for understanding both phage and bacterial ecology. There are not many direct visualization methodologies to assess Phage-host interactions in biofilms. The use of nucleic acid mimics (peptide nucleic acid (PNA) and locked nucleic acid (LNA)) with fluorescence in situ hybridization (FISH) can be a good option for the detection of microbial cells within a biofilm.

Microscopy-based Phage-Biofilm Visualization Assays

Many microscope-based imaging modalities can be used to analyze biofilms. Several of these approaches have already been used to examine phage-biofilm interactions, namely epifluorescence microscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), field emission SEM, and atomic force microscopy.

• Light microscopy remains a useful baseline technique to provide visual identification of biofilm formation. The advantages of light microscopy are simple sample preparation, low cost, and ease of operation.
• For fluorescence microscopy, biofilm elements need to be labeled with fluorescent probes. Microbial cells are usually stained with DAPI or Live/Dead to determine viability.
• CLSM is the most versatile and powerful microscopic technique to decipher biofilm spatial structure and associated functions. Biofilm CLSM imaging can be performed with a series of fluorescent probes with unique specificity.
• SEM is the preferred method for biofilm visualization because it provides information about the spatial structure and detects the presence of EPS. It is a very useful comparative analysis tool in biofilm research.

FISH-based Phage-Biofilm Assay

The FISH technique is based on molecular probes to target a specific sequence within a cell. The use of selective probes associated with FISH is state-of-the-art technology in biofilm research, showing many advantages. This technology can be exploited to characterize phage/biofilm interaction since it is possible to target phage mRNA during replication inside their hosts making infected cells fluorescent. Although phage FISH was designed to detect Pseudoalteromonas using polynucleotide probes, more recent techniques using locked nucleic acid probes as an alternative to DNA probes proved to be very successful when applied on biofilm. These probes allow the discrimination of phage-infected cells and the visualization of their spatial distribution within single-species or multi-species biofilms.

Fig.1 Growth temperature influences biofilm architecture in P. aeruginosa.¹

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Reference:

1. Bisht, Karishma, et al. "Impact of temperature-dependent phage expression on Pseudomonas aeruginosa biofilm formation." npj Biofilms and Microbiomes 7.1 (2021): 22. Under Open Access license CC BY 4.0, without modification.