DETERMINATION OF COMPLEMENT-MEDIATED KILLING OF BACTERIA BY VIABILITYSTAINING AND BIOLUMINESCENCE

Complement-mediated killing of bacteria was monitored by Bow cytometri c, luminometric, and conventional plate counting methods, A flow cytom etric determination of bacterial viability was carried out by using du al staining with a LIVE/DEAD BacLight bacterial viability kit, In addi tion to the viable cell population, several other populations Emerged in the fluorescence histogram, and there was a dramatic decrease in th e 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 seru m complement can be measured rapidly and conveniently by using viabili ty stains or bioluminescence, In principle, any bacterial strain can b e used for viability staining and Bow cytometric analysis. For the bio luminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a lar ge number of samples.