Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems
Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems
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DOI：
10.1016/j.mimet.2017.02.006
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发表时间：
2017-04-01
影响因子：
2.2
通讯作者：
French, Christopher E.
中科院分区：
文献类型：
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作者：
Duedu, Kwabena O.;French, Christopher E.
监测细菌生长是许多应用所需的一项重要技术，例如针对化合物（例如药物）测试细菌、评估环境中的细菌组成（例如污水和废水或食物悬浮液）以及测试工程细菌的各种功能（例如纤维素降解）。 T?= 1,FigItem(1) ReloadFigure=Yes 传统上，使用 600 nm (OD600) 的分光光度测量来快速估计细菌生长，但这种估计不能区分活细胞和死细胞或其他碎片。菌落计数可计数活细胞，但该过程很费力且不适合大量样本。通过流式细胞术进行活细菌计数是一种更合适的快速方法，使用 SYBR I Green 核酸凝胶染色剂和碘化丙啶 (SYBR-I/PI) 双重染色。然而，流式细胞术设备和维护成本相对较高，并且许多可能需要快速方法来评估细菌生长的实验室无法使用该技术。因此，我们寻求调整和评估 SYBR-I/PI 活细菌细胞计数技术，以获得更便宜的平台（荧光计）。采用荧光测定法对混浊生长培养基中的细菌进行 SYBR-I/PI 计数，与 OD600 直接相关 (p > 0.001)。为了能够比较不同实验室和仪器的荧光结果，提出了一种荧光强度标准单位，即等效荧光 DNA (EFD)，经过评估并发现它很有用。进一步评估了该技术在含有不溶性颗粒的混浊介质中计数细菌的有效性。获得了 OD600 无法给出的可重复结果。还评估和比较了基于使用 Pierce Coomassie Plus (Bradford) Assay 评估总蛋白的替代方法。总之，SYBR-I/PI 方法被发现是最快、最可靠的。该协议可用于高通量应用，例如监测 96 孔微孔板中活细菌细胞的生长以及评估纤维素降解酶系统的体内活性。 (C) 2017 Elsevier B.V. 保留所有权利。
Monitoring bacterial growth is an important technique required for many applications such as testing bacteria against compounds (e.g. drugs), evaluating bacterial composition in the environment (e.g. sewage and wastewater or food suspensions) and testing engineered bacteria for various functions (e.g. cellulose degradation). T?= 1,FigItem(1) ReloadFigure=Yesraditionally, rapid estimation of bacterial growth is performed using spectrophotometric measurement at 600 nm (OD600) but this estimation does not differentiate live and dead cells or other debris. Colony counting enumerates live cells but the process is laborious and not suitable for large numbers of samples. Enumeration of live bacteria by flow cytometry is a more suitable rapid method with the use of dual staining with SYBR I Green nucleic acid gel stain and Propidium Iodide (SYBR-I/PI). Flow cytometry equipment and maintenance costs however are relatively high and this technique is unavailable in many laboratories that may require a rapid method for evaluating bacteria growth. We therefore sought to adapt and evaluate the SYBR-I/PI technique of enumerating live bacterial cells for a cheaper platform, a fluorimeter. The fluorimetry adapted SYBR-I/PI enumeration of bacteria in turbid growth media had direct correlations with OD600 (p > 0.001). To enable comparison of fluorescence results across labs and instruments, a fluorescence intensity standard unit, the equivalent fluorescent DNA (EFD) was proposed, evaluated and found useful. The technique was further evaluated for its usefulness in enumerating bacteria in turbid media containing insoluble particles. Reproducible results were obtained which OD600 could not give. An alternative method based on the assessment of total protein using the Pierce Coomassie Plus (Bradford) Assay was also evaluated and compared. In all, the SYBR-I/PI method was found to be the quickest and most reliable. The protocol is potentially useful for high throughput applications such as monitoring of growth of live bacterial cells in 96-well microplates and in assessing in vivo activity of cellulose degrading enzyme systems. (C) 2017 Elsevier B.V. All rights reserved.