Sam:LabNotes/Microbione/2009-3-6: Difference between revisions

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>Sam Chiang
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>Sam Chiang
 
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='''Bacteria labeling using SYBR gree'''
='''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:Example.ogg]]
**[[Media:Rapid Bacteria staining with laser cytometry.pdf|Rapid Bacteria staining with laser cytometry]]
 


==Samples & Materials==
==Samples & Materials==
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*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
          No-treat 100uL 200uL 400uL
             ---------------------------
             ---------------------------
   H2O      400      0    0    0  uL
   H2O      400      0    0    0  uL
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*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 1000 mL for each tube, the new concentration for E. coli -> 1000 CFU/uL)
*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 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]]
  '''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 (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?)

Latest revision as of 00:44, 7 March 2009

Bacteria labeling using SYBR green[edit]

Objective[edit]

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?)