9/9/97

work wants some pretty pictures of his yeast, Schizosaccharomyces pombe,

for use in seminars. I have read up on some techniques to use but have

two main queries:

1. To collect the yeast onto filter paper ( a method used in

several publications), do I just drop a suspension of the yeast onto the

filter paper? What sort of filter paper do I use? Is there a "better"

way of collecting these cells such as settling them onto poly-l-lysine

coverslips?

2. Is there a preferred fixative that works? The literature

suggests a plethora of fixative cocktails! For TEM, I slam the cells

onto a liquid nitrogen cooled copper mirror and process them via

substitution in methanol and embed them in lowicryl HM20.

We do not have an ESEM so please don't suggest I view them unfixed.

Thanking you all in advance, this really is a great way of learning and

sharing information!

Sarah Ellis

sarahe@raid.res.petermac.unimelb.edu.au

fixation. Then process it as usual for dehydration and critical point dry.

Best regards,

* Ming H. Chen, PhD *

* Medicine/Dentistry Electron Microscopy Unit *

* University Of Alberta. *

* Edmonton, Alberta, Canada *

* *

* Visit My Page At: *

* http://www.ualberta.ca/~mingchen

Collect the yeast on to membrane filters with nice circular pores (e.g.

Nucleopore), not a torturous-path filter like filter paper (or e.g.

Millipore). This will give a nice smooth background against which to view

the yeast. Filters with 0.22 micron holes are maybe best, although 0.45

micron will work. The pores will also give an *approximate!* size standard

for the yeast cells.

*Before* collecting the yeast, coat both sides of the filters in a sputter

coater. This gives better conductivity for viewing. If you're rich, use

silver filters instead.

For fixation, I'd use the recipe the article(s) that show SEMs of yeast

most like what you're trying to achieve (including 'scope kV), and is the

simplest.

Phil

oshel@ux1.cso.uiuc.edu

alcohol): coat with l mg/ml aqueous solution of poly-L-lysine for several

minutes, rinse in distilled wate.

Take a (preferably) aqueous suspension of the yeast and place onto the

microscope slide and allow the cells to settle for about one hour at RT.

Carefully tip the slide and allow the unattached cells to flow off. Now,

gently dropper some fixative (2% glut/4% formald in buffer of choice) onto

the slide to completely cover the smear. Allow to set undisturbed overnight

at RT in a humid chamber (petri dish with filter paper or Tupperware

container with moist paper towels). Transfer the slide into a rinse

solution (Coplin jar of distilled water or petri of distilled water). With

some yeasts, the aldehyde fix is adequate to preserve the integrity of the

cells (others may collapse without osmium post-fixation). The slides are

then slowly dehydrated in ethanol (20-40-60-80-100-100%) for 10 min each.

Critical point dry using liquid carbon dioxide as transitional solvent. You

will need to break the slide into 1 inch squares to fit into the CPD

device. Do this by scoring the slide with a diamond marker pen and pressing

down gently onto an applicator stick. Mark an "X" the back side with the

diamond marker to help identify the good side later. Freeze drying from

the ethanol should also work well.

IF osmium is needed place slide/specimen into 2% aqueous osmium tetroxide

overnight - take care to avoid evaporation by either immersion of the slide

into a shallow container of osmium or by vapor fixation of the

slide/specimen. Vapor fixation (CAUTION WITH OS FUMES - DO IN PROPERLY

OPERATING FUME HOOD) may be accomplished by placing the wet slide/specimen

into a plastic petri dish and then placing a small volume (4-5 ml) of 2%

osmium solution nearby in the dish. Rinse in distilled water and dehydrate

and CPD as described above.

Caveats: do not overload the slides with culture since you do not want a

heaped up mess but isolated cells. A suspension that is quite turbid should

work well - not a paste or milky/opaque solution.

Contact me if you have any other questions. I have done a lot of imaging of

yeasties by TEM and SEM.

Cheers,

####################################################################

John J. Bozzola, Ph.D., Director

Center for Electron Microscopy

Neckers Building, Room 146 - B Wing

Southern Illinois University

Carbondale, IL 62901

U.S.A.

Phone: 618-453-3730

Fax: 618-453-2665

Email: bozzola@siu.edu

Web: http://www.siu.edu/departments/shops/cem.html