User:Roberta Diaz Jimenez/Notebook/CHEM 472/2016/03/23

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Objectives

  1. Perform first disc diffusion assay
  2. Make agar to plate petri dishes

Materials

  1. Agar mix
  2. Three 100mL falcon tubes
  3. 2.8L flask
  4. Hot plate
  5. Two 1L screw-cap bottles
  6. 30 eppendorf tubes
  7. Protein-nanoparticle solutions
  8. Ampicilin
  9. Whatman Membrane Filter Paper (6mm discs punched out from larger discs) – autoclaved in dry cycle
  10. 20mL Ag and Au protein nanoparticle reactions done on March 15

Procedure

  • Making Agar
  1. Followed instructions on agar mix packaging
  • Dilutions of Protein Nanoparticle Samples and Ampicillin Control
Concentration (x)
1
0.5
0.25
0.125
0.0625
  • Ampicillin Control Dilutions
Concentration (mg/L) Concentration (µg/µL) Volume (µl) Mass (µg)
4 0.004 25 0.1
8 0.008 25 0.2
16 0.016 25 0.4
32 0.032 25 0.8
64 0.064 25 1.6
  • Disc Diffusion
  1. Agar disc diffusion specific dilution data, results and pictures can be found at the link SRB Lab Notebook Data for this entry's date
  2. Thaw E. Coli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice
  3. Plate 100µl sample of thawed E. Coli solution onto Mueller Hinton agar plates by pipetting volume and then spreading with a triangular sterile spreader in a circle motion
  4. Load 20µl of sample dilutions or control dilution onto their own individual 6mm Whatman membrane filter paper discs with tweezers
  5. Place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover plate
  6. Incubate at 37˚C overnight and check results after 24 hours incubation
  7. After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs
  • Note: plating was conducted on the bench top. Between each loading of a disc on a plate, the tweezers used were washed with EtOH to minimize contamination. However, the plates were open to the air for a period of time during plating and loading of discs. Because we are testing for bacterial inhibition by the nanoparticles and control, if there is contamination, but inhibition we may never know that there was contamination in the first place. Discussed with Dr. Hartings and protocol was approved.