Jeff:LabNotes/Microbiome/2009-4-8
Bacterial loading into Microwells[edit]
Purpose[edit]
To test whether the cells are loading properly into the wells by using a blank slide as a posite control. All three stains will be used as well as very high concentrations of cells.
Microarray Preparation[edit]
- 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[edit]
- Prepare two 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, 2x SYBR Green, and a 1:1 mixture of 1x YOYO and 2x SYBR Green 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)
- Do a 1:10 dilution with 1x YOYO, 2x SYBR Green, and a 1:1 mixture of 1x YOYO and 2x SYBR Green for a final conc of 10000 cell/uL
- Pipette thouroughly to mix
- Incubate in the dark for 5 min
Loading onto Microwells[edit]
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/stain to 1 square each (Sam did a duplicate experiment)
- This will give cell counts of 10000 and 50000 cells per square
- Pipette to ensure entire square is covered
- Allow to dry (this could take several minutes)
Results[edit]
The following were taken with 20x magnification using a flourescent microscope.
File:M-h-bw-1.jpeg Mixed 50000 B/W
File:M-h-cc-1.jpeg Mixed 50000 Color
File:,m-l-bw-1.jpeg Mixed 10000 B/W
File:M-l-cc-1.jpeg Mixed 10000 color
File:Y-h-bw-1.jpeg YOYO 50000 B/W
File:Y-h-cc-1.jpeg YOYO 50000 color
File:Y-l-bw-1.jpeg YOYO 10000 B/W
File:Y-l-cc-1.jpeg YOYO 10000 color
File:S-h-bw-1.jpeg SYBR 50000 B/W
File:S-h-cc-1.jpeg SYBR 50000 color
File:S-l-bw-1.jpeg SYBR 10000 B/W
File:S-l-cc-1.jpeg SYBR 10000 color
File:Slide-m-h-bw-1.jpeg Mixed 50000 B/W
File:Slide-m-h-cc-1.jpeg Mixed 50000 Color
File:Slide-m-l-bw-1.jpeg Mixed 10000 B/W
File:Slide-m-l-cc-1.jpeg Mixed 10000 color
File:Slide-y-h-bw-1.jpeg YOYO 50000 B/W
File:Slide-y-h-cc-1.jpeg YOYO 50000 color
File:Slide-y-l-bw-1.jpeg YOYO 10000 B/W
File:Slide-y-l-cc-1.jpeg YOYO 10000 color
File:Slide-s-h-bw-1.jpeg SYBR 50000 B/W
File:Slide-s-h-cc-1.jpeg SYBR 10000 B/W
File:Slide-s-l-bw-1.jpeg SYBR 50000 color
File:Slide-s-l-cc-1.jpeg SYBR 10000 color
Discussion[edit]
The cells appeared much more clearly on the slides without the wells. Both the flourescent and black and white images were very clear and displayed a large number of cells. In the slides with the wells, cells appeared but in a much smaller concentrations. Furthermore, it was very difficult to see the cells in the flourescent light. This suggests that the cells are not loading properly into the wells. Explanations include that the Tween 20 is preventing the cells from loading. Thus an experiment must be carried out where no Tween 20 is used. If the hypothesis is correct, the cells will be easily seen in the wells. Also, some particles that were not bacteria were observed. This could be chemicals from the different stains. Another experiment where the staining is done prior to dilution will confirm this hypothesis.