Sam:LabNotes/Microbione/2009-3-6: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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='''Bacteria labeling using SYBR | ='''Bacteria labeling using SYBR green'''= | ||
==Objective== | ==Objective== | ||
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* Using SYBR to stand E. coli and confirm using inverted flourescent microscope | * Using SYBR to stand E. coli and confirm using inverted flourescent microscope | ||
* Reference | * Reference | ||
**[[Media:Bacteria staining protocol.pdf]] | **[[Media:Bacteria staining protocol.pdf|Bacteria staining protocol]] | ||
**[[Media:Rapid Bacteria staining with laser cytometry.pdf]] | **[[Media:Rapid Bacteria staining with laser cytometry.pdf|Rapid Bacteria staining with laser cytometry]] | ||
==Samples & Materials== | ==Samples & Materials== | ||
Line 19: | Line 20: | ||
*Prepare four 1.5-mL tube, labeled and wrapped with foil. | *Prepare four 1.5-mL tube, labeled and wrapped with foil. | ||
*Set up as following exp design: | *Set up as following exp design: | ||
No-treat 100uL 200uL 400uL | |||
--------------------------- | --------------------------- | ||
H2O 400 0 0 0 uL | H2O 400 0 0 0 uL | ||
Line 27: | Line 28: | ||
*Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min. | *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 | *After incubation, add H2O to 1 mL for each tube, the new concentration for E. coli -> 1000 CFU/uL) | ||
*Prepare two microscopy slide with labeling. | *Prepare two microscopy slide with labeling. | ||
*load 1 uL stained E. coli sample from each tube on the slide and place with cover slips. | *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. | *Keep the slide in dark storage box for microscope visualizing. | ||
==Results== | ==Results== | ||
[[Image:40X - B&W - 400 SYBG_(c1).jpg|500px]] [[Image:40X - B&W -N treatment.jpg|500px]] | [[Image:40X - B&W - 400 SYBG_(c1).jpg|500px]] [[Image:40X - B&W -N treatment.jpg|500px]] | ||
'''Left: 40X, 400 uL 2XSYBR treatment, B&W picture. Right | '''Left: 40X, 400 uL 2XSYBR treatment, B&W picture. Right: 40X, 400 uL No-treat, B&W picture''' | ||
==Discussion== | ==Discussion== | ||
*No flourresecnce was observed using Grean light | *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. | *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 | *A higher magnification such 100X~200X may be necessary for our application | ||
**E. coli size may be around 3~10 uM | **E. coli size may be around 3~10 uM (according previous studies) | ||
**The common practice for visulizing microbes is based on the microscopy with 200X magnification and Epi-flourescent microscope type. For better flourescence capturing?) | **The common practice for visulizing microbes is based on the microscopy with 200X magnification and Epi-flourescent microscope type. For better flourescence capturing?) |
Latest revision as of 00:44, 7 March 2009
Bacteria labeling using SYBR green[edit]
Objective[edit]
- Using SYBR to stand E. coli and confirm using inverted flourescent microscope
- Reference
Samples & Materials[edit]
- 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[edit]
- 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 1 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[edit]
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[edit]
- 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 (according previous studies)
- The common practice for visulizing microbes is based on the microscopy with 200X magnification and Epi-flourescent microscope type. For better flourescence capturing?)