Sam:LabNotes/Micro-manipulation/2011-4-12
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Single E.coli cell micromanipulation test on 04-12-'11[edit]
Testing objectives[edit]
- Focus on the performance of fluorescently stained cells.
- Question 1: Can we see the same number of samples under the BF GFP/DAPI filters? (If not, what do we missed, E.coli or unknown particles?)
- Question 2: Do we observe flourescently stained particles in negative control (blank slide)?
Cell resource[edit]
- Using old stored E.coli K-12 culture (4C, 1XPBS, March, 2010)
- Estimated concentration = 10^9 CFU/mL
E.coli stain (SYBR-safe / DAPI)[edit]
- Prepare dye working solution (10X)
- Add 1uL SYBR-safe(10,000X) with 50uL 1X TE in a 1.7mL tube wrapped in foil => 200X
- Add 10uL SYBR-safe(200X) with 190uL 1X TE in a 1.7mL Amber tube => 10X
- Transfer 2uL E.coli culture into the 200uL dye working solution
- Same method was prepared for DAPI staining.
E.coli cell microscopy analysis[edit]
- Comparison types:
1. DAPI-stained cell(2uL, 10^7 CFU/mL) on regular slide with coverslip File:041211 dapi stained ecoli-bf1.png File:041211 dapi stained ecoli-dapi100.png
2. DAPI-stained cell(2uL, 10^7 CFU/mL) on micro-petri-dishes File:041211 dapi stained dish-bf1.png File:041211 dapi stained dish-dapi200.png
3. no cell on micro-petri-dishes File:041211 no cells dish-bf1.png File:041211 no cells dish-dapi200.png
4. no cell on regular slide File:041211 no cells slide-bf1.png File:041211 no cells slide-gfp500.png
Discussions[edit]
- The number of particles (probably E.coli cells) are not consistent between BF and DAPI modes.
- The missing particles have the similar size as non-missing particles.
- It seems like the larger particles were stained better with DAPI and more observable under DAPI mode.
- There are observable particles on no cell slide under both DAPI and SYBR modes. These particles showed similar size as the expected E.coli size (1~2um).
Conclusion[edit]
- How do we tell if they are E.coli or non-Ecoli particles?
- Based on the cell floating motion
- Use high concentration of cells (density changed with our serial dilution)
- Higher magnification of objective with phase-contrast setting.
Next[edit]
- Try to improve the dye staining protocol
- Using fixed cell
- SSC hybridization buffer?
- Higher dye concentration and longer staining time.
- Use AntiFade reagent?
- Optimize phase-contrast condition.