Available online 11 November 2023, 126473

Author links open overlay panel, , Abstract

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.

Section snippetsUncited references

Kubota et al., 2006, Moraru and Amann, 2012, Schlafer and Meyer, 2017, van de Corput et al., 1998, Zuriani et al., 2013.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by a Grant-in-Aid for Challenging Research (Exploratory) (KAKENHI Grant Number JP22K18818) from the Japan Society for the Promotion of Science (JSPS). KU was supported by a Grant-in-Aid for JSPS Fellows (JP21J21027) and the MEXT WISE Program for Sustainability in Dynamic Earth (SyDE), Japan.

References (23)Y. Chen et al.Key factors improving the stability and the loading capacity of nitrogen removal in a hydroxyapatite (HAP)-enhanced one-stage partial nitritation/anammox process

J. Chem. Eng.

(2023)

K. Kubota et al.Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridizatio with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA–FISH)

J. Microbiol. Methods

(2006)

K. Kubota et al.Niche differentiation of phenol-degrading Microbial cells in UASB granular sludge as revealed by fluorescence in situ hybridization

Eng.

(2022)

C. Moraru et al.Crystal ball: fluorescence in situ hybridization in the age of super-resolution microscopy

Syst. Appl. Microbiol.

(2012)

S. Schlafer et al.Confocal microscopy imaging of the biofilm matrix

J. Microbiol. Methods

(2017)

P. Travascio et al.DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex

Chem. Biol.

(1998)

Brüwer, J. D., Orellana, L. H., Sidhu, C., Klip, H. C., Meunier, C. L., Boersma, M., Wiltshire K. H., Amann R., Fuchs,...H. Daims et al.Daime, a novel image analysis program for microbial ecology and biofilm research

Environ Microbiol.

(2006)

P. Greenspan et al.Nile red: a selective fluorescent stain for intracellular lipid droplets

J. Cell Biol.

(1985)

A.H. Hopman et al.Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification

J. Histochem. Cytochem

(1998)

K. KubotaCARD-FISH for environmental microorganisms: technical advancement and future applications

Microbes Environ.

(2013)

View full text

© 2023 Elsevier GmbH. All rights reserved.