Recovery and quantification of bacterial cells associated with streambed sediments
Recovery and quantification of bacterial cells associated with streambed sediments
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DOI：
10.1016/j.mimet.2008.06.004
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发表时间：
2008-10-01
影响因子：
2.2
通讯作者：
Fazi, Stefano
中科院分区：
文献类型：
--
作者：
Amalfitano, Stefano;Fazi, Stefano
需要有效地分离和提纯与河床沉积物相关的细菌细胞，以便通过应用荧光显微镜技术来量化细胞丰度和评估群落组成。我们采用了化学处理(即焦磷酸钠和聚山梨酸酯)和物理处理(即摇动和超声)，然后尼科登兹密度梯度离心法有效地回收了底栖细菌。这一过程产生了高度纯化的细胞悬浮液，允许通过应用荧光染料进行精确的细胞定量。从原始沉积物中回收了约93%的总细胞，从细粒级(90%)中的回收率(90%)高于从粗粒级(69%)中的回收率。在经过预过滤的水对照中，评估了应用程序对细胞完整性的潜在损害作用。由于提取的细菌纯度高，成功地将流式细胞术应用于沉淀物细胞悬液的计数方法。然而，通过估计~H-亮氨酸掺入率，纯化样品的蛋白质合成显著减少，这增加了在Nycodenz纯化后应用潜在代谢分析的可能性的不确定性。(C)2008爱思唯尔B.V.保留所有权利。
Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the 3 H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification. (C) 2008 Elsevier B.V. All rights reserved.