Sam:LabNotes/Microbione/2009-3-6
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Bacteria labeling using SYBR green
Objective
- Using SYBR to stand E. coli and confirm using inverted flourescent microscope
- Reference
Samples & Materials
- Bacteria: E.coli suspension in PBS (OD600=0.913 -> around 10^9 CFU/mL = 10^6 CFU/uL)
- SYBR 2X: Diluted from SYBR 50X stock solution
Procedures
- Dilute SYBR 50X to 2X with RNAse-free H2O: 40 uL of 50X SYBR + 960 uL H2O -> 2X SYBR, 1000 uL.
- Prepare four 1.5-mL tube, labeled and wrapped with foil.
- Set up as following exp design:
No-treat 100uL 200uL 400uL --------------------------- H2O 400 0 0 0 uL 2X SYBR 0 100 200 400 uL E. coli 1 1 1 1 uL ---------------------------
- Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min.
- After incubation, add H2O to 1000 mL for each tube, the new concentration for E. coli -> 1000 CFU/uL)
- Prepare two microscopy slide with labeling.
- load 1 uL stained E. coli sample from each tube on the slide and place with cover slips.
- Keep the slide in dark storage box for microscope visualizing.
Results
File:40X - B&W - 400 SYBG (c1).jpg File:40X - B&W -N treatment.jpg Left: 40X, 400 uL 2XSYBR treatment, B&W picture. Right: 40X, 400 uL No-treat, B&W picture
Discussion
- No flourresecnce was observed using Grean light excitation.
- The suspecious dot might be the E. coli, which is not too small to confirm by morphology.
- A higher magnification such 100X~200X may be necessary for our application
- E. coli size may be around 3~10 uM
- The common practice for visulizing microbes is based on the microscopy with 200X magnification and Epi-flourescent microscope type. For better flourescence capturing?)