User:Roberta Diaz Jimenez/Notebook/CHEM 472/2016/04/13

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Objective

  1. Conduct UV/Vis and Fluorescence analysis for the reacted 20mL samples prepared the day before.
  2. Perform a fifth disc diffusion assay using newly made metal-protein NP samples.

Materials

  1. Mueller Hinton Agar self-prepared (March 2)tored in fridge
  2. Whatman Filter Paper (6mm discs punched out from larger discs)*
    1. Switched from Whatman membrane filter paper discs, to these which are much more absorbent, and show no leakage whatsoever, we probably should have been using these from the beginning
  3. Autoclaved in dry cycle
  4. Dilutions of Protein Nanoparticle Samples and Ampicillin Control
    1. Used the metal-protein nanoparticle solutions synthesized on April 12.

Methods

  • Silver and gold protein nanoparticle dilutions performed following the dilution factors below:
Concentration (x)
1
0.5
0.25
0.125
0.0625
  • AMP control dilutions
Concentration (mg/L) Concentration (µg/µL) Volume (µL) Mass (µg)
40.004250.1
80.008250.2
160.016250.4
320.032250.8
640.064251.6
  • Disc Diffusion
  1. Thaw E. Coli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 mins over ice
  2. Dilute E. Coli thawed stock to OD600 (absorbance at 600nm) of 0.08-.1 (specifically 0.1A)
  3. Plate 100µl sample of diluted thawed E. Coli solution onto Mueller Hinton agar plates by first pipetting the volume and then using a triangular spreader in circles
  4. Load 20µl with pipette onto 6mm Whatman membrane filter paper discs for each sample and load onto plates 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 in incubator 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 these diameters and photograph the discs for future analysis.
  • Note: on March 30, noticed that loading 10µl onto the membrane filter paper discs resulted in significantly decreased inhibition which is likely due to such a small volume loaded onto the discs. Increased the amount for that assay to 15µl and note changes in inhibition. 15µl were chosen since it is a middle point between 10µl (which was too little), and 20µl (which was too much).
  • Note: plating of the discs 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.

Results

Agar disc diffusion specific dilution data, results and pictures can be found at the link SRB Lab Notebook Data for this entry's date.



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