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.