New Device for High-Throughput Viability Screening of Flow Biofilms
New Device for High-Throughput Viability Screening of Flow Biofilms
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DOI：
10.1128/aem.03065-09
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发表时间：
2010-07-01
影响因子：
4.4
通讯作者：
Matin, A.
中科院分区：
文献类型：
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作者：
Benoit, Michael R.;Conant, Carolyn G.;Matin, A.
生物膜的控制需要快速的方法来鉴定对它们有效的化合物，并分离抗性受损的突变体以鉴定参与增强的生物膜抗性的基因。虽然可获得用于微量滴定板孔（“静态”）生物膜的快速筛选方法，但不存在用于连续流动生物膜（“流动生物膜”）的这种筛选的方法。由于后者生物膜更接近天然生物膜，因此需要开发一种高通量（HTP）方法来筛选它们。我们在这里描述了一种新的方法，该方法使用由微流体通道和分布式气动泵（BioFlux）组成的装置，该装置为96个单独的生物膜提供流体流。该装置允许在宽的流速范围内对连续或间歇流体流进行精细控制，并且使用标准孔板格式提供了与板读取器的兼容性。我们表明，使用绿色荧光蛋白（GFP）表达的细菌，碘化丙啶染色，并与读板荧光测量允许快速和准确的测定生物膜的活力。用酶标仪测量的生物膜活力与使用平板计数确定的生物膜活力以及荧光显微镜图像分析的结果一致。使用BioFlux和酶标仪，我们能够快速筛选几种抗菌剂对铜绿假单胞菌PAO 1流动生物膜活力的影响。
Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well ("static") biofilms are available, there are no methods for such screening of continuous flow biofilms ("flow biofilms"). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.