Cell growth quantification of mercury resistant Escherichia coli DH5α in differents Hg concentrations by Optical Density

Reagents:

- Mercury chloride 20mg/ml;

- Chloramphenicol 34 µg/ml;

- Luria-Bertani + Mercury (LM) medium.

Steps:

1. Pre-inoculum: Inoculate bacteria (DH5α transformed with BBa_K1355004) in Luria-Bertani + Mercury (LM) 0.05µg/ml + chloramphenicol 34µg/ml. Repeat the process for bacteria control with no high Hg resistance.

2. Incubate in shaker overnight at 37 °C.

3. Measure optical density (O.D.) of cultures in spectrophotometer at 600 nm wavelength.

4. Aliquot bacteria suspension in six tubes (50 mL) with 10 mL LM medium in the following concentrations: 0µg/ml, 1 µg/ml; 2.5 µg/ml; 5 µg/ml; 10 µg/ml; and 20 µg/ml. Use aliquots quantity according to the optical density.

5. Incubate in shaker at 37 °C for 48 hours. Measure optical density (O.D.) of cultures in: 2.5 hours – 4.5 hours – 6.5 hours – 17 hours – 24 hours – 43 hours and 48 hours.

6. After 48 hours of growth, measure the final concentration of mercury present in each sample in Direct Mercury Analyzer (DMA-80).