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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.

Fluorescence in situ hybridisation (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localisation 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 multi-labelled FISH approach (MiL-FISH) that: 1) enables the targeting of up to seven microbial groups simultaneously using multi-spectral labelling of a single oligonucleotide probe, 2) is applicable for the isolation of unfixed environmental microorganisms via fluorescence activated cell sorting (FACS), and 3) improves signal and imaging quality of tissue sections in acrylic resin for precise localisation 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 localisation of bacteria associated with animal tissue as well as their isolation from host tissues using FACS. To further increase the number of potential target organisms a streamlined “combinatorial labelling and spectral imaging-FISH (CLASI-FISH)” concept with MiL-FISH probes is presented. Through the combination of increased probe signal, the possibility to target hard-to-detect taxa and isolate these from an environmental sample, the identification and precise localisation of microbiota in host tissues and the simultaneous multi-labelling of up to seven microbial groups, we show here that MiL-FISH is a multi-faceted alternative to standard mono-labelled FISH that can be used for a wide range of biological and medical applications.