Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge.
Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge.
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DOI：
10.3389/fmicb.2016.00903
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发表时间：
2016
影响因子：
5.2
通讯作者：
Gessner MO
中科院分区：
文献类型：
--
作者：
Frossard A;Hammes F;Gessner MO
细菌丰度是微生物学中的一个基本指标，但其评估往往是繁琐的，特别是对于土壤和沉积物样品。为了克服这一局限性，我们采用了一种时间效率高的流式细胞术（FCM）计数方法，该方法涉及通过在接收样品悬浮液和Histodenz®溶液的试管中离心使细胞脱离并与基质颗粒分离。我们使用这种方法来评估细菌丰度在不同的土壤（自然和农业），沉积物（河流和湖泊）和污泥从砂过滤器在饮用水处理厂和比较的结果，细菌丰度确定两个既定的方法，荧光显微镜（EM）和三磷酸腺苷（ATP）定量。FCM和EM确定的细胞丰度相关性相当好，虽然绝对细胞丰度一般较低时，由FCM确定。FCM也显示出显着的关系，从ATP浓度转换的细胞计数，虽然来自ATP测定的估计值通常较高，表明存在的ATP来源以外的细菌。土壤和沉积物有机质（OM）含量的影响与EM和FCM获得的计数之间的拟合优度。特别是，在含有少于10% OM的样品中，如河流沉积物中，通过FCM测定的细菌丰度与EM评估的细胞计数特别相关。总之，这些结果表明，细胞分离和纯化后的FCM是一种有用的方法，以增加样品的吞吐量，用于确定土壤，沉积物和污泥中的细菌丰度。然而，显着的分散和只有部分一致性之间的FCM和参考方法表明，协议需要进一步改进的评估要求高精度，特别是当OM含量的样品是高的。
Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz® solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high.