Growing and analyzing static biofilms

Abstract

Many bacteria can exist as surface-attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small- or relatively large-scale studies. Unlike assays involving continuous-flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.

Figure 1B.1.1

(A) Schematic of angled plate for air-liquid interface (ALI) assay (back view). (B) Side view of ALI assay setup. An angled plate (a) is supported by paper clips (b). The ALI occurs at the center of the bottom of each well, where a meniscus forms.

Figure 1B.1.2

Phase-contrast micrographs taken at the air-liquid interfaces of biofilms formed by the wild-type Pseudomonas aeruginosa PA14 strain and a nonflagellated mutant (flgK), both grown as described in Basic Protocol 2.

Figure 1B.1.3

Side view of a colony biofilm assay.

Figure 1B.1.4

Side view of modified 6-well plate lid for Kadouri system.

Figure 1B.1.5

Diagram of Kadouri drip-fed biofilm system. Fresh culture medium is pumped onto a biofilm grown in the bottom of a well in a 6-well plate, while planktonic bacteria and spent medium are removed through a needle placed on the opposite side of the well. Numbers are explained in Basic Protocol 4, step 4.

Figure 1B.1.6

Microtiter plate biofilm assay of Pseudomonas aeruginosa. (A) Crystal violet–stained wells from a microtiter dish at 0 and 10 hr postinoculation. The bacteria were grown on glucose supplemented with casamino acids. Wells were inverted to facilitate photography. (B) Quantification of staining at various time points over 10 hr, based on absorbance readings made at 600 nm. (C) The wells show formation of a biofilm when P. aeruginosa is grown on LB (left; requires aerobic growth) or on arginine (right; can be utilized anaerobically).

Figure 1B.1.7

Using the Kadouri system, a biofilm of P. aeruginosa was grown in minimal medium supplemented with arginine at room temperature and photographed by phase-contrast microscopy at 24, 48, and 118 hr. Microcolonies can be observed as early as 24 hr, with large macrocolonies formed by 118 hr.