[3dem] negative staining artifact

[Reinhard Rachel]

Dear Linda,
besides the points discussed already, I am curious to learn why you use copper
grids with formvar, instead of using grids purely coated with carbon films
(prepared by evaporation onto mica, floating off, and then depositing the
c-film onto the grids). Formvar can be nice, can be nasty. 
Second, I wonder whether it is your sample what we are looking at. Does it
contain "phosphate-containing compounds" like PBS, ribosomes or remnants, etc
etc? all PO4-containing substances - bio or not bio - will "attract"
Uranylacetate. Many carboxyl-groups do attract Uranylacetate. Think about the
positive staining effect of DNA, ribosomes, P-lipid-membranes, etc etc. 
How "pure" is the sample? What is the pH of the sample? and did you wash for a
fraction of a second with bi-distilled water? we often do this - and hope the
best that the sample survives. It may help. 
just my 2 cents ... kind regards - Reinhard
 ============================
 
> I believe its your stain precipitate . I too had this problem once (my UA
> was old). I used a mild centrifuge rotation and took the supernatant for
> staining and it worked. I hope this will solve your issue.
> 
> Regards,
> 
> Subhomoi B.
> 
> On Mon, Sep 25, 2017 at 12:31 PM, Johanna Höög <johanna.hoog at gu.se> wrote:
> 
>> Hej Linda!
>>
>>
>>
>> I had similar precipitates a while ago and it took me forever to find the
>> source. For us, it was that the oil filter on the glow discharger that
>> needed to be changed. Try just putting an empty grid in the glow
discharger
>> and then have a look at it to exclude this possibility.
>>
>>
>>
>> Good luck!
>>
>>
>>
>> Johanna
>>
>>
>>
>> *From: *3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Linda Sandblad <
>> linda.sandblad at umu.se>
>> *Date: *Sunday 24 September 2017 23:27
>> *To: *"3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
>> *Subject: *[3dem] negative staining artifact
>>
>>
>>
>> Hi all experts!!
>>
>> I know, this do not look nice, but I would like to hear your opinion about
>> this artifact. Tiny dark spots covering both sample and background, do you
>> know what it is, were it comes from and how to avoid it? We have this bad
>> background time to time from different labs…. It is a “normal” negative
>> staining protocol: copper grids, formvar, carbon, glow discharge, UA.
>>
>> I’m happy for your suggestions, Best Linda