Sam:LabNotes/Micro-manipulation/2011-4-15
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Single E.coli cell micromanipulation test on 04-15-'11
Objective
- Further improve the DAPI staining protocol
Experiment design
- Test the following conditions:
- The effect of filtering the fixed E.coli (by 5um-filter)
- Washing and resuspend in PBTw to remove background.
- Using higher amount of DAPI dye.
Procedures
Test of pre-filtering the EtOH-fixed samples
- 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. Wash 1X in 1mL PBS(0.2um-filtered).
- One of the samples was filtered through 5um-filter, and labeled as "F".
- The other one was labeled as "R" following the regular procedure.
- Resuspend in 0.5 mL 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 1uL dye for "R" tube. Add 4uL dye for the "F" tube.
- Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400rpm for 15min.
Test of adding final washing with PBTw
- Started from the DAPI-stained sample on 4-14-'11
- Separate the sample into two tubes(250uL/tube)
- Pellet stained cells at 8000rpm, 5min, RT.
- Prepare the PBTw (PBS containing 0.1% Tween20) by adding 40uL Tween20 into 40mL PBS(0.2um filtered).
- Pick up one of the samples. Using 1mL PBTw to wash cells twice. Resuspend the cell pellet in 250uL PBTw. Label the tube as "washed".
- Label the other tube as "non-washed".
Results
Comparisons of different conditions on DAPI-stained E.coli File:041511 R nofilter-dapi200 annotatede.png File:041511 F filter-dapi200 annotatede.png File:041511 Unwashed-dapi200 annotated.png File:041511 PBTw washed-dapi200 annotated.png
Discussion
- Effect of Filter:
- It's a good idea to filter the fixed E.coli sample before DAPI staining. The fixed-E.coli suspsend in 1mL PBS can still pass through the 5um filter.
- The picture of filtered sample showed much uniformed particles than non filtered samples.
- Although it looks like non filtered sample is brighter. However, these bright particles mostly stained large debris. The small particle (probably read E.coli cells) are still dim.
- Effect of higher dye concentration:
- Although not seen in pictures, it seems like 4uL dye stained sample("F") showed a better signal/noise than 1uL dye staining sample ("R") if we compared under the same microscope setting.
- It's effective but result is not significant.
- Effect of final washing:
- Add finally washing using PBTw affect the signal intensity significantly, and I can't tell if the background is decreased or not in the washed sample.
- I am planing to confirm this again in the next experiment.
Next
- Test if I can do TryphenBlue + DAPI staining at the same time.
- Confirm the effect of adding final-washing.
- Test the effect of glycerol on cell floating mobility. Confirm the background reflection problem.