Does anyone have a solution ?? ANy advise?

Dr. Theresa A. Fassel
fassel@post.its.mcw.edu

Hi Teresa,
I have done Yeast, lichen, soybean and tobacco seedlings with microwave fixation and processing, and have been quite pleased with the vast improvement in results.

I have used both a standard microwave and a nice one from Ted Pella specifically for EM.

There are many ways to do this, but the following is what we follow in our lab:

Microwave rules:

1. Always use at least a 250 ml water load to collect the heat and keep it off your sample. I change this every 16 seconds of use ( 2 fixations). **** Always set the water load in the same place each and every time.

2. Calibrate the Microwave. Set the water load/ beaker in it's designated spot. Use liquid crystal sheets and microwave about 15 seconds. -- Observe where the hot spots show up, and avoid them. Pick a place preferable inbetween hot spots.

3. Always do a pilot study first.

4. Things to avoid like the plague:
--- Always chill your sample in wetted down crushed ice for at least 30-60 seconds before microwaving. This will allow MW, but keep heat off your sample.
**** As soon as you are finished microwaving , put back into the ice for a moment.M
---- Always wipe off the outside of your vial, for water draws heat ( hence the water load situations) Also never allow water to be anywhere but in the waterload beaker.
--- Using the same principle, only use enough fixative/ solution to just cover the sample. I usually use about 1-2 mm high. Greater volumes will EXPONENTIALY (sp) increase the heat load to your sample. The difference between 6 drops and 1 ml can be 40C!!!!!

Processing times: ==============

You have to experiment with this.

I'd start out with possibly the following for things with thick walls and for plants:

1. Ice sample, wipe, microwave for 7 seconds, ice.
2. Repeat the above for a total of 3 times.
3. Rinse in buffer.
4. Put into OsO4---2%aq. Ice, MW 7 seconds, let set 10 min................change solution and repeat once more. And save the last OsO4 on the sample.

****** it is amazing how MW speeds up processing times, for the first time ever after starting microwave, I have to really watch OVER staining enbloc. So...... repeat a 3rd time if you don't like the staining of the first, but try doing only 2 OsO4's at first.

5. To the last OsO4 solution on sample, Add equal volume of 3% Potassium Ferricynide. Set for 20 minutes only.

6. Rinse with water.

7. Add filtered saturated aq Uranyl Acetate, no older than 10 days. ( Might be our water problem) Ice, MW 7 seconds Ice, and let set for 15 minutes at room temperature. REPEAT

8. Dehydrate as norm. 8-10 min. each step.

9. Infiltration: -- these will get warm, it's OK, and even helps greatly.

1:1 MW 10 seconds, no ice, incubate (rotating)15 minutes and REPEAT
1:4 MW 10 seconds, no ice, incubate 1 hour and repeat.
Pure epoxy: MW 10 seconds, no ice Incubate 1 hour intervals and overnight.
Repeat MW in am, make at least 2 changes of epoxy.

** Also helps to spend overnight in vacuum in a desicator hooked up to house vacuum.

The difference I've seen in structure, staining and non-pitting out of the specimens vs the more traditional methods I've used is incredible. We like microwave so much we now use it for all of our samples.

I'm going to be leaving for a week, but if you'd like to know more, I can get you references etc when I'm at work when I get back. Our methods are based on Logan's, Ted Pella's and my 2 years of working through techniques.

Lou Ann Miller
lmiller@ux1.cso.uiuc.edu