Sam:LabNotes/Micro-manipulation/2011-4-26

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Revision as of 17:37, 27 April 2011 by >Sam Chiang (→‎Next)
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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:
  1. Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
  2. Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL PBS.
  3. Transfer into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. Resuspend in 1mL PBS.
  • Prepare 100uL sample for staining.
  1. Prepare six 1.5-mL tubes. Transfer 100uL cell suspension into each of tubes.
  2. Fresh-Ecoli x3 (SYBR-gold, YoYO, DAPI), Dead-Ecoli x3 (SYBR-gold, YoYO, DAPI)
    1. 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.
  3. 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
  1. Add 2uL of diluted dye into sample suspension (0.5mL). Repeat pipetting slowly ~20X to mix the dye with sample.
  2. 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)
  1. The DNA in dead cell are degraded?
  2. 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.