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EM Negative Staining Protocol for V

时间:2006-01-22 15:56来源:ihcworld.com 作者:bioguider 阅读:
EM Negative Staining Protocol for Virus and Macrophages

 

Fixation:

 

100 ul of virus/phages suspension was fixed with 10ul 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:

·          Fixed or unfixed virus/phages suspension ----- 10 ul

·          Latex spheres (for quantitative study) ------------ 10 ul

·          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:

 

Ten fields for each sample are randomly photographed at 5000x after first examining the grid for uniformity. The negatives are enlarged 2.5x to a final magnification of 12,500x.

 

Ten photographs for each sample are examined for the number of virus/phages particles and the number of beads. Beads are recognized as being much larger than virus (about 1 ½ times larger). Both beads and virus/phages particles are marked as they are counted to avoid duplicate counting. Beads are marked in black, and virus/phages in red.

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