Jeff:LabNotes/Microbiome/2009-4-3
Bacterial loading into Microwells
Purpose
To test different concentrations of bacteria using YOYO and observe how they load into the wells.
Microarray Preparation
- Wash the array with isopropanol and allow to dry
- Carefully apply the silicon gasket and ensure a tight seal
- Add 15 uL of the .1% Tween 20 soluton to each square and allow to dry
Bacterial Dilution and Staining
- Prepare three different concentrations of bacteria
- Begin with E coli of OD=1.0 (10^6 cell/uL)
- Add 10 uL cell to 90 uL PBS (10^5 cell/uL)
- Add 20 uL of diluted cells to 20 uL PBS (20000 cell/uL)
- Do a 1:10 dilution with 1x YOYO for a final conc of 2000 cell/uL
- Begin with E coli of OD=1.0 (10^6 cell/uL)
- Add 10 uL cell to 90 uL PBS (10^5 cell/uL)
- Add 10 uL of diluted cells to 90 uL PBS (10000 cell/uL)
- Do a 1:10 dilution with 1x YOYO for a final conc of 1000 cell/uL
- Begin with E coli of OD=1.0 (10^6 cell/uL)
- Add 10 uL cell to 90 uL PBS (10^5 cell/uL)
- Add 10 uL of diluted cells to 90 uL PBS (10000 cell/uL)
- Add 40 uL of diluted cells to 60 uL PBS (4000 cell/uL)
- Do a 1:10 dilution with 1x YOYO for a final conc of 400 cell/uL
- Pipette thouroughly to mix
- Incubate in the dark for 5 min
Loading onto Microwells
Each square has 3600 wells. Ideally, we want less than 1 cell per well. However in this case we want to ensure that the bacteria loads properly into the wells. Therefore, we will load 10000 cells/square.
- Add 5 uL cells with each concentration to 2 squares each
- This will give cell counts of 10000, 5000, and 2000 cells per square
- Pipette to ensure entire square is covered
- Allow to dry (this could take several minutes)
Results
The following were taken with 20x magnification using a flourescent microscope.
File:1000-bw-1.jpeg 10000 B/W
File:1000-cc-1.jpeg 10000 Color
File:5000-bw-1.jpeg 5000 B/W
File:5000-cc-1.jpeg 5000 color
File:2000-bw-1.jpeg 2000 B/W
File:2000-cc-1.jpeg 2000 color
Discussion
The most effective stain seems to be YOYO as you can view the bacteria in both black and white as well as color. However, in the black and white pictures, you can see that the bacteria is loading into the well with all three stains. The next step would be to use different concentrations of cells stained with YOYO and view how they load into the wells.