[3dem] Carbon peeling from C flat gold grids

[Reinhard Rachel]

Hi Janet, 

> I’m having issues with C flat grids that I recently purchased.  The grids
are 
> holey carbon 2/2 and 1.2/1.3 gold 200 mesh grids purchased from EM Sciences.

> Prior to seeding cells on grids, they are glow discharged, UV sterilized in
a 
> tissue culture hood, dipped briefly in 70% ethanol followed by water and 
> placed in a culture dish. Poly lysine is added to the dish and grids sit 
> overnight at 4C before being rinsed several times with water. Media and
cells 
> are added. Grids are plunge frozen using a Leica GP2 and are clipped to be 
> imaged in a Krios microscope.  In the light microscope, carbon films appear

> intact prior to freezing but I think the carbon is very fragile and will
tear 
> upon the slightest perturbation.  In the last experiment, every grid (8) had

> large areas of carbon missing.  I have used Quantifoils for this same 
> protocol with no issues at all.

I am just trying hard to understand this protocol - not only as an electron
microscopist (yes, I do have quite some experience), but as a microbiologist. 
If you do the glow discharging: did you test those grids after glow
discharging, to which extent you are likely to contain any further
microbiological contamination, in the follow-up procedures? means: live, viable
cells? Glow discharging is likely to result in quite some reduction of viable
microorganisms on these grids - if there were any viable microbes (consider:  -
a) vacuum, b) UV). Whether this is sufficient? (you may test this). 
The urgent question for me arises whether you really need further steps like 
(b) UV sterilization, and (c) ethanol treatment. My gut feeling: at least one
of these steps is not needed, in terms of sterile surfaces on grids. Probably
both?
I am just curious: how long (minutes) is the exposure to the Poly-Lys (which
is sterile, I suppose)?
Which media?
which kind of cells are added, and then how long is the 'incubation'? 

My argument: any type of grids for cryo (or: for bio-TEM) are highly labile
structure, 10-20 nm thin carbon films, with holes. Whatever you do, they are
likely to break, and the more steps you "do" with treatments, the more are they
likely to break. - 
second argument: sterile is sterile. One treatment to reach sterility is fine.
- and: the number of viable (!) cells on grids is low anyway. 
just my 2 cents. 
Kind regards,
Reinhard

-- 
Prof. Dr. Reinhard Rachel
University of Regensburg
TEM+SEM / Anatomy
Faculty of Biology & Preclin. Med.
Universitaetsstrasse 31
D-93053 Regensburg - Germany
tel +49 941 943 -2837, -1666
mail reinhard.rachel at biologie.uni-regensburg.de
office: VKL 3.1.29 
member of the IFSM board

Next microscopy conference:
- IMC21, The 21st Intl. Microscopy Congr 2026 in Liverpool, UK: Aug 31-Sept 4,
2026
- next Microbiol. conference:  VAAM 22.3.-25.3.2026 - Berlin