The Bead Assay for Biofilms: A Quick, Easy and Robust Method for Testing Disinfectants

Bacteria live primarily in microbial communities (biofilms), where they exhibit considerably higher biocide tolerance than their planktonic counterparts. Current standardized efficacy testing protocols of disinfectants, however, employ predominantly planktonic bacteria. In order to test the efficacy of biocides on biofilms in a standardized manner, a new assay was developed and optimized for easy-handling, quickness, low running costs, and above all—repeatability. In this assay, 5 mm glass- or polytetrafluoroethylene beads in 24 well microtiter plates served as substrate for Pseudomonas aeruginosa biofilms. After optimizing result-relevant steps, the actual performance of the assay was explored by treating P. aeruginosa biofilms with glutaraldehyde, isopropanol, or peracetic acid in predefined concentrations. The aspired 5 log10 reduction in CFU counts was achieved by glutaraldehyde at 5% (30 min), and by peracetic acid at 0.3% (10 min). In contrast, 80% isopropanol (30 min) failed to meet the reduction goal. However, the main accomplishment of this study was to unveil the potential of the array itself; most noteworthy here, a reliable repeatability of the results. The new bead assay for biofilms is a robust, quick and cost-effective method for assessing the efficacy of biocides against biofilms

Comparison of the bead assay, the MBEC^™ assay and the CDC biofilm reactor.

<p>Comparison of the bead assay, the MBEC^™ assay and the CDC biofilm reactor.</p

Reduction of planktonic bacteria and biofilm in comparison.

<p>(a) CFU counts after 30 min glutaraldehyde treatment of planktonic bacteria of <i>P</i>. <i>aeruginosa</i>. (b) Reduction after 30 min glutaraldehyde treatment of <i>P</i>. <i>aeruginosa</i> biofilm and planktonic bacteria (<b>O</b>: biofilm (mean of three independent experiments); <b>Δ</b>: planktonic (mean of three independent experiments); dashed line: 5log₁₀ reduction goal).</p

Microscopic characterization.

<p>(a) SEM image: overview on a glass bead after 24 h cultivation with <i>P</i>. <i>aeruginosa</i>. (b) The bead surface is evenly covered with biofilm. (c) The bacteria are densely arranged in a monolayer. (d) Overview on a glass bead after the biofilm had been removed by sonication. (e, f) The bead surface is virtually empty, except for some residual debris. (g) CLSM image: The sugar-matrix of the <i>P</i>. <i>aeruginosa</i> biofilm was stained with Concanavalin A (assigned color: magenta), and the bacteria with Syto60 (assigned color: green). (h) LIVE/DEAD^® staining of the biofilm on a glass bead. (i) LIVE/DEAD^® staining after the biofilm had been removed from the bead by sonication.</p

Testing for repeatability.

<p>CFU counts of three independent experiments with untreated biofilm on glass beads. (The horizontal lines through the data points represent mean and standard deviation).</p

Assay standardization.

<p>(a) CFU counts of 3 and 5 mm glass beads. (b) CFU counts of 5 mm glass and polytetrafluoroethylene (PTFE) beads. (c) CFU counts of 5 mm glass beads cultivated on shakers with 8 and 25 mm orbit (The horizontal lines through the data points represent mean and standard deviation).</p

Disinfectant efficacy testing on biofilm.

<p>(a) CFU counts after 30 min isopropanol treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (b) CFU counts after 10 min peracetic acid treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (c) CFU counts after 30 min glutaraldehyde treatment of <i>P</i>. <i>aeruginosa</i> biofilm. (The horizontal lines through the data points represent mean and standard deviation).</p