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Applied and Environmental Microbiology, January 2003, p. 468-474, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.468-474.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Online Monitoring of Escherichia coli Ghost Production

W. Haidinger,1,2* M. P. Szostak,1,3 W. Jechlinger,1,2 and W. Lubitz2

Apovia AG, D-82152 Martinsried, Germany,1 Institute of Microbiology and Genetics, University of Vienna, UZAI, A-1090 Vienna,2 BIRD-C GmbH & Co. KEG, A-1080 Vienna, Austria3

Received 19 June 2002/ Accepted 31 October 2002

Controlled expression of cloned {phi}X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.

* Corresponding author. Mailing address: Institute of Microbiology and Genetics, University of Vienna, UZAI, Althanstrasse 14, A-1090 Vienna, Austria. Phone: 43 1 4277 54651. Fax: 43 1 4277 54655. E-mail: haiding{at}gem.univie.ac.at.

Applied and Environmental Microbiology, January 2003, p. 468-474, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.468-474.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ebensen, T., Paukner, S., Link, C., Kudela, P., de Domenico, C., Lubitz, W., Guzman, C. A. (2004). Bacterial Ghosts Are an Efficient Delivery System for DNA Vaccines. J. Immunol. 172: 6858-6865 [Abstract] [Full Text]  
  • Haidinger, W., Mayr, U. B., Szostak, M. P., Resch, S., Lubitz, W. (2003). Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease. Appl. Environ. Microbiol. 69: 6106-6113 [Abstract] [Full Text]  


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