[3dem] Re: 3dem Digest, Vol 49, Issue 20

[DocDave50 at aol.com]

Hi Liz,
 
I would agree with Rachael's question  re: vesicle blebbing from the cell 
or, if there is a serum component in your  media, there could be excess lipid 
desorbing from the serum.  Given the  density of the blobs and the relative 
amount of them over the image space, they  appear very much like detergent 
or amphiphile globules that one would see in  water that isn't cleaned via a 
"clean" reverse phase column in the water  purification system used to 
prepare the buffers.  A second source of these  little 'buggers'...since I am 
moving to London, I have to start using some  appropriate phraseology I'd 
heard while there in July...is the glassware  cleaning process.  If glassware 
used to prepare buffers is washed in a  dishwasher and the wash cycles are not 
appropriately set OR the glassware is not  rinsed frequently enough...12 
times to remove regular dishwashing soap...a lot  huh?...then 'soap' is left 
to dry on the glass surface and then you prepare your  buffers, media, 
sterilize it and what to your wondering eyes should appear in  every prep and 
every time you try to make a change...blobs!
 
I'd suggest this one option.   First, don't solvent wash your grids.  If 
they need solvent washing, get  fresh grids.  If you are going to solvent wash 
them, wash with Chloroform  and then pure hexane.  After washing, you must 
leave your grids under  vacuum for ~3 min to ensure that the solvent is no 
longer on/in the hydrophobic  carbon surface.  If you are concerned about 
contaminants coming from the  grids, you could try very short carbon 
evaporation on your holey carbon  grids...both sides...prior to sample addition.  My 
suggestion is that you  get some of your purified water...often times if you 
have a glass bottle  reservoir you can see it there.  Take a surface angle 
glance...since fat  floats...and see if you can see any sheen on the surface 
of the water.   Remember that double glass distillation does not remove 
organics just  inorganics.  If there is a sheen, there is your problem. When it 
gets  really bad, there is a slight soap ring on your reservoir.  I would 
then  take your water being used to prepare buffers and just use this to 
prepare a  grid with the very same freeze plunge process you are  currently 
using.  Are the blobs there in this image?  Yet another  potential source of 
these blobs...and I've seen them myself...are you possibly  maintaining your 
grids under vacuum using either house vacuum or an oil vacuum  pump to 
establish vacuum.  Both of these systems provide very low vacuum  AND, most notably, 
TONS of oil back-streaming which, over time, contaminate  your grids.  I 
found this out the hard way when I was starting the  process of making our own 
periodic holey carbon with the PDMS  stamping procedure.  If I stored the 
grids long enough under a vacuum  pump driven by oil or house vacuum, you 
could actually see 'puddles' of oil on  the grid surface when you looked at 
them from the side.  You now have a  very prominent source of 'blobbish' 
looking things all over the grid and in  the ice.
 
I would be interested to know the  storage conditions you are using for 
your grids...remember, that in many  Biochemistry labs, there are folks using 
house vacuum or oil vacuum pumps such  that there is oil contaminants in the 
lab air...been there, done that...and  whether a quick look at your base 
water supply yields the same  'blobs'.
 
Hope this helps...and, yes, I have seen  them and these were the causes in 
my case.
 
David W. Chester, Ph.D.
Department of Biochemistry
Center for Structural  Biology
Imperial College of London
 
 
 
In a message dated 9/28/2011 3:00:07 P.M. Eastern Daylight Time,  
3dem-request at ncmir.ucsd.edu writes:

http://dl.dropbox.com/u/23095990/Caulobacter_2sept2011.tif 
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