User:Roberta Diaz Jimenez/Notebook/CHEM 472/2016/04/06: Difference between revisions
From OpenWetWare
Line 54: | Line 54: | ||
#Dilute E. Coli thawed stock to OD600 (absorbance at 600nm) of 0.08-0.1 (specifically 0.1A) | #Dilute E. Coli thawed stock to OD600 (absorbance at 600nm) of 0.08-0.1 (specifically 0.1A) | ||
#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 | #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 | ||
#Soak individual 6mm Whatman membrane filter paper discs in | #Soak individual 6mm Whatman membrane filter paper discs directly in sample for 30 mins and load onto plates with tweezers | ||
#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 of incubation | #Incubate at 37˚C overnight in incubator and check results after 24 hours of incubation | ||
#After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs. Results noted and discs photographed for later analysis. | #After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs. Results noted and discs photographed for later analysis. | ||
*Note: on March 30, loaded 10ul onto the membrane filter paper discs which resulted in significantly decreased inhibition very likely due to such low volume. | *Note: on March 30, loaded 10ul onto the membrane filter paper discs which resulted in significantly decreased inhibition very likely due to such low volume. Soaking directly in sample for 30mins and note if there is inhibition; this would allow for a method that prevents any leaking to occur once the discs are placed on the plate. | ||
*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 13:43, 4 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||
ObjectivePerform fourth agar diffusion disc assay with increased volume loaded onto discs. Materials
Procedure
Results
|