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Detecting inactivated endospores in fluorescence microscopy using propidium monoazide

Published online by Cambridge University Press:  17 January 2012

Alexander Probst
Affiliation:
Department for Microbiology and Archaea Centre, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany
Alexander Mahnert
Affiliation:
Department for Microbiology and Archaea Centre, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany
Christina Weber
Affiliation:
Compliance – Advice and Services in Microbiology GmbH, Robert-Perthel-Straße 49, 50739 Cologne, Germany Deutsches Wollforschungsinstitut DWI, Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Interactive Materials Research, Pauwelsstraße 8, 52056 Aachen, Germany
Klaus Haberer
Affiliation:
Compliance – Advice and Services in Microbiology GmbH, Robert-Perthel-Straße 49, 50739 Cologne, Germany
Christine Moissl-Eichinger*
Affiliation:
Department for Microbiology and Archaea Centre, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany

Abstract

The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2012

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References

Anonymous (2002). COSPAR/IAU Workshop on Planetary Protection. Committee on Space Research (COSPAR), International Council for Science, Paris, France (Amended 2011). http://cosparhq.cnes.fr/Scistr/PPPolicy%20%2824Mar2011%29.pdf.Google Scholar
Beaman, T.C., Pankratz, H.S. & Gerhardt, P. (1972). J. Bacteriol. 109, 11981209.CrossRefGoogle Scholar
Bechtel, D.B. & Bulla, L.A. Jr. (1976). J. Bacteriol. 127, 14721481.CrossRefGoogle Scholar
Blocher, J.C. & Busta, F.F. (1985). Appl. Environ. Microbiol. 50, 274279.CrossRefGoogle Scholar
Boulos, L., Prevost, M., Barbeau, B., Coallier, J. & Desjardins, R. (1999). J. Microbiol. Methods 37, 7786.CrossRefGoogle Scholar
Coleman, W.H., Zhang, P., Li, Y.-Q. & Setlow, P. (2010). Lett. Appl. Microbiol. 50, 507514.CrossRefGoogle Scholar
Cook, A.M. & Brown, M.R.W. (1964). J. Pharm. Pharmacol. 16, 725732.CrossRefGoogle Scholar
Driks, A. (1999). Microbiol. Mol. Biol. Rev. 63, 120.CrossRefGoogle Scholar
Fast, P.G. (1972). J. Invertebr. Pathol. 20, 139140.CrossRefGoogle Scholar
Foerster, H.F. & Foster, J.W. (1966). J. Bacteriol. 91, 11681177.CrossRefGoogle Scholar
Ghosh, S. & Setlow, P. (2009). J. Bacteriol. 191, 17811797.Google Scholar
Kondo, M. & Foster, J.W. (1967). J. Gen. Microbiol. 47, 257271.CrossRefGoogle Scholar
La Duc, M., Dekas, A., Osman, S., Moissl, C., Newcombe, D. & Venkateswaran, K. (2007). Appl. Environ. Microbiol. 73, 26002611.CrossRefGoogle Scholar
LaFlamme, C., Verreault, D., Lavigne, S., Trudel, L., Ho, J. & Duchaine, C. (2005). Front. Biosci. 10, 16471653.CrossRefGoogle Scholar
Liu, H., Bergman, N.H., Thomason, B., Shallom, S., Hazen, A., Crossno, J., Rasko, D.A., Ravel, J., Read, T.D., Peterson, S.N., et al. (2004). J. Bacteriol. 186, 164178.CrossRefGoogle Scholar
Magge, A., Setlow, B., Cowan, A.E. & Setlow, P. (2009). J. Appl. Microbiol. 106, 814824.CrossRefGoogle Scholar
Mohapatra, B.R. & La Duc, M.T. (2011). Microbiol. Immunol., Epub ahead of print, doi: 10.1111/j.1348-0421.2011.00404.x.Google Scholar
Nicholson, W. L. & Setlow, P. (1990). Molecular biological methods for Bacillus, Sporulation Germination and outgrowth, pp. 391450.Google Scholar
Nocker, A., Cheung, C.Y. & Camper, A.K. (2006). J. Microbiol. Methods, 67, 310320.CrossRefGoogle Scholar
Nocker, A., Mazza, A., Masson, L., Camper, A.K. & Brousseau, R. (2009). J. Microbiol. Methods, 76, 253261.CrossRefGoogle Scholar
Nocker, A., Richter-Heitmann, T., Montijn, R., Schuren, F. & Kort, R. (2010). Int. Microbiol. 13, 5965.Google Scholar
Nocker, A., Sossa, K.E. & Camper, A.K. (2007a). J. Microbiol. Methods, 70, 252260.CrossRefGoogle Scholar
Nocker, A., Sossa-Fernandez, P., Burr, M.D. & Camper, A.K. (2007b). Appl. Environ. Microbiol. 73, 51115117.CrossRefGoogle Scholar
Preston, R.A. & Douthit, H.A. (1984). J. Gen. Microbiol. 130, 10411050.Google Scholar
Probst, A., Facius, R., Wirth, R. & Moissl-Eichinger, C. (2010). Appl. Environ. Microbiol. 76, 51485158.CrossRefGoogle Scholar
Probst, A., Facius, R., Wirth, R., Wolf, M. & Moissl-Eichinger, C. (2011). Appl. Environ. Microbiol. 77, 16281637.CrossRefGoogle Scholar
Puleo, J.R., Fields, N.D., Bergstrom, S.L., Oxborrow, G.S., Stabekis, P.D. & Koukol, R. (1977). Appl. Environ. Microbiol. 33, 379384.CrossRefGoogle Scholar
Raso, J., Gongora-Nieto, M.M., Barbosa-Canovas, G.V. & Swanson, B.G. (1998). Int. J. Food Microbiol. 44, 125132.CrossRefGoogle Scholar
Rawsthorne, H., Dock, C.N. & Jaykus, L.A. (2009). Appl. Environ. Microbiol. 75, 29362939.CrossRefGoogle Scholar
Setlow, B., Loshon, C.A., Genest, P.C., Cowan, A.E., Setlow, C. & Setlow, P. (2002). J. Appl. Microbiol. 92, 362375.CrossRefGoogle Scholar
Smoot, L. & Pierson, M.D. (1982). J. Food Prot. 45, 8492.CrossRefGoogle Scholar
Turner, L., Ryu, W.S. & Berg, H.C. (2000). J. Bacteriol. 182, 27932801.CrossRefGoogle Scholar
Vasin, V.B. & Trofimov, V.I. (1995). Adv. Space Res. 15, 273276.CrossRefGoogle Scholar
Venkateswaran, K., Satomi, M., Chung, S., Kern, R., Koukol, R., Basic, C. & White, D. (2001). Syst. Appl. Microbiol. 24, 311320.CrossRefGoogle Scholar
Waring, M.J. (1965). J. Mol. Biol. 13, 269282.CrossRefGoogle Scholar
Wirth, R., Bellack, A., Bertl, M., Bilek, Y., Heimerl, T., Herzog, B., Leiner, M., Probst, A., Rachel, R., Sarbu, C., et al. (2011). Appl. Environ. Microbiol. 77, 15561562.CrossRefGoogle Scholar