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.
- Pellet at 5min, 8000 rpm. Remove supernatant. Wash 1X in 1mL PBS(0.2um-filtered).
- Prepare TryphanBlue working solution (0.5mL 0.4% TryphanBlue + 0.3mL PBS)
- Resuspend washed cells 100uL TryphanBlue working sol. Repeat pipetting several times.
- Incubate in the Eppendorf rocking incubator at RT (25C), 500rpm for 10min.
- Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 1mL PBS(0.2um-filtered).
- Filter the cell suspension through 5um-filter.
- Pellet at 5min, 8000 rpm. Remove supernatant.
- Resuspend in 0.5mL PBS(0.2um-filtered).Transfer sample in a 1.7mL amber tube.
- Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
- Add DAPI working solution in the E.coli
- Add 2uL dye for the tube labeled as "2". Add 4uL dye for the tube labeled as "4".
- 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.