A rapid population method for action spectra applied to Halobacterium halobium.

We have developed a simple and rapid technique for measuring the action spectra for phototaxis of populations of microorganisms and applied it to halobacteria. A microscope with a dark-field condenser was used to illuminate the cell suspension in a sealed chamber with light of wavelength greater than 750 nm; in this region of the spectrum, the halobacteria show no phototactic response. A 150-micron spot of light from a xenon arc lamp, whose wavelength and intensity can be varied, was projected through the objective lens into the center of the dark field. The objective lens imaged this measuring spot through a 780-nm cut-off filter on an aperture in front of a photomultiplier. The intensity of the scattered 750-nm light, and therefore the photomultiplier current, is proportional to the number of cells in the measuring spot. A third lamp provided background light of variable wavelength and intensity through the dark-field condenser. To minimize secondary effects due to large changes in cell density, we recorded the initial changes in the photomultiplier current over 1 min after the actinic light had been switched on. By plotting the rate of change against wavelength, we obtained action spectra after the proper corrections for changes in light intensity with wavelength were applied and saturation effects were avoided.