8. Add 100 μL of Substrate Solution

8. Add 100 μL of Substrate Solution to each well. Incubate for 30 minutes at room temperature on the benchtop. Protect from light.9. Add 100 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings madedirectly at 450 nm without correction may be higher and less accurate.