Sam:LabNotes/Micro-manipulation/2011-4-18

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Single E.coli cell micromanipulation - Dual labeling test (TB + DAPI)[edit]

Objective[edit]

  • Test the dual staining (TryphanBlue plus DAPI) protocol
    • If it works the TryphanBlue staining can help Andy to locate E.coli cell even easier.

Procedures[edit]

  • Starting with EtOH fixed cell (stored in -80C) x2
This cell are about 100uL of OD600=1.0 E.coli, fixed in 500uL 100% EtOH for 3hr.
  1. Pellet at 5min, 8000 rpm. Remove supernatant. Wash 1X in 1mL PBS(0.2um-filtered).
  2. Prepare TryphanBlue working solution (0.5mL 0.4% TryphanBlue + 0.3mL PBS)
  3. Resuspend washed cells 100uL TryphanBlue working sol. Repeat pipetting several times.
  4. Incubate in the Eppendorf rocking incubator at RT (25C), 500rpm for 10min.
  5. Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 1mL PBS(0.2um-filtered).
  6. Filter the cell suspension through 5um-filter.
  7. Pellet at 5min, 8000 rpm. Remove supernatant.
  8. Resuspend in 0.5mL PBS(0.2um-filtered).Transfer sample in a 1.7mL amber tube.
  9. Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
  10. Add DAPI working solution in the E.coli
    1. Add 2uL dye for the tube labeled as "2". Add 4uL dye for the tube labeled as "4".
  11. Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 500rpm for 15min.

Results[edit]

File:04-19-11-Dual stained 2uL Ecoli BF-10X.jpg  File:04-19-11-Dual stained 2uL Ecoli DAPI-10X.jpg

File:04-19-11-Dual stained 4uL Ecoli BF-10X.jpg  File:04-19-11-Dual stained 4uL Ecoli DAPI-10X.jpg

Discussion[edit]

  • The staining of TryphanBlue on E.coli didn't have significant improvement on target observation under BF field.
  • However, the staining of TryphanBlue didn't effect the DAPI staining.
  • No significant difference either between different amount of DAPI dye dose (2uL vs 4uL of DAPI working sol.)

Next[edit]

  • Test how to improve TryphenBlue staining.
  • Maybe I can test Crystal Violet staining.