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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|400px]] [[File:041511_F_filter-dapi200_annotatede.png|400px]] [[File:041511_Unwashed-dapi200_annotated.png|400px]] [[File:041511_PBTw_washed-dapi200_annotated.png|400px]] ==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 decrease 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.
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