User:Benjamin Friedel/Notebook/CHEM 471/2016/04/13

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Objectives

  1. Perform 5th Agar Disc Diffusion Assay


Procedure

Materials for Agar Disc Diffusion

  1. Mueller Hinton Agar self-prepared 03/2/16 and stored in fridge
    1. Whatman Filter Paper (6mm discs punched out from larger discs)*

-changed from Whatman membrane filter paper discs, these regular Whatman filter paper discs are MUCH more absorbent, with no leakage whatsoever, we probably should have been using these from the beginning -autoclaved in dry cycle

    1. Dilutions of Protein Nanoparticle Samples and Ampicillin Control

-used 4/12/16 reaction products for this set of disc diffusion tests

      1. Protein-Nanoparticle samples and Ampicillin Control Dilutions

AMP Table

Concentration (mg/L) Concentration (ug/uL) Volume (ul) Mass (ug)
40.004250.1
80.008250.2
160.016250.4
320.032250.8
640.064251.6


Example Nanoparticle Sample Dilutions

Concentration (x)
1
0.5
0.25
0.125
0.0625


  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

Disc Diffusion Procedure

  1. Thaw Ecoli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice
  2. Dilute Ecoli thawed stock to OD600 (absorbance at 600nm) of .08-.1 (specifically .1A)
  3. Plate 100ul sample of diluted thawed Ecoli solution onto mueller hinton agar plates by first pipetting the volume and then using a triangular spreader in circles
  4. Load 20ul with pipette ontol 6mm Whatman membrane filter paper discs for each sample and load onto plates with tweezers

-it was noted on 03/30/16 that loading 10ul onto the membrane filter paper discs did not result much less inhibition may be due to less volume loaded , this time load 15ul and see if there is still inhibition as to lessen the load on the filter paper discs from 20ul as well as increase load from 10ul

  1. place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover plate
  2. incubate at 37˚C overnight in incubator and check results after 24 hours incubation

Note that this technique was conducted on the benchtop. 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.

Checking the discs

  1. After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs


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