Figure 2

Visualizing the extracellular matrix of Haloferax volcanii biofilms. (A) Development of a GFP-expressing strain for use in biofilm visualization: colonies formed by the parental strain H. volcanii H1206 under transmitted light (left), under blue excitation (center), and by the strain H. volcanii H1206(pJAM1020) under blue excitation (right). Scale bar equals 250 μm. (B) Seven-day H. volcanii H1206(pJAM1020) SL-biofilm stained with concanavalin A-Texas Red collected with blue excitation (left), green excitation (center), and shown as an overlay (right). Scale bar equals 20 μm. (C,D,E) Top-down projections of a Z-stack for a H. volcanii H1206(pJAM1020) SL-biofilm stained with DAPI. (C) GFP signal under blue excitation. (D) DAPI-stained material with violet excitation. (E) Overlay of (C) and (D). Orthogonal views from CLSM analysis are shown below each panel and the plane of the orthoslice is shown in (E). Scale bars equal 20 μm. (F,G,H) Seven-day H. volcanii H1206(pJAM1020) SL-biofilm stained with Congo red with blue excitation (F), and green excitation for CR fluorescence (G), with an overlay of (F) and (G) shown in (H). Scale bar equals 20 μm. (I) Bright-field view of 7-day CR-stained SL-biofilm within a chamber slide. Scale bar equals 1 mm. Area outlined by black box is shown in (J). (K,L) Ten-day SL-biofilm within Petri dish grown at 25°C in medium containing CR with CR-stained string or web-like structures magnified in (L). Scale bar equals 1 cm. (M) Seven-day H1206 SL-biofilms grown in Hv-YPC medium stained with thioflavin T under blue excitation (top down 3D projection of a Z-stack; scale bar equals 20 μm). CLSM, confocal laser scanning microscopy; CR, Congo red; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; SL-biofilm, static liquid biofilm.