Fluorescence-based protocol for revealing cellular arrangement in biofilms

Abstract

Standardized assays have greatly advanced the understanding of multicellular bacterial biofilms, but they lack cell-scale detail. Here, we present a fluorescence-based protocol that builds on past assays to reveal the cellular-scale arrangement within biofilms. We describe steps for growing biofilms on cover glass, followed by imaging and visualization of cellular arrangements in biofilms. We have applied this protocol to study Escherichia coli biofilms, though it could also be adapted to study biofilm formation in other species. For complete details on the use and execution of this protocol, please refer to Puri et al. (2023).¹.

Keywords: Cell-based Assays; Microbiology; Microscopy.

Graphical abstract

Figure 2

Illustration of modifications to standardized assay to image cellular arrangement in biofilms

Figure 3

Representative images for experimental set up (A) Prepared 96-well plate for biofilm growth. Inset depicts cover glass pieces inserted in wells for the biofilm to grow on. Plates were loosely covered with a lid and incubated at 37°C. (B) Image of cover glass with biofilm prepared for imaging. Cover glass pieces taken out from wells were placed on a glass slide and a 22 × 50 mm cover glass was fixed on top by taping the edges.

Figure 1

Illustration of standardized hydrostatic assay for biofilm formation in microtitre plates

Figure 4

Imaging cellular arrangement in biofilms (A) Representative image of biofilm imaged through the described procedure. Biofilms were inoculated from a 1:10 mixture of cells with fluorescence (red and green combined in equal amounts; green is represented with cyan in this image) and cells with no fluorescence. (B and C) Highlighted region is magnified in (B) (multi-fluorescence) and (C) (bright field). The altered cell-mixing ratio for this biofilm illustrates the case described in the notes, where the inoculum contains cells with no fluorescence (in higher amounts) and fluorescence (in lower amounts; containing an equal ration of red and green fluorescent cells). In this case, cells without fluorescence served as background to visualize red and green (depicted in cyan here) cellular chains.

Figure 5

Clonal communities washed from biofilm (A) Illustration of procedure for washing biofilms. (B and C) Representative image of clonal communities washed off of a biofilm: (B) wider field of view of the agarose pad and (C) a higher magnification example. Biofilm was grown from a 1: 10 mixture of cells containing green (represented as cyan here) and red fluorescence respectively. Washing procedure is described in steps 12–15.