[urtula]

Dear all,

I often have to sterilise liquid media by use of an autoclave. Now I just open the autoclave when the pressure is down again (at 99°C).
I however did read somewhere this is not good.
It said that one should not open the autoclave untill the temperature is below 90-85°C. I wonder why is this?

Some stuff I found: due to opening it and the sudden temperature difference you can get "boiling over" effect. I dont get it? If you close the bottles right after opening the autoclave, than how can it boil over?
Also: it said somehwere that because of the high temperature difference the liquid will boil longer !
Is this possible? Seems a bit weird.

Any insights in this?

(I am not sure this is the right subforum for this question)

[billnotgatez]

Have you read this WIKI?

http://en.wikipedia.org/wiki/Autoclave

[urtula]

I dont understand your point. THis page does not state anything about what I asked?

[billnotgatez]

My apologies the WIKI was too sparse on information. I thought reading it would allow you to deduce your answers.

This GOOGLE result seems better but I am not sure it is vetted like WIKI.

http://oomyceteworld.net/protocols/autoclave%20operation.pdf

Also a phase diagram of water might help in understanding

http://en.wikipedia.org/wiki/Properties_of_water#mediaviewer/File:Water_phase_diagram.svg
note the temp is in K not C

In any case basically you have liquid in a steam chamber under higher pressure than 1 Atmosphere. Also the chamber has been taken to a temperature higher than boiling water. So now you got very hot liquid at a very high temp and pressure and you release the pressure. What do you think will happen? I know you said 99 C, but lets take my question first.

[urtula]

Well, if the pressure drops (and the temperature stays the same) than it would become a vapour. This I get.

If I look at the phase diagram it seems that by releasing the temperature and pressure together you will not have problems taking the bottles out.
The pressure at 99°C stays the same (compared to 23°C outside) , its the temperature that drops and looking at the graph, it should stay a liquid.
So I dont see any problem really by opening it at 99°C

[Yggdrasil]

I would not necessarily trust the temperature reading on the autoclave to give an accurate measurement of the temperature of the liquids inside.  The air around the bottles will likely cool much faster than the liquid inside the bottles, so even though the reading may say 99^oC, the liquids may still be above 100^oC and could boil over if the pressure drops back down to 1 atm.

[urtula]

I would not necessarily trust the temperature reading on the autoclave to give an accurate measurement of the temperature of the liquids inside.  The air around the bottles will likely cool much faster than the liquid inside the bottles, so even though the reading may say 99^oC, the liquids may still be above 100^oC and could boil over if the pressure drops back down to 1 atm.

There is also a pressure gauge, you not open the autoclave if there is still overpressure inside.
So the pressure is not going to drop back down when you open it, its already at the normal room pressure.
Or you think that inside the bottles the pressure is stil higher than outside the bottles?

After googling a bit I came up with this:  superheating. This is what I think those people mean. Should I be worried about this?
I once saw it: I took a bottle out of the autoclave (seemed ok, the autoclave was 98°C), but when I placed the bottle on the bench I could clearly see it started to "cook").

[Yggdrasil]

I would not necessarily trust the temperature reading on the autoclave to give an accurate measurement of the temperature of the liquids inside.  The air around the bottles will likely cool much faster than the liquid inside the bottles, so even though the reading may say 99^oC, the liquids may still be above 100^oC and could boil over if the pressure drops back down to 1 atm.

There is also a pressure gauge, you not open the autoclave if there is still overpressure inside.
So the pressure is not going to drop back down when you open it, its already at the normal room pressure.
Or you think that inside the bottles the pressure is stil higher than outside the bottles?

After googling a bit I came up with this:  superheating. This is what I think those people mean. Should I be worried about this?
I once saw it: I took a bottle out of the autoclave (seemed ok, the autoclave was 98°C), but when I placed the bottle on the bench I could clearly see it started to "cook").

Yes, I would worry about superheating.  Laboratory glassware is generally very smooth and does not provide nucleation points for superheated liquids to boil.  Shaking the liquid or otherwise handling the bottles, however, could cause superheated liquid inside the bottles to boil which would be very dangerous.

Is there any reason why you want to remove the bottles so soon after autoclaving? 

[urtula]

There is also a pressure gauge, you not open the autoclave if there is still overpressure inside.
So the pressure is not going to drop back down when you open it, its already at the normal room pressure.
Or you think that inside the bottles the pressure is stil higher than outside the bottles?

After googling a bit I came up with this:  superheating. This is what I think those people mean. Should I be worried about this?
I once saw it: I took a bottle out of the autoclave (seemed ok, the autoclave was 98°C), but when I placed the bottle on the bench I could clearly see it started to "cook").
[/quote]

Yes, I would worry about superheating.  Laboratory glassware is generally very smooth and does not provide nucleation points for superheated liquids to boil.  Shaking the liquid or otherwise handling the bottles, however, could cause superheated liquid inside the bottles to boil which would be very dangerous.

Is there any reason why you want to remove the bottles so soon after autoclaving?
[/quote]

Hallo,

Well, I often remove them pretty fast in order to load another batch.
Is this than so dangerous ? If you close the bottles right after removing them, I can not imagine something going wrong?
So your advice would be to wait till its 90°C or even lower?

On a side note: I am not really an expert on superheating. If I understand you correct than there have to some rough spots in the bottle to form "bubbles" to cook? So because its smooth you will form less of these bubbles (nucleation)?
This superheating, how come it still happens when you take the bottles out of the autoclave ? I would think that the liquid boiled inside (and already formed bubbles there) and thus it would not superheat anymore?

[Yggdrasil]

Well, I often remove them pretty fast in order to load another batch.
Is this than so dangerous ? If you close the bottles right after removing them, I can not imagine something going wrong?

What might you think can happen if the liquid starts boiling in a sealed container?

So your advice would be to wait till its 90°C or even lower?

  Yes, I would follow the recommended procedures for operating your autoclave.

On a side note: I am not really an expert on superheating. If I understand you correct than there have to some rough spots in the bottle to form "bubbles" to cook? So because its smooth you will form less of these bubbles (nucleation)?
This superheating, how come it still happens when you take the bottles out of the autoclave ? I would think that the liquid boiled inside (and already formed bubbles there) and thus it would not superheat anymore?

A superheated liquid is a liquid that exists at a temperature above its boiling point.  This can occur because boiling (the formation of bubbles of water vapor) requires some "activation energy" to break the surface tension of water and allow bubbles to form.  Scratches in the glassware can aid in bubble formation because the help to break the surface tension of water and will aid in the nucleation of bubbles.  These bubbles can nucleate the formation of new bubbles which will catalyze a chain reaction and lead to rapid boiling of the solution (a similar phenomenon happens when you add mentos to a carbonated beverage).  Heating relatively pure liquids in laboratory glassware often results in superheating, and disturbing the liquid by shaking it or adding an impurity can cause rapid boiling.

[urtula]

It seems weird that this can happen in a bottle that has been in an autoclave.
The liquid must have boiled in the autoclave, right?
So after boiling it cools down but yet it can be superheated? Seems weird, difficult to understand.


Answering your question: I pressume the pressure in the bottle can become higher, but can it become so high that the bottle could explode?

[Yggdrasil]

It seems weird that this can happen in a bottle that has been in an autoclave.
The liquid must have boiled in the autoclave, right?
So after boiling it cools down but yet it can be superheated? Seems weird, difficult to understand.

The point of an autoclave is to pressurize the contents before heating to above 100^oC.  Because the boiling point of a liquid depends pressure, pressurizing the autoclave prior to heating will keep the liquid below its boiling point during sterilization.

Answering your question: I pressume the pressure in the bottle can become higher, but can it become so high that the bottle could explode?

Yes.

[urtula]

Ah yes, indeed, I forgot about the fundamentals of autoclaving!

Seems amazing a bottle can still explode!
Have never heard about this.

I will keep your advice in mind.


However one more question: if the liquids should not boil, than why is boil over such a big problem? You are not supposed to fill the bottles completely, because of boil over, but why? If it should not happen?

[Arkcon]

We seem to be going around in circles, however -- if you heat something hotter than its boiling point, but pressurize it so it doesn't boil.  Won't it boil when you reduce the pressure?  And doesn't that have to happen, when you open the autoclave?

I remember moving this thread from Physical chemistry, where you initially posted it, to Biochemistry where it is now.  This was because I thought you could use some tips, from some people with experience.

However one more question: if the liquids should not boil, than why is boil over such a big problem? You are not supposed to fill the bottles completely, because of boil over, but why? If it should not happen?

The reason why is because its a good idea, at all times, to be generous with safety and handling procedures to avoid making a mess.  Consider, if the pressure on a bottle is just slightly high, if it hasn't cooled off just slightly not enough, if it is just slightly overfilled, if a surface nucleation site not present in 99 other bottles is present -- hot media boils over, onto the user, into the autoclave, making a mess. 

You seem to have been going though this entire thread, under the theory that the phase diagram for water applies precisely to a media solution (it doesn't, of course, given coligative properties), and that the temperature and  pressure gauges are accurate and precise to 0.1 units, and you should be able to handle the entire autoclave contents without concern at the very instant your instruments read at the correct point.  Basically to save time?  It seems like this is an interesting thought problem, or a creative argument, but rapidly becoming an impractical discussion, as we continue to try to "logic" away real world concerns.    Are you seeing where I'm coming from?

[Babcock_Hall]

Not all glassware is suitable for autoclaving.  I seem to recall one broken bottle many years ago, but I don't recall the details.