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Author Topic: Negative Staining For Virus and Phages  (Read 4738 times)

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Offline ihcwor2

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Negative Staining For Virus and Phages
« on: March 12, 2003, 10:58:20 PM »
Negative Staining For Virus and Phages


Fixation (not necessary):

100 ul of virus/phages suspension was fixed with 10 ul fixative (10% formaldehyde and 5% glutaraldehyde in deionized water) to give a final concentration in the virus/phages suspension of 1% formaldehyde and 0.5% glutaraldehyde.


Procedure:

1.   Mix the followings:
a) Fixed or unfixed virus/phages suspension --- 10 ul
      b) Latex spheres (for quantitative study) -------- 10 ul
      c) 1% BSA in distilled water ---------------------- 10 ul
      Pipette up and down at least 30 times. For qualitative study, no latex spheres are needed.  

2.   Hold the grid in forceps and apply about 6-10 ul virus/phages suspension onto the grid and leave on for 30 seconds to 1 minute, and then draw off from the edge of the grid with filter paper (watch grid surface to make sure a thin layer of liquid film is formed).

3.   Apply immediately (before sample is dried) 6-10 ul of 2% aqueous phosphotungstic acid (adjust pH to 7.3 using 1N NaOH) and leave on for 30 seconds, and draw off from the edge of the grid with filter paper (watch grid surface to make sure a thin layer of liquid film is formed) and place the grid directly into grid box and allow them air-dry for 30 minutes before observation.


EM Observation:

Negative Staining For Virus and Phages
« on: March 12, 2003, 10:58:20 PM »