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[Yeast] TEM procedure using cell wall removal!

Z.J. Zhang ZZhang at uwyo.edu
Fri Sep 1 12:30:18 EST 2006


Hilal:
 
I assume that the reason you want to remove the cell wall is for osmium
fixation (cell wall has to be removed in order for osmium getting into
the cell). There is an alternative for fixing the yeast cells using
potassium permanganate, instead of osmium. Permanganate preserves
membrane structure very well (see references below). Here is the
protocol - 
 
1.	Harvested yeast cells are fixed first with 2.5% (v/v)
glutaraldehyde in PBS for 40 min at room temperature.
2.	Rinse with PBS 3X
3.	Cells are further fixed with 2% potassium permanganate in water
for 1 h at room temperature.
4.	Rinse with water 
5.	Dehydration as usual 
6.	.....
 
The potassium permanganate will stain cells black, as osmium, so that
you can easily find cells for section.
 
If you DO need to remove the cell wall for reasons I am unaware of. Here
is the protocol
1.	Harvested yeast cells are fixed first with 2.5% (v/v)
glutaraldehyde in PBS for 40 min at room temperature.
2.	Rinse
3.	Cells are digested with Zymolyase 100T (0.3mg/L, final
concentration) for 10 - 30 min to remove the cell wall.
4.	Cells are further fixed with 1% osmium in water or buffer for 1
hour at room temperature.
5.	Rinse
6.	Dehydration
7.	....
 
The time on step 3 is critical for your success (i.e. remove the cell
wall without damaging the cell). For the purpose of fixation (of
osmium), you really don't need remove 100% of the cell wall. Partially
remove it would be best. When you do the digestion, for every 10 min or
so, take some cells and check under a compound microscope. Once you see
the cell shaped changed (such as from football shape to round), it is
time to stop the digestion. Otherwise, it might be too much (even though
it may not be enough for making spheroplast).
 
Hope this helps.
 
Zhaojie 
 
References:
ROBIN WRIGHT (2000) Transmission Electron Microscopy of Yeast.
MICROSCOPY RESEARCH AND TECHNIQUE 51:496-510
Hui Yang, Qun Ren & Zhaojie Zhang (2006) Chromosomeor chromatin
condensation leads tomeiosis or apoptosis in stationary yeast
(Saccharomyces cerevisiae ) cells FEMS Yeast Res xx (2006) 1-10
 
Zhaojie Zhang, Ph.D.
Director, Microscopy Core Facility
University of Wyoming
Laramie, WY 82071
zzhang at uwyo.edu
http://www.uwyo.edu/microscopy
 
 
________________________________

From: Hilal Ilarslan [mailto:ihilal at iastate.edu] 
Sent: Wednesday, August 30, 2006 5:43 AM
To: yeast at magpie.bio.indiana.edu
Subject: [Yeast] TEM procedure using cell wall removal!
 
Hi,

I am working on Saccharomyces cerevisiae. I would like to get help about
TEM technique with cell wall removal without giving any damage to Yeast
cell. Is any one wiilling to give me protocol which I can be succesfull
on that. Thanks, Hilal


 
Hilal Ilarslan  Ph. D. 
Depart. of Genetics, Development and Cell Biology 
Iowa State University 
Room 1 Bessey Hall 
Ames, Iowa  50011-1020 USA 
Phone: (515)-294-3872 
Fax:     (515)-294-1337 
Email: ihilal at iastate.edu
http://www.public.iastate.edu/~ihilal/ 
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