An Introduction to Fluorescence in situ Hybridization in Microorganisms

Abstract

Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms, and the clinical, for the rapid diagnostic of infectious agents. The FISH method is based on the hybridization of a fluorescently-labelled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, a hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell.In here, we will review the major steps involved in a standard FISH protocol, namely fixation/permeabilization, hybridization, washing and visualization/detection. For each step the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail. .

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

“…In a FISH protocol, the variables temperature and formamide concentration must be well adjusted in order to optimize the hybridization performance [ 46 ]. Formamide destabilizes double-stranded molecules by interfering with hydrogen bonds [ 47 , 48 ], and therefore, reducing the hybridization temperature.…”

“…Formamide destabilizes double-stranded molecules by interfering with hydrogen bonds [47,48], and therefore, reducing the hybridization temperature. It is expected that for each 1% (vol/vol) formamide added, the temperature decreases about 1 • C. It is common to test formamide concentrations ranging from 5% to 70% (vol/vol), depending on the type of microorganism, and to use a range of temperatures near 50-60 • C [46]. Here, we tested 55 and 60 • C with 5, 30 and 50% (vol/vol) formamide.…”

Imaging biofilms using fluorescence in situ hybridization: seeing is believing

“…Briefly, the probe length and GC content (due to the effect of the GC triple hydrogen bonds) influences the melting temperature (temperature at which 50% of the double-stranded of nucleic acid strands is changed to single-stranded) ( Almeida and Azevedo, 2021 ; Teixeira et al., 2021 ). The effect of GC content is more pronounced when the probes are shorter, and it is recommended should be between 40% and 60% ( Aquino de Muro, 2008 ; Almeida and Azevedo, 2021 ). In addition, the probe length also influences the diffusion of the probe through cellular envelope (a shorter the probe provides a better the diffusion) and its discrimination power (a shorter the probe provides higher the discrimination).…”

“…Finally, the FISH performance may also be affected by the number of mismatches found in sequences of the nontarget organisms. The presence of mismatches delays the hybridization rate and, therefore, the designed probe should present none mismatches for the target sequences and, as much as possible mismatches for the nontarget sequences ( Almeida and Azevedo, 2021 ).The FISH protocol involves the following four steps ( Figure 1 -I): 1) cells fixation and permeabilization, 2) hybridization of the probe with the target, 3) washing the residual probe, and 4) visualization of the fluorescence emitted by hybridized cells. In the first step, chemical fixatives commonly used in bacterial and human cells are used, to inactivate enzymes and stabilize nucleic acids’ structures.…”

“…The next step is the sample washing to remove all loosely bound or unbound labelled probes, conferring a higher detection specificity. Lastly, but still of huge importance, the result of hybridization is visualized by epifluorescence microscopy or CLSM ( Cerqueira et al., 2008 ; Almeida and Azevedo, 2021 ; Nácher-Vázquez et al., 2022 ).…”