Detection of Microorganisms by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid

  • Ricardo Oliveira
  • Carina Almeida
  • Nuno F. AzevedoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2105)


Fluorescence in situ hybridization (FISH) is a 30-year-old technology that has evolved continuously and is now one of the most well-established molecular biology techniques. Traditionally, DNA probes are used for in situ hybridization. However, synthetic molecules are emerging as very promising alternatives, providing better hybridization performance and making FISH procedures easier and more efficient. In this chapter, we describe a universal FISH protocol, using nucleic acid probes, for the detection of bacteria. This protocol should be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and their distribution patterns in different types of samples.

Key words

Microorganisms PNA FISH Pure cultures Enriched samples Histological samples Biofilms 



This work was financially supported by: the project UID/EQU/00511/2019—Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE funded by national funds through FCT/MCTES (PIDDAC); the project POCI-01-0145-FEDER-030431, the project PTDC/DTP-PIC/4562/2014—POCI-01-0145-FEDER-016678, the project POCI-01-0145-FEDER-029961, and the project POCI-01-0145-FEDER-031011 funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; and the project “LEPABE-2-ECO-INNOVATION”—NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors also thank FCT for the PhD Fellowship SFRH/BD/138883/2018.


  1. 1.
    Guimarães N, Azevedo NF, Figueiredo C et al (2007) Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens. J Clin Microbiol 45:3089–3094. Scholar
  2. 2.
    Bayani J, Squire JA (2007) Application and interpretation of FISH in biomarker studies. Cancer Lett 249:97–109. Scholar
  3. 3.
    Rogers SW, Moorman TB, Ong SK (2007) Fluorescent in situ hybridization and micro-autoradiography applied to ecophysiology in soil. Soil Sci Soc Am J 71:620. Scholar
  4. 4.
    Dmochowski IJ, Tang X (2007) Taking control of gene expression with light-activated oligonucleotides. Biotechniques 43:161, 163, 165 passimCrossRefGoogle Scholar
  5. 5.
    Catalina P, Cobo F, Cortés JL et al (2007) Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review. Cell Biol Int 31:861–869. Scholar
  6. 6.
    DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243:1360–1363CrossRefGoogle Scholar
  7. 7.
    Levsky JM, Singer RH (2003) Fluorescence in situ hybridization: past, present and future. J Cell Sci 116:2833–2838. Scholar
  8. 8.
    Almeida C, Azevedo NF, Iversen C et al (2009) Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (enterobacter sakazakii) in powdered infant formula. Appl Environ Microbiol 75:2925–2930. Scholar
  9. 9.
    Cerqueira L, Azevedo NF, Almeida C et al (2008) DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int J Mol Sci 9:1944–1960. Scholar
  10. 10.
    Rohde A, Hammerl JA, Appel B et al (2015) FISHing for bacteria in food – a promising tool for the reliable detection of pathogenic bacteria? Food Microbiol 46:395–407. Scholar
  11. 11.
    Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6(5):339–348CrossRefGoogle Scholar
  12. 12.
    Felix H (1982) Permeabilized cells. Anal Biochem 120:211–234CrossRefGoogle Scholar
  13. 13.
    Thavarajah R, Mudimbaimannar VK, Elizabeth J et al (2012) Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol 16:400–405. Scholar
  14. 14.
    Azevedo N (2005) Survival of Helicobacter pylori in drinking water and associated biofilms. Dissertation for PhD degree in Chemical and Biological Engineering. University of MinhoGoogle Scholar
  15. 15.
    Amann R, Kühl M (1998) In situ methods for assessment of microorganisms and their activities. Curr Opin Microbiol 1:352–358. Scholar
  16. 16.
    Jasson V, Jacxsens L, Luning P et al (2010) Alternative microbial methods: an overview and selection criteria. Food Microbiol 27:710–730. Scholar
  17. 17.
    Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500CrossRefGoogle Scholar
  18. 18.
    Stender H, Mollerup TA, Lund K et al (1999) Direct detection and identification of Mycobacterium tuberculosis in smear-positive sputum samples by fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes. Int J Tuberc Lung Dis 3:830–837PubMedGoogle Scholar
  19. 19.
    Azevedo NF, Vieira MJ, Keevil CW (2003) Establishment of a continuous model system to study Helicobacter pylori survival in potable water biofilms. Water Sci Technol 47:155–160CrossRefGoogle Scholar
  20. 20.
    Juhna T, Birzniece D, Larsson S et al (2007) Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks. Appl Environ Microbiol 73:7456–7464. Scholar
  21. 21.
    Bragança SM, Azevedo NF, Simöes LC et al (2007) Use of fluorescent in situ hybridisation for the visualisation of Helicobacter pylori in real drinking water biofilms. Water Sci Technol 55:387–393CrossRefGoogle Scholar
  22. 22.
    Gião MS, Wilks SA, Azevedo NF et al (2009) Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms. Biofouling 25:335–343. Scholar
  23. 23.
    Almeida C, Azevedo NF, Santos S et al (2011) Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One 6:e14786. Scholar
  24. 24.
    Azevedo NF, Jardim T, Almeida C et al (2011) Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples. Antonie van Leeuwenhoek 100:463–470. Scholar
  25. 25.
    Almeida C, Azevedo NF, Bento JC et al (2013) Rapid detection of urinary tract infections caused by Proteus spp. using PNA-FISH. Eur J Clin Microbiol Infect Dis 32:781–786. Scholar
  26. 26.
    Almeida C, Azevedo NF, Fernandes RM et al (2010) Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of salmonella spp. in a broad spectrum of samples. Appl Environ Microbiol 76:4476–4485. Scholar
  27. 27.
    Almeida C, Sousa JM, Rocha R et al (2013) Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method. Appl Environ Microbiol 79:6293–6300. Scholar
  28. 28.
    Machado A, Almeida C, Carvalho A et al (2013) Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples. Int J Food Microbiol 162:64–70. Scholar
  29. 29.
    Almeida C, Cerqueira L, Azevedo NF, Vieira MJ (2013) Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples. Int J Food Microbiol 161:16–22. Scholar
  30. 30.
    Rocha R, Almeida C, Azevedo NF (2018) Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria. PLoS One 13:e0196522. Scholar
  31. 31.
    Cerqueira L, Fernandes RM, Ferreira RM et al (2011) PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori. BMC Microbiol 11:101. Scholar
  32. 32.
    Nielsen PE (2001) Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Curr Opin Biotechnol 12:16–20CrossRefGoogle Scholar
  33. 33.
    Perry-O’Keefe H, Rigby S, Oliveira K et al (2001) Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods 47:281–292CrossRefGoogle Scholar
  34. 34.
    Orum H, Nielsen PE, Jørgensen M et al (1995) Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection. Biotechniques 19:472–480PubMedGoogle Scholar
  35. 35.
    Mendes L, Rocha R, Azevedo AS et al (2016) Novel strategy to detect and locate periodontal pathogens: the PNA-FISH technique. Microbiol Res 192:185–191. Scholar
  36. 36.
    Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J (2002) PNA for rapid microbiology. J Microbiol Methods 48:1–17. Scholar
  37. 37.
    Wagner M, Horn M, Daims H (2003) Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6:302–309. Scholar
  38. 38.
    Drobniewski FA, More PG, Harris GS (2000) Differentiation of Mycobacterium tuberculosis complex and nontuberculous mycobacterial liquid cultures by using peptide nucleic acid-fluorescence in situ hybridization probes. J Clin Microbiol 38:444–447PubMedPubMedCentralGoogle Scholar
  39. 39.
    Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770CrossRefGoogle Scholar
  40. 40.
    Stevens KA, Jaykus LA (2004) Bacterial separation and concentration from complex sample matrices: a review. Crit Rev Microbiol 30:7–24. Scholar
  41. 41.
    Yilmaz LS, Noguera DR (2004) Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization. Appl Environ Microbiol 70:7126–7139. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Ricardo Oliveira
    • 1
    • 2
  • Carina Almeida
    • 1
    • 2
    • 3
  • Nuno F. Azevedo
    • 1
    Email author
  1. 1.LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of EngineeringUniversity of PortoPortoPortugal
  2. 2.INIAV - National Institute for Agrarian and Veterinarian ResearchVairaoPortugal
  3. 3.CEB - Centre of Biological EngineeringUniversity of MinhoBragaPortugal

Personalised recommendations