Schimak Mario P, Kleiner Manuel, Wetzel Silke, Liebeke Manuel, Dubilier Nicole, Fuchs Bernhard M
Max Planck Institute for Marine Microbiology, Bremen, Germany
Max Planck Institute for Marine Microbiology, Bremen, Germany Energy Bioengineering and Geomicrobiology Research Group, University of Calgary, Calgary, Alberta, Canada.
Appl Environ Microbiol. 2015 Oct 16;82(1):62-70. doi: 10.1128/AEM.02776-15. Print 2016 Jan 1.
Fluorescence in situ hybridization (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localization, and isolation of defined microbial taxa. However, fluorescence signal strength is often a limiting factor for targeting all members in a microbial community. Here, we present the application of a multilabeled FISH approach (MiL-FISH) that (i) enables the simultaneous targeting of up to seven microbial groups using combinatorial labeling of a single oligonucleotide probe, (ii) is applicable for the isolation of unfixed environmental microorganisms via fluorescence-activated cell sorting (FACS), and (iii) improves signal and imaging quality of tissue sections in acrylic resin for precise localization of individual microbial cells. We show the ability of MiL-FISH to distinguish between seven microbial groups using a mock community of marine organisms and its applicability for the localization of bacteria associated with animal tissue and their isolation from host tissues using FACS. To further increase the number of potential target organisms, a streamlined combinatorial labeling and spectral imaging-FISH (CLASI-FISH) concept with MiL-FISH probes is presented here. Through the combination of increased probe signal, the possibility of targeting hard-to-detect taxa and isolating these from an environmental sample, the identification and precise localization of microbiota in host tissues, and the simultaneous multilabeling of up to seven microbial groups, we show here that MiL-FISH is a multifaceted alternative to standard monolabeled FISH that can be used for a wide range of biological and medical applications.
荧光原位杂交（FISH）已成为环境和医学微生物学的重要工具，常用于鉴定、定位和分离特定的微生物分类群。然而，荧光信号强度往往是针对微生物群落中所有成员的限制因素。在此，我们介绍一种多标记FISH方法（MiL-FISH）的应用，该方法（i）通过单个寡核苷酸探针的组合标记能够同时针对多达七个微生物群体，（ii）适用于通过荧光激活细胞分选（FACS）分离未固定的环境微生物，以及（iii）提高丙烯酸树脂中组织切片的信号和成像质量，以精确定位单个微生物细胞。我们展示了MiL-FISH利用海洋生物模拟群落区分七个微生物群体的能力，及其在定位与动物组织相关细菌并使用FACS从宿主组织中分离这些细菌方面的适用性。为了进一步增加潜在目标生物的数量，本文提出了一种使用MiL-FISH探针的简化组合标记和光谱成像-FISH（CLASI-FISH）概念。通过增强探针信号、针对难以检测的分类群并从环境样本中分离这些分类群的可能性以及在宿主组织中鉴定和精确定位微生物群，以及同时对多达七个微生物群体进行多标记，我们在此表明MiL-FISH是标准单标记FISH的多方面替代方法，可用于广泛的生物学和医学应用。
