- Prepare the bacterial culture as described in protocol #4, and then adjust the bacterial population to 106 - 107 cfu/ml.
- For the end-point measurement of HMC/PPO:
(a) incubate the bacteria under test at 106 cfu/ml with 60 μg purified HMC plus 100 nmol 4-methylcatechol (4ME) in 100 μl PBS at 37 °C for 1 h.
(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 μl of serially diluted samples on nutrient agar plates and incubating at 37 °C overnight. Perform the colony count and calculate the bacterial cfu/ml.
(c) add 10 nmol of phenylthiourea (PTU) to the reactions in order to evaluate the impact of PO activity.
(d) also set up controls by incubating bacteria with HMC/PPO or 4ME or PTU separately, or in combinations.
- For real-time imaging of the bacterial clearance elicited by HMC/PPO:
(a) mix each bacterial strain at 107 cfu/ml with 60 μg HMC/PPO and 100 nmol 4ME in 100 μl PBS.
(b) apply one μl of the mixture to fluorescence microscopy and examine it at magnification of 63 x 1.6, with time-elapse method.
(c) capture images at intervals of 30 s for 1 h, and make a movie.
- For the end-point measurement of metHb:
(a) incubate the bacteria under test at 106 cfu/ml with 36 μg metHb and 1.6 μmol H2O2 in 200 μl PBS at 37 °C for 1 h.
(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 μl of serially diluted samples on nutrient agar plates and incubating at 37 °C overnight. Perform the colony count and calculate the bacterial cfu/ml.
(c) to further prove that the antibacterial activity was indeed attributable to ROS, apply 2 μmol GSH to the incubation mixture to quench the superoxide ions.
(d) set up controls by incubating bacteria with metHb, H2O2 or GSH separately or in combinations.