[3dem] uneven staining/contamination on negative stained grids

Berith Isaac bisaac at mcb.harvard.edu
Mon Aug 2 13:47:26 PDT 2010

Dear all, 

We are experiencing repeated problems of uneven staining on our negative stained grids. 

Here is a link to some examples of micrographs with the uneven staining/contamination:

Since this problem seems to return every summer (our lab is located in cambridge, MA), we assumed it is related to our thin carbon being exposed to higher humidity (we work with both home made holey grids and quantifoil grids). 

We have tried storing our grids& carbon in a dessicator, connecting our carbon evaporator to nitrogen so it doesn't vent with air, keeping our evaporator bell jar extremely clean, installing a dehumidifier in our grid preparation room and finally baking (at 80C) our grids& carbon prior to use (followed by glow discharging). Nothing seems to help however. 

We have previously ruled out other sources of contamination (such as protein, chloroform, uranyl formate). 

We would be grateful for any suggestions. 


Berith Isaac, PhD
Dept. of Molecular and Cellular Biology
Room 305
Harvard University
52 Oxford street
Cambridge, MA 02138, USA
Phone: 617-496-3808
Fax: 617-496-2712

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