Cost-Effective and Versatile Analysis of Archaeal Surface Adhesion Under Shaking and Standing Conditions.

Biofilms are aggregates of cells surrounded by an extracellular matrix providing protection from external stresses. While biofilms are commonly studied in bacteria, archaea also form such cell aggregates both in liquid cultures and on solid surfaces. Biofilm architectures vary when in liquid cultures versus on surfaces as well as when incubated under static conditions versus under shear forces of flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe surface adhesion assays employing a cost-effective, 3D-printed coverslip holder that can be used under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows the same culture to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analysis of the dynamics of biofilm formation. Additionally, a traditional surface adhesion assay in a 12-well plate under standing conditions is outlined as well. We anticipate the combination of these protocols to be useful for analyzing a wide array of biofilms and answering a multitude of biological questions.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.