Summary

Микротитровальные Блюдо образование биопленки Пробирной

Published: January 30, 2011
doi:

Summary

Анализ описывает быстрый способ для измерения раннего образования биопленки у бактерий и грибков. Этот метод использует планшет в качестве субстрата для микробной образование биопленки и биопленки визуализируется использованием штамма кристаллический фиолетовый. Препарат обеспечивает либо качественных или количественных тест для раннего образования биопленки.

Abstract

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.

The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.

This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.

In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

Protocol

1. Рост биопленки Рост культуры дикого типа синегнойной палочки или мутантный штамм в течение ночи в богатой среде (т.е. LB) Развести более 1:100 культуры ночь в свежую среду для биопленки анализов. Стандартной среде анализа биопленки для P. палочки М63 является минимальн…

Discussion

Этот метод может быть модифицирована для использования в самых разнообразных видов микроорганизмов. Подвижные микробы обычно придерживаются стен и / или днища колодцев, в то время как неподвижные микроорганизмы обычно придерживаются на дно скважины. Оптимальные условия для образова?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Спасибо Шерри Кучма, Пит Ньюэлл и Роберт Шанкс для обеспечения изображения на рисунке 1. Эта работа была поддержана NIH грант R01AI083256 в ГАО

Materials

Material Name Type Company Catalogue Number Comment
1 X M63       Prepare as a 5X M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature. Dilute 5X stock 1:5, autoclave, cool, then add the desired components.
KH2PO4   Fisher P285-500  
K2HPO4   Fisher P288-500  
(NH4)2SO4   Sigma A5132  
Magnesium sulfate   Fisher M63-500 Add to 1 mM final concentration. Prepare as a 1 M stock in water and autoclave.
Glucose   Fisher D16-3 Add to 0.2% final concentration. Prepare as a 20% stock in water and autoclave.
Casamino acids   Beckton-Dickinson 223050 Add to 0.5% final concentration. Prepare as a 20% stock in water and autoclave.
Arginine   Sigma A5131 Add to 0.4% final concentration. Prepare as a 20% stock in water and filter sterilize. This alternative carbon/energy source can replace glucose and casamino acids
Microtiter plates   Beckton-Dickinson 353911 Falcon 3911, Microtest III, Flexible assay plates, 96 well, U-bottom, non-sterile, non-tissue-culture treated.
Microtiter plate lids   Beckton-Dickinson 353913 The lids can be reused by cleaning with 95% ethanol in water.
Crystal violet   Sigma 229641000 Prepare as a 0.1% solution in water.

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Cite This Article
O’Toole, G. A. Microtiter Dish Biofilm Formation Assay. J. Vis. Exp. (47), e2437, doi:10.3791/2437 (2011).

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