Biofilms are cellular aggregates encased in extracellular polymeric substances and are commonly formed by single-celled eukaryotes, bacteria, and archaea. In addition to attaching to solid surfaces, these cellular aggregates can also be observed floating on or immersed within liquid cultures. While biofilms on surfaces have been studied in some archaea, little is known about liquid biofilms. Surprisingly, immersed liquid biofilms of the model archaeon Haloferax volcanii do not require the same set of machinery needed to form surface-attached biofilms. In fact, to date not a single gene has been identified that is involved in forming immersed liquid biofilms. Interestingly, after an immersed liquid biofilm forms, removal of the Petri dish lid induces rapid, transient, and reproducible honeycomb patterns within the immersed liquid biofilm itself, triggered by a reduction in humidity. In this chapter, we outline a protocol for both immersed liquid biofilm and honeycomb pattern formations. This protocol will be essential for determining the novel components required for the formation of immersed liquid biofilms and honeycomb patterns.
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