[Histonet] Vacuum processing question

[Rene J Buesa]

Vacuum processing has been advocated in the past as a way to process, but the penetration of liquid is not a function of negative pressure ("vacuum") but of positive pressure.
In the initial experiments with the Peloris instrument in Australia (by BioSystems before it was bought by Leica Microsystems) they subjected the tissues that were dehydrated with ethanol to high temperature under vacuum because they needed to eliminate the ethanol before the hot paraffin wax infiltration because ethanol absolutely does not mix with paraffin.
Once they changed to isopropyl alcohol (that mixes with melted paraffin) the vacuum and heat step was reduced in time and intensity.
The VIP uses vacuum in their retort as the only way the have to empty the retort but that vacuum step has effect on the tissues inside only during the few minutes the retort is empty, that is when the tissues as subjected to vacuum. At that moment the liquids inside the tissues will exit them and "facilitate" the penetration of the next liquid that will enter the retort under pressure.
Vacuum per se has no place in tissue processing except to facilitate the elimination of some liquid that has already penetrated the tissue and as an intermediate step between other steps involving a liquid.
I just do not know why you expect to obtain with your vacuum oven. They are usually used when you want to evaporate some liquid at a temperature below the boiling point. In the same way that a pressure cooker allows you to heat water at above 100ºC without boiling, a vacuum oven will allow you to boil water at less than 100ºC. Both variations of the boiling point (above or below) are an inverse relation with the (+) or (-) pressure.
If you leave your specimens without any liquid in your vacuum oven, you will just dehydrate them.
It seems that you have been asked to do this without any further instructions. Ask whomever requested you to do this what for you are supposed to do this.
Never fear to ask anybody who instructs you to do something: why!
René J.

--- On Thu, 3/15/12, Kara Lee <karabou76 <@t> hotmail.com> wrote:


From: Kara Lee <karabou76 <@t> hotmail.com>
Subject: [Histonet] Vacuum processing question
To: histonet <@t> lists.utsouthwestern.edu
Date: Thursday, March 15, 2012, 10:10 AM



Hello all,
I have a question about vacuum processing. 

We have ligament tissues that are processed over night, then are to be vacuum processed.  I've never done this before, but was able to get ahold of a vacuum oven.  
>From what I understand, I just put the tissues in a metal container and turn on the vacuum oven for 1 hour.  But what are they supposed to be in?  Parafin?  Alcohol?  Do I just leave them in the cassettes and not put them in any sort of liquid? 

Is there a specific temperature or vacuum that any of you have used that works particularly well?

Also, is it possible to let the tissues sit over the weekend after the regular processing before putting them in the vacuum oven for processing?

Any advice would be greatly appreciated as I am new to this world of histology, having a biochemistry background.

Many thanks in advance,
Kara
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