Measurements of cell density by spectrophotometry
A 1 mL sample of culture was centrifuged at 3,000 × g for 10 seconds to
separate sulfur from the solution. The remaining solution was then
centrifuged at 17,000 × g for 1 minute to pellet cells. The supernatant
was removed, and the cells were resuspended in 1 mL of 5 mM Tris-HCl,
adjusted to pH 8.5. The suspended cells were incubated at 95 °C for 10
minutes. In a 96-well plate, 200 µL of the cell solution was added to 50
µL of a 5× SYBR Green I solution diluted with 5 mM Tris-HCl and
incubated at room temperature for 10 minutes. The fluorescence was
measured at an excitation wavelength of 497 nm and an emission
wavelength 520 nm using a Molecular Devices SpectraMax M2
spectrophotometer after a background subtraction using 200 µL
ddH2O with the SYBR stain. Any fluorescence values below
the background were set at zero.
The fluorescence measurements were converted to OD600measurements using a power function regressing known
OD600 concentrations of A. ferrooxidanssuspensions with the above protocol (Fig. S1).