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

Abstract

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.

Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin

“…5 μL of each well was spot deposited with a multichannel pipette on square tryptic soy agar plates. The plates were incubated overnight at 37°C and spotting areas with 5 to 50 CFU counted [ 29 ].…”

Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

“…Since we used a system in which water droplets were stabilized in oil using surfactant, there were no solid surfaces or crevices where bacteria/clumps could attach to form a biofilm, leaving them in a pre-biofilm clumping stage. Future investigations could add extra beads 40 or surfaces 41 into droplets to encourage bacterial attachment. With capabilities beyond those of traditional microscopy and well-plate systems, our high-throughput experimental design could be advantageous for further basic research into clumping, as well as research into the initiation and…”

An in-vitro evaluation of C-di-GMP mediated biofilm dispersal as a potential strategy to restore antimicrobial efficacy

“…CBD was found here not to be suitable for routine high-throughput CFU/mL count. The bead biofilm technique employed here has high reproducibility that can be used to measure parameters such as CFU/mL in multiple conditions in relatively high throughput (Konrat, Schwebke et al 2016). It is suggested that as long as beads remain submerged in media, it is possible to use as many beads as necessary as technical replicates (Sternberg, Bjarnsholt et al 2014).…”

“…The suggested protocols (Ceri, Olson et al 1999, Moskowitz, Foster et al 2004 involve mechanical disruption of biofilms to detach bacteria and constituted in this research a high variance in preliminary experimental results. To overcome this problem, a novel method exists that utilizes biofilms grown on 5 mm glass beads (Konrat, Schwebke et al 2016). Beads-grown biofilms show a high level of repeatability, essentially the same techniques can be applied to bead biofilms, but beads can be more readily collected and handled.…”

“…Biofilm time-kill kinetics to assess MDK 99 : Antibiotic susceptibility was determined as a factor of time. Here, biofilms were grown for 3 days using 5 mm glass beads (Konrat, Schwebke et al 2016) in MHB. Two beads (representing two technical replicates, verifying reproducibility) were placed into each well of a 24-well microtiter plate and exposed to fresh MHB containing 1% (w/v) arabinose or arabinose free medium (control samples).…”