FISH for Probiotic Enumeration

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Overview

Epifluorescence microscopy has been described as an effective method to enumerate total bacteria in a sample. Fluorescence in situ hybridization (FISH) is considered one of the most powerful techniques in modern microbial ecology, as it has the clear advantage of direct cell counting in their natural environment without the need for cultivation. The popularity of FISH is mainly due to its ability to provide information about the distribution of micro-organisms in a specific matrix and the possibility of performing highly specific detection, in some cases to the strain level.

General Principles of FISH

FISH, or whole cell probes, is an advanced cytogenetic technique that uses fluorescent probes (typically 15 to 30 nucleotide lengths with a reporter, known as a fluorochrome) to detect specific DNA sequences on chromosomes in combination with highly similar, fluorescent, microscopic detection. FISH involves the preparation of two main components: the DNA probe and the target DNA with which the probe will be hybridized. FISH facilitates the rapid and specific identification of individual microbial cells in their natural environments by detecting nucleic acid sequences with a fluorescently labeled probe that hybridizes specifically to its complementary target sequence within the intact cell.

FISH Techniques at Creative Biolabs

Non-culture methods such as FISH are promising as a means to enumerate bacteria in probiotic products quickly and accurately. FISH experiments usually use several probes with different specificities, which are labeled with different fluorophores. Domain-, group- and strain-specific probes are the most commonly used for the analysis of mixed bacterial communities. FISH provides the ability to visualize and quantify bacterial strains, and to reliably enumerate and accurately describe products. The protocol for species-specific bacterial enumeration in mixed cultures consists of the following main steps:

  • The cells are fixed and permeabilized.
  • The fluorescent-labeled probes are incubated with permeable cells to hybridize the probe with the target sequence.
  • Quantification of hybridized cells using FC or epifluorescence microscopy.

Epifluorescence microscopy images of the crop microbial biostimulant product.Fig.1 Epifluorescence microscopy images of the crop microbial biostimulant product. (Pasulka, 2021)

Advantages

FISH has the potential to more accurately reflect bacterial growth dynamics in the environment in which the product is applied, without the need for additional growth restrictions imposed by plate counting. Hence, the FISH technique is often used for enumerating targeted microorganisms in composite microbial communities with single-cell resolution. In addition, FISH technology has made significant advances in speed, and safety, and paved the way for the development of simultaneous detection, quantitative analysis, and live-cell imaging for multiple targets. The FISH method can visualize and quantitatively analyze the strain. In addition, it can make it possible to characterize the growth dynamics of bacteria in the environment of a given probiotic product.

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Reference

  1. Pasulka, A.L.; et al. Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH). Journal of Microbiological Methods. 2021, 182: 106151.

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