Sam:LabNotes/Micro-manipulation/2011-4-26
Jump to navigation
Jump to search
Single E.coli cell micromanipulation - Fresh vs. dead sample test
Objective
- I compare DAPI-staining performance on fixed(EtOH) and non-fixed E.coli cells(using the old E.coli sample stored from 03-10-'10 in 4C). The fixed cell still give me bright DAPI signal, but the non-fixed E.coli cell showed very dim single as previous tests I did.
- I start doubting if the E.coli samples in the non-fixed samples are too old or the DNA were degraded in these cells.
- When bacteria are dying the DNA will be degraded slowly, however I thought the degradation is too slow to be regarded as a concern.
- I grew the fresh E.coli cell on 04-25-'11, and will compare fresh cells with old cells on DNA dye staining again.
Procedures
- Sample preparation:
- Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
- Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL PBS.
- Transfer into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. Resuspend in 1mL PBS.
- Prepare 100uL sample for staining.
- Prepare six 1.5-mL tubes. Transfer 100uL cell suspension into each of tubes.
- Fresh-Ecoli x3 (SYBR-gold, YoYO, DAPI), Dead-Ecoli x3 (SYBR-gold, YoYO, DAPI)
- Dead E.coli were from the same source as on 4-21-'11's test. The E.coli cells cultured on 3-10-'10, stored in 4C.
- Pellet cells at 8000rpm, 5min, RT. Remove supernatant, resuspend in 0.5mL PBS.
- Prepare following DNA staining dye
SYBR-GOLD(10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X YOYO-1 (10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X DAPI (pre-diluted 5mg/mL) -> 1/5 dilution with H2O -> 1mg/mL
- Add 2uL of diluted dye into sample suspension (0.5mL). Repeat pipetting slowly ~20X to mix the dye with sample.
- Incubate in the Eppendorf rocking incubator at 25C, 400~500rpm for 15min.
Results
File:04-26-11 deadcell-sybrgold-GFP200.png File:04-26-11 livecell-sybrgold-gfp400.png File:04-26-11 livecell-sybrgold-a-bf1.png File:04-26-11 deadcell-yoyo-GFP200.png File:04-26-11 livecell-yoyo-gfp300.png File:04-26-11 livecell-yoyo-bf1.png File:04-26-11 deadcell-dapi-DAPI200.png File:04-26-11 livecell-dapi-dapi200.png
Discussion
- Using fresh cell (live cells) significantly improve the staining performance. However, the YOYO dye staining is not very successful even on the live cells.
- I am wondering why DNA staining doesn't work on old cells (dead cells)
- The DNA in dead cell are degraded?
- The cell form some biofilm that avoid the dye to penetrate into cell wall?
Next
- Modify the YOYO non-fixed cells staining protocol?
- Combine the current non-fixed cell staining protocol with pre-filtering steps and glycerol adding
- Since I will need to use fresh cell, I will check if there is a way to cyropreseve fresh cell for long term usage.
- The cell storage protocols used in Bigelow's lab will be useful. They cyropreserve fresh cells in -80C with either glycerol or Betaine suspension.