Protocol abstract
Nature Protocols 1, - 2253 - 2256 (2006)
Published online: 14 December 2006 | doi:10.1038/nprot.2006.397
Subject Categories: Cell and tissue culture | Model organisms
Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red
Arthur F J Ram1 & Frans M Klis2
Abstract
The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4–6 h (excluding the incubation time required for fungal growth).
- Institute of Biology Leiden, Leiden University, Molecular Microbiology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
- Swammerdam Institute for Life Sciences, University of Amsterdam, BioCentrum Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
Correspondence to: Arthur F J Ram1 e-mail: A.F.J.Ram@biology.leidenuniv.nl
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A-Z product listing
- 10% potassium hydroxide(Sigma)
- 860956(Sigma)
- 87% glycerol(Sigma)
- Bürker-Türk(Marienfeld)
- F-3543(Sigma)
- MES-NaOH(Sigma)
