Abstract
Fluorescence in situ hybridization (FISH) has been widely used for the detection and identification of microorganisms in their natural environments. In this chapter we describe the use of a simple FISH-based protocol to detect and identify clinically relevant yeast species in culture and biological samples using Cryptococcus neoformans as a model. After fixation of cells with paraformaldehyde, the same are embedded in hybridization buffer containing specific fluorochrome-labeled oligonucleotide probes. After incubation and a subsequent washing step for removing unbound probes, samples are analyzed by epifluorescence microscopy.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Amann R, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
Moter A, Göbel UB (2000) Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Methods 41:85–112
Zwirglmaier K (2005) Fluorescence in situ hybridisation (FISH)—the next generation. FEMS Microbiol Lett 246:151–158
Alexander BD, Ashley ED, Reller LB et al (2006) Cost savings with implementation of PNA FISH testing for identification of Candida albicans in blood cultures. Diagn Microbiol Infect Dis 54:277–282
Martins ML, Ferreira AS, Sampaio A et al (2010) Direct and specific identification of Cryptococcus neoformans in biological samples using fluorescently-labelled DNA probes. Eur J Clin Microbiol Infect Dis 29:571–576
Stender H, Kurtzman C, Hyldig-Nielsen JJ et al (2001) Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes. Appl Environ Microbiol 67:938–941
Tavares S, Inácio J, Fonseca Á et al (2004) Direct detection of Taphrina deformans on peach trees using molecular methods. Eur J Plant Pathol 110:973–982
Xufre A, Albergaria H, Inácio J et al (2006) Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations. Int J Food Microbiol 108:376–384
Andrews JH, Spear RN, Nordheim EV (2002) Population biology of Aureobasidium pullulans on apple leaf surfaces. Can J Microbiol 48:500–513
Li S, Spear RN, Andrews JH (1997) Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscopic slides and leaf surfaces. Appl Environ Microbiol 63:3261–3267
Inácio J, Ludwig W, Spencer-Martins I et al (2010) Assessment of phylloplane yeasts on selected Mediterranean plants by fluorescence in situ hybridisation (FISH) with group- and species-specific oligonucleotide probes. FEMS Microbiol Ecol 71:61–72
Behrens S, Rühland C, Inácio J et al (2003) In situ accessibility of small subunit ribosomal RNA of members of the domains bacteria, archaea and eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microbiol 69:1748–1758
Inácio J, Behrens S, Fuchs BM et al (2003) In situ accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-labelled oligonucleotide probes comprising the D1 and D2 domains. Appl Environ Microbiol 69:2899–2905
Casadevall A, Perfect JR (1998) Cryptococcus neoformans. ASM Press Books, Washington, DC
Amann R, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770
Fuchs BM, Wallner G, Beisker W et al (1998) Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 64:4973–4982
Fuchs BM, Syutsubo K, Ludwig W et al (2001) In Situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 67:961–968
Tavares A, Inácio J, Melo-Cristino J et al (2008) Use of fluorescence in situ hybridization for rapid identification of staphylococci in blood culture samples collected in a Portuguese hospital. J Clin Microbiol 46:3097–3100
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Inácio, J., da Luz Martins, M. (2013). Microscopic Detection of Yeasts Using Fluorescence In Situ Hybridization. In: O'Connor, L., Glynn, B. (eds) Fungal Diagnostics. Methods in Molecular Biology, vol 968. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-257-5_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-257-5_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-256-8
Online ISBN: 978-1-62703-257-5
eBook Packages: Springer Protocols