Sam:LabNotes/Microbiome-new/2011-3-31
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Single E.coli cell micromanipulation new test initiation
Background
- Collabrate with Andy Chen
- Adapt the successful experience of micromanipulation on single human lymphocyte
Fresh E.coli cells recovery
- Recovery fresh E.coli K-12 cell from glycerol stock (-80C)
- Prepare 10mL pre-warmed LB in 10mL culture tube (x4)
- Innoculate E.coli K-12 into the LB mediua(using 10p tips)
- Cap the tube. Vortexing briefly. Incubate at 37C over night (3:00PM~9:00AM next day)
- (The medium look cloudy in all of four tubes)
- Washing cells
- Pellet the cell by centrifuging.
- Centrifuge the 10mL culture tubes at 3000rpm, 4C, 10min. Discard supernatant.
- Resuspend the pellet in 1.5mL 1X PBS. Transfer into a 1.7mL tube.
- Centrifuge the tube at 8000g, RT, 3min. Discard supernatant.
- Resuspend the pellet in 1.0mL 1X PBS.
- Repeat the last washing step again (Two times PBS washing in total).
- Based on the culture cloudy extent, I estimate it's about 10^9 CFU/mL
E.coli stain (SYBR-green / DAPI)
- Prepare dilued staining dye
- Dilute SYBR(200X) into 10X by 1/20 dilution in a 1.7mL tube wrapped in foil
- Thaw DAPI(10,000X)stock solution. Dilute DAPI into 10X by 1/1000 dilution in a 1.7mL tube wrapped in foil.
- Cell staining
- Add 2uL E.colu culture(10^9 CFU/mL) with 200uL 10X dye (SYBR / DAPI). Incubate at 37C on the shaker for >30min.
- Dilute the stained cell from 10^7 CFU/mL to 10^5 CFU/mL with 1X PBS.
E.coli cell micromanipulation test
- Test of non-stained cell at BF, 10X condition
- Pipette 8~10uL sample on the slide and put a cover slip to keep moisture.
- Able to target (see) cells with a drop (8~10uL) of 10^4 CFU/mL ~ 10^5 CFU/mL diluted E.coli cells
- 10^5 CFU/mL is still a little bit too dense for targeting a single cells. 10^4 CFU/mL is probably the best density.
- Challenges:
- E.coli cell is floating around and difficult to locate
- Contaminaiton of debris (some of them have size larger than an E.coli cell)
- Not able to tell if the target is E.coli or dust or debris.
- Compared to non-stained E.coli, SYBG-stained E.coli is more difficult to target since we need to adjust contrast and focus more carefully to decrease the backgound noise.
- The E.coli number observed in BF is different than that in GFP.
- Lessons learned:
- It seems possible to direcetly target non-stained E.coli using 10X under BF condition. The challenge is how do we confirm they are E.coli cells or not.
- SYBR or DAPI staining: need more tests on condition adjustments. It seem like most debris are also stained by SYBR too.
- Decrease the debris or dust during sample preparation is important. Use 0.2um-filtered PBS for all of the steps in sample preparation. Clean the microscope station carefully. Prepare the extra clean slice and cover slip.
- Next:
- Adjust the setting for SYGB and DAPI stined E.coli cells
- Find a way efficiently switch microscope setting between BF and GFP/DAPI conditions.
- Compare the following condition: 1 concentration (10^5) x 4 types of smaple (non-stained, SYBG, DAPI, Trypheblue stained). And see if we the number we observed is same within different samples.