User:Benjamin Friedel/Notebook/CHEM 471/2016/04/05: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 64: Line 64:
# Thaw Ecoli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice  
# Thaw Ecoli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice  
# Dilute Ecoli thawed stock to OD600 (absorbance at 600nm) of .08-.1 (specifically .1A)
# Dilute Ecoli thawed stock to OD600 (absorbance at 600nm) of .08-.1 (specifically .1A)
###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
#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
####Load 10ul of sample dilutions or control dilution onto their own individual 6mm Whatman membrane filter paper discs with tweezers
#Load 10ul of sample dilutions or control dilution onto their own individual 6mm Whatman membrane filter paper discs with tweezers
-it was noted on 03/30/16 that loading 10ul onto the membrane filter paper discsdid 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
-it was noted on 03/30/16 that loading 10ul onto the membrane filter paper discsdid 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
##### place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover plate
# place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover plate
###### incubate at 37˚C overnight in incubator and check results after 24 hours incubation
# 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.
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.

Revision as of 14:57, 3 May 2016

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Objectives

  1. Perform 3rd 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 Membrane Filter Paper (6mm discs punched out from larger discs)

-autoclaved in dry cycle

    1. Dilutions of Protein Nanoparticle Samples and Ampicillin Control
      1. Protein-Nanoparticle samples and Ampicillin Control Dilutions

AMP Table

Concentration (mg/L) Concentration (ug/uL) Volume (ul) Mass (ug)
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


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 10ul of sample dilutions or control dilution onto their own individual 6mm Whatman membrane filter paper discs with tweezers

-it was noted on 03/30/16 that loading 10ul onto the membrane filter paper discsdid 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

-Bacterial growth was very spotty and inconsistent this week, not growing in lawns but many separate colonies so even though it looked like there were inhibition diameters, they were impossible to accurately measure due to the growth - see pictures at link above


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Benjamin_Friedel/Notebook/CHEM_471/2016/04/05&oldid=940693"