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Appl Environ Microbiol, February 1998, p. 515-519, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence

Marko Virta,^1,2 Sanna Lineri,¹ Pasi Kankaanpää,¹ Matti Karp,² Karita Peltonen,¹ Jari Nuutila,¹ and Esa-Matti Lilius^1,*

Department of Biochemistry, University of Turku, FIN-20014 Turku,¹ and Department of Biotechnology, University of Turku, FIN-20520 Turku,² Finland

Received 23 September 1997/Accepted 13 November 1997

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.

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^* Corresponding author. Mailing address: Department of Biochemistry, University of Turku, Arcanum, Vatselankatu 2, FIN-20014 Turku, Finland. Phone: 358-2-333 6888. Fax: 358-2-333 6860. E-mail: esamatti.lilius{at}utu.fi.

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