Microtiter Dish Biofilm Formation Assay
Microtiter Dish Biofilm Formation Assay
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DOI：
10.3791/2437
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发表时间：
2011-01-01
影响因子：
1.2
通讯作者：
O'Toole, George A.
中科院分区：
文献类型：
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作者：
O'Toole, George A.
生物膜是附着在表面的微生物群落，可以在医疗、工业和自然环境中找到。事实上，生物膜中的生命可能代表了大多数环境中微生物的主要生长模式。成熟的生物膜有几个明显的特点。生物膜微生物通常被一种细胞外基质包围，这种基质为群落提供结构和保护。在生物膜中生长的微生物也有一个特征的结构，通常由大菌落(包含数千个细胞)组成，周围环绕着充满液体的通道。生物被膜微生物也因其对包括临床相关抗生素在内的一系列抗菌剂的耐药性而臭名昭著。微量培养皿试验是研究生物被膜形成早期阶段的重要工具，主要用于细菌生物被膜的研究，尽管该方法也被用于研究真菌生物被膜的形成。因为这项试验使用静态、批量生长条件，所以它不允许形成通常与流动细胞系统相关的成熟生物膜。然而，该方法已经有效地确定了许多启动生物膜形成所需的因子(即鞭毛、菌毛、粘附素、参与环二GMP结合和代谢的酶)以及与胞外多糖产生有关的基因。此外，已发表的研究表明，在微量培养皿中生长的生物膜确实具有成熟生物膜的某些特性，例如对抗生素的耐受性和对免疫系统影响的抵抗力。这种简单的微量培养皿试验允许在微量培养皿的壁上和/或底部形成生物膜。该方法的高通量特性使其适用于遗传筛选，以及测试多个菌株在不同生长条件下的生物膜形成。这种方法的变体已经被用于评估各种微生物的早期生物膜形成，包括但不限于假单胞菌、霍乱弧菌、大肠杆菌、葡萄球菌、肠球菌、分枝杆菌和真菌。在这里描述的方案中，我们将重点使用这种方法来研究模式生物铜绿假单胞菌的生物膜形成。在这项试验中，生物膜的形成程度是用染料结晶紫(CV)来测量的。然而，已经报道了一些其他比色和代谢染色用于使用微量平板分析来定量生物被膜的形成。微量平板法具有操作简便、成本低、操作灵活等优点，已成为生物被膜研究的重要工具。
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.