Fluorescence In Situ Hybridization (FISH) for Gut Microbiota Research

Fluorescence in situ hybridization (FISH) not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms and can therefore help in understanding complex microbial communities. Creative Biolabs is a diverse, knowledgeable, and agile team focusing in live biotherapeutic products (LBP) development. With our complete suite of gut microbiota solutions, we can be your one-stop-shop.

Introduction of FISH

FISH is based on hybridization between specific fluorescent labeled probes and complementary target RNA or DNA sequences. This technology is used to study gene expression, detect, and locate nucleotide sequences in a variety of samples (environment, human or animal) while maintaining cell integrity. FISH can be combined with flow cytometry for high-resolution automatic analysis of mixed microbiota.

A schematic representation of FISH.Fig.1 A schematic representation of FISH. (Kumar, 2020)

The FISH Procedure

FISH consists of four steps: sample fixation, permeabilization, hybridization, detection, and analysis of the target by microscopic visualization. The pretreatment of the sample can be performed to facilitate the entry of FISH probes and thus increase their hybridization efficiency. The actual hybridization reaction is then performed by incubating the FISH probe with the sample for several hours at a temperature that provides specificity for the hybridization reaction. The concentration of formamide in the hybrid buffer changes the severity of the hybridization reaction and requires specific probe selection. The composition of the buffer as well as the hybridization temperature determines the sensitivity and specificity of the reaction, usually based on the length of the probe and the sample of interest. Several rigorous washing steps after hybridization ensure the removal of unbound FISH probes and the specificity of detection signals. In addition, nuclear staining is usually performed before detection of FISH signals by epifluorescence microscopy or confocal microscopy, or super-resolution microscopy.

Schematic representation of the experimental FISH assay procedure.Fig.2 Schematic representation of the experimental FISH assay procedure. (Huber, 2018)

Advantages and Disadvantages of FISH

FISH is very attractive because of its speed, low cost, and proven feasibility. It reduces the time for operation and analysis from a few hours to one or two. Live bacteria can be detected, and the technique is sensitive enough to detect only one microbial cell. FISH can be used not only on slides but also in conjunction with flow cytometry. However, the FISH technique has several drawbacks. Like other in situ techniques, FISH requires critical reading, so learning-interpreting slides remain key to ensuring the validity of the results. In addition, although PNA probes are effective, they are still expensive to use. The average price of these probes is four times that of conventional probes.

FISH for Analysis of Gut Microbiota

One of the most direct methods for the identification of single cells within complex ecosystems is FISH, with specific 16S rRNA-based oligonucleotide probes. FISH has been applied to the molecular evaluation of microbiota in newborns delivered vaginally. If we can gain a better overall understanding of fecal microbiota throughout early childhood, then not only will the pathophysiology of intestinal disease be better understood, but treatment can also become more specific and effective. A fresh faecal sample has to be thoroughly homogenized by rough shaking and the use of glass beads. Fresh stool samples must be thoroughly homogenized by rough shaking and the use of glass beads. This is a very important step because only a small number of samples are analyzed, so it must be representative of the entire sample. Subsequently, large particles are removed by brief centrifugation at low centrifugal force (≤300 g). The aliquots of the supernatant were then fixed with paraformaldehyde in parallel with ethanol.

The most widely used probes for the characterization of the human gut microbiota.Fig.3 The most widely used probes for the characterization of the human gut microbiota. (Namsolleck, 2004)

FISH has yielded important information into the microbiologic diagnosis as it bears great advantage in the analysis of microbial communities and the information is not biased by the limits of classic culture techniques. This may help illuminate the role of microorganisms in their niche and provide interesting insights into the ecology of the gastrointestinal system. Creative Biolabs has expertise and capabilities in providing high-quality gut microbiota solutions. If you are interested in our FISH services, please contact us for more.

References

  1. Kumar, G.; et al. Application of molecular techniques in biohydrogen production as a clean fuel. Science of The Total Environment. 2020, 722: 137795.
  2. Huber, D.; et al. Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH? Micro and Nano Engineering. 2018, 1: 15-24.
  3. Namsolleck, P.; et al. Molecular methods for the analysis of gut microbiota. Microbial ecology in health and disease. 2004, 16(2-3): 71-85.

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