The demonstration of photochemical reducing sites in Chromatium sp.

DYAn [I] used blue tetrazolium to demonstrate intracellular particles exhibiting marked reducing activity in illuminated and in non-illuminated plant tissues. The blue-black reduced tetrazolium appeared in granules dispersed throughout the cytoplasm of blue-green algae and within the intracellular chloroplasts of green algae and higher plants. Since the dye was reduced more rapidly and at more numerous sites in illuminated than in non-illuminated cells, she concluded that these stained particles probably represented grana. We have demonstrated similar sites in isolated bean leaf chloroplasts [4]. The present investigation was undertaken to adapt the blue tetrazolium technique to the study of the photosynthetic bacterium, Chromatium sp., and to demonstrate, if possible, the intracellular loci of photochemical reduction. The preliminary results reported here indicate that this technique is more convenient and may prove to be more specific and more reliable than growth or respirometric methods for studying the photoskythetic and dark metabolism of specific substrate by these organisms. Cells were grown in liquid media containing either sodium sulfide or sodium thiosulfate [3], harvested by centrifugation, washed twice with and finally resuspended in 0.01 M phosphate buffer, pH 7.0. The indicator solution was prepared by dissolving 1 mg of tetrazolium blue (Nutritional Biochemicals Corporation) in 30 ml of 0.03 M phosphate buffer, pH 7.0. Solutions of substrate were prepared in tap water and contained either 0.5 per cent Na,S * 9 H,O or 1.0 per cent Na,S,O, * 5 H,O. One ml of the indicator solution was mixed in a Thunberg tube with 1 ml of a IO per cent suspension of cells and 1 ml of either tap water or substrate solution. The tubes were evacuated for three minutes with a water aspirator and sealed. All tubes were prepared in duplicate. One series was placed in a water bath at 27°C and illuminated from below with a loo-watt incandescent bulb in a reflector desk lamp. The second series was completely wrapped with aluminium foil to exclude light and placed in the same bath. The incubation period was varied from I to 24 hours. Although we have not been able to prevent photoreduction of tetrazolium in illuminated cells lacking exogenous subtrate, we have succeeded in delaying for more than an hour any detectable evidence for the reduction by using cells which had been depleted of interfering endogenous materials (starved cells). Usually, intense illumination for two hours was sufficient to starve buffered suspensions of cells which had been harvested from thiosulfate medium. Cells grown in sulfide medium normally contained intracellular sulfur droplets which disappeared only after several days of illumination. Consequently, sulfid-grown cells were not starved for the tetrazolium tests.