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Sam:LabNotes/Micro-manipulation/2011-4-26
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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: #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|400px]] [[File:04-26-11 livecell-sybrgold-gfp400.png|400px]] [[File:04-26-11 livecell-sybrgold-a-bf1.png|400px]] [[File:04-26-11 deadcell-yoyo-GFP200.png|400px]] [[File:04-26-11 livecell-yoyo-gfp300.png|400px]] [[File:04-26-11 livecell-yoyo-bf1.png|400px]] [[File:04-26-11 deadcell-dapi-DAPI200.png|400px]] [[File:04-26-11 livecell-dapi-dapi200.png|400px]] ==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. *If we compare the same sample under BF filter, DAPI-staining still showed a better representation rate on of whole population than SYBR-GOLD staining (Some cells stained in SYBR-GOLD are too dim to been seen). *I am wondering why DNA staining doesn't work on old cells (dead cells) #The DNA in dead cell are degraded? #The cell formed some kind of biofilm that avoids the dye to penetrate 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 cyropreserve fresh cell for long term usage. *Cyropreservation protocol from Bigelow's lab: #[[Media:Bigelow_lab_Sample_cryopreservation_glycerol.pdf|Sample_cryopreservation_glycerol]] #[[Media:Bigelow_lab_Sample_cryopreservation_BeTE.pdf|Sample_cryopreservation_BeTE]]
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