Caged oxygen compound (4.37 mM final concentration) was added to B. subtilis and E. coli cells in LB media and to B. halodurans in Horikoshi II media (OD600= 0.35). Flat capillary tubes (0.1×1.0 mm, #5010, VitroCom, NJ, USA) were then half-filled with the bacterial suspension/caged oxygen mixture. The capillaries were sealed at both ends with Critoseal (Monoject. Sci. Division, MO, USA), placed on the microscope stage, and observed with a 40× glass phase-contrast objective. A green interference filter was interposed between the sample and light source to enhance video contrast and facilitate tracking of motile cells. Data collection occurred 30 to 45 min after preparation of the capillary tubes. Duration of the near-UV light flash was 900 ms for B. subtilis and 100 ms for E. coli and B. halodurans. Images of swimming bacteria were captured with a CCD camera and digitized with a VP110 video digitizer (VP120 digitizer, Expert Vision 2D/AT Release 3.1 version software; Motion Analysis, Santa Rosa, CA, USA). The digitizer was equipped with a −5 V event tone marker that signals the opening of the shutter (UniBlitz, Vincent Associates, Rochester, NJ, USA) with a 5 s delay. A custom-made NOT gate circuit connected to a Grass stimulator was interposed between the digitizer and shutter driver/timer (UniBlitz, model T132) to convert the event tone to a +35 V pulse needed to activate the shutter system. Illumination from a 100-W mercury short arc lamp (HBO 100 W/2, Osram, Munich, Germany) was passed through the electronic shutter and filtered through a Nikon fluorescence cube (360±20 nm excitation, >400 nm barrier filter, 400 nm dichroic mirror). Intensity of the UV light, measured with a silicon photodiode (XRL340B #5348, International Light, Newburyport, MA, USA), was 1.60×10−4 W cm−2. The amount of oxygen released from 4.37 mM caged oxygen was estimated to be approximately 3.27× 10−13 mol (0.046 pmol) for a 900-ms flash and 3.63×10−15 mol (0.0051 pmol) for a 100-ms flash (calculations not shown) using a quantum yield of 0.1.
When flash photolysis was performed immediately after bacteria were introduced into the capillary tube, wild-type B. subtilis failed to respond because receptors were saturated with oxygen. A 30–60 min incubation period was used to assure a semi-anaerobic environment. A field of view (approximately 150×150 μm) containing 20–40 cells with a rate of change of direction (rcd) = 700–800° s−1 for B. subtilis and 700–1200° s−1 for E. coli was selected for observation. Data collection began 5 s before the flash, and an average of 1000 paths (30 repetitive assays) were recorded for 30 s. A stage micrometer was used to calibrate image measurements (scale factor = 0.795 μm/pixel). Although UV light acts as a repellent, it does not prevent a positive response to oxygen to occur.