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Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays.

ABSTRACT: Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth. While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells. We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model. Scanning electron microscopy imaging suggests that the treated surfaces could provide points of attachment to facilitate the recruitment of bacteria in the initial phase of biofilm colony establishment. The sandblast treated polypropylene, but not polystyrene, plates were found suitable in studying the impact of flavonoid quercetin on the biofilm formation in Bacillus subtilis FB17. Further investigation revealed that due to the hydrophobicity of the polystyrene surfaces, a greater amount of quercetin was adsorbed on the plate surface, effectively lowering the concentration of the flavonoid in solution.

SUBMITTER: Bordeleau E

PROVIDER: S-EPMC9086168 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays.

Bordeleau Emily E Mazinani Sina Atrin SA Nguyen David D Betancourt Frank F Yan Hongbin H

RSC advances 20180919 57

Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth. While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells. We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a <i>Pseudomonas fluorescens</i> Pf0-1 mod ...[more]

PMID: 35547717

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Project description:BackgroundConventional crop protection has major drawbacks, such as developing pest and pathogen insensitivity to pesticides and low environmental compatibility. Therefore, alternative crop protection strategies are needed. One promising approach treats crops with chemical compounds that induce the primed state of enhanced defense. However, identifying priming compounds is often tedious as it requires offline sampling and analysis. High throughput screening methods for the analysis of priming-active compounds have great potential to simplify the search for such compounds. One established method to identify priming makes use of parsley cell cultures. This method relies on measurement of fluorescence of furanocoumarins in the final sample. This study demonstrates for the first time the online measurement of furanocoumarins in microtiter plates. As not all plants produce fluorescence molecules as immune response, a signal, which is not restricted to a specific plant is required, to extend online screening methods to other plant cell cultures. It was shown that the breathing activity of primed parsley cell cultures increases, compared to unprimed parsley cell cultures. The breathing activity can by monitored online. Therefore, online identification of priming-inducing compounds by recording breathing activity represents a promising, straight-forward and highly informative approach. However, so far breathing has been recorded in shake flasks which suffer from low throughput. For industrial application we here report a high-throughput, online identification method for identifying priming-inducing chemistry.ResultsThis study describes the development of a high-throughput screening system that enables identifying and analyzing the impact of defense priming-inducing compounds in microtiter plates. This screening system relies on the breathing activity of parsley cell cultures. The validity of measuring the breathing activity in microtiter plates to drawing conclusions regarding priming-inducing activity was demonstrated. Furthermore, for the first time, the fluorescence of the priming-active reference compound salicylic acid and of furanocoumarins were simultaneously monitored online. Dose and time studies with salicylic acid-treated parsley cell suspensions revealed a wide range of possible addition times and concentrations that cause priming. The online fluorescence measuring method was further confirmed with three additional compounds with known priming-causing activity.ConclusionsDetermining the OTR, fluorescence of the priming-active chemical compound SA and of furanocoumarins in parsley suspension cultures in MTPs by online measurement is a powerful and high-throughput tool to study possible priming compounds. It allows an in-depth screening for priming compounds and a better understanding of the priming process induced by a given substance. Evaluation of priming phenomena via OTR should also be applicable to cell suspensions of other plant species and varieties and allow screening for priming-inducing chemical compounds in intact plants. These online fluorescence methods to measure the breathing activity, furanocoumarin and SA have the potential to accelerate the search for new priming compounds and promote priming as a promising, eco-friendly crop protection strategy.

Project description:Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.

Dataset Information

Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays.

Publications

Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays.

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