User:Melissa Novy/Notebook/CHEM-572/2013/02/27

Objectives

• Conduct 15-hour study of DH5α-T1 cell growth in different concentrations of Ag⁺.

DH5α-T1 Cell Growth Inhibition Pilot Study

• Protocol
  1. Six 250-mL Erlenmeyer flasks each containing 50 mL of sterile LB media, made on 2013/02/19 were obtained.
  2. According to the data and calculations made on 2013/02/20, a specific volume of LB media was removed from each flask and then replaced with the same volume of 0.1 M or 0.0105 M AgNO₃. Please refer to the entry on 2013/02/20 for the calculated volumes. Note that there were five experimental solutions in total.
  3. Then, to each of these five flasks, 1 mL of DH5α-T1 starter culture, made on 2013/02/19 was added.
  4. To one of the remaining flasks containing LB media, 5000 μL of solution was removed and replaced with 5000 μL 0.1 M AgNO₃ was added, to produce a final AgNO₃ concentration of 10 mM. This flask was one of the control solutions.
  5. To the last flask containing LB media, 1 mL of DH5α-T1 starter culture was added. This flask was another control solution.
  6. The seven flasks were placed in an orbital shaker set to 37°C and 230 rpm and shaken for about 1 min.
  7. Then, 3 mL of solution was pipetted into a plastic cuvette using a sterile pipet tip, and the absorbance of the solution at 600 nm was recorded.
  8. The solution was then poured back into the flask from which it was obtained.
  9. Steps 7 and 8 were repeated for all seven flasks, upon which shaking of all the flasks resumed.
  10. The process of measuring the absorbance of the solutions at 600 nm was repeated every hour for a period of 15 hours.

• Observations
  • Immediately after AgNO₃ was added to the LB media, a slight pale gray or white precipitate was observed in the flask. This is likely due to the precipitation of Ag out of solution. However, the precipitate dissipated upon swirling and subsequent shaking of the flasks.
  • The Ag present in the control flask containing LB media and 10 mM AgNO₃ and the experimental flask containing LB media, DH5α-T1 cells, and 10 mM AgNO₃ both oxidized over time from a pale white color to a dark purple gray color.