bugging me

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Mike7704

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Okay, this has been bugging me for a little while and no one in my lab seems to know...when you autoclave a pipette box how are the pipettes inside the closed box sterilized? The box is completely closed so I can't figure out how the steam would get inside the box. If steam gets into the box what is stopping contaminated air from getting into the box as well?

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Idk why, put this post made me LOL. Sorry OP, but cant help you there..
 
Hmm...I've never questioned it, but I have appreciated not getting any uninvited nasties in my cell cultures.

... Several minutes have passed since I wrote the above, and I have given the problem a bit more thought. First, the steam does get in the containers. I often autoclave fresh tips at the end of the day and leave them in the warm autoclave over night to make sure they are dry.

As for the problem of air getting back in, well, there really isn't a problem. If you aren't already familiar with Pasteur's work which helped shed doubt on the spontaneous generation theory of life, this Wikipedia link will probably explain it nicely for you. Essentially, air is not the problem, and just because air can get in does not mean bacteria can as well.
 
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hmm... I don't think its the "steam getting inside the box" that kills bacteria persay, but rather the high temps/pressure. Regardless if the steam gets in, the high temps/pressure will purge those bacteria.

Here's a little thing you can do though. Take a piece of autoclave tape (normally no black lines; turns into black stripes at high temp/pressure) and put it inside your tip box. After that, autoclave your tips, then bring your tips somewhere sterile like a culture hood I guess and check if that tape changed color to soothe your worries. If it did, your samples are sterile. Congratulations!

I'm no expert on this so someone correct me before poor OP has crap growing in his cultures. 🙂

edit: OP if your lab has preracked tips those are sold sterile (RNase/DNase free) and will remain so if you are careful and use them under sterile conditions.
 
hmm... I don't think its the "steam getting inside the box" that kills bacteria persay, but rather the high temps/pressure. Regardless if the steam gets in, the high temps/pressure will purge those bacteria.

Here's a little thing you can do though. Take a piece of autoclave tape (normally no black lines; turns into black stripes at high temp/pressure) and put it inside your tip box. After that, autoclave your tips, then bring your tips somewhere sterile like a culture hood I guess and check if that tape changed color to soothe your worries. If it did, your samples are sterile. Congratulations!

I'm no expert on this so someone correct me before poor OP has crap growing in his cultures. 🙂

That's what I thought, too. Actually, the air is purged from the system and replaced with steam since air is pretty tough to sterilize.
 
That's what I thought, too. Actually, the air is purged from the system and replaced with steam since air is pretty tough to sterilize.

hmm interesting, I did not know that it was the air that had to be purged. Thanks for that. Just curious, do you use preracked tips in your lab? I think those should be ok for the OP? 😕
 
hmm interesting, I did not know that it was the air that had to be purged. Thanks for that. Just curious, do you use preracked tips in your lab? I think those should be ok for the OP? 😕

We've been racking our own tips lately...budget cuts 😡
I haven't had any problems racking/sterilizing on my own.
 
We've been racking our own tips lately...budget cuts 😡
I haven't had any problems racking/sterilizing on my own.

I agree. if the autoclave is working properly, even if the boxes are closely completely, they're not airtight therefore steam can still get in and sterilize the tips.

and yes that means bacteria CAN can in after the tips are sterilized(although to an very limited extent since the box prevents most air flow into the box under normal conditions, whereas the autoclave cycles forces air/steam into the box). so if you want to be safe you can store sterilized tips in the hood.

but anyway. if you have good sterile techniques when doing cell culture, contamination is not likely.
 
I think in this case the sterilization factor is simply the high temperature. The steam makes sterilization quicker but is not necessary from a theoretical perspective.

That's what I thought, too. Actually, the air is purged from the system and replaced with steam since air is pretty tough to sterilize.

The goal is not to sterilize the air. The goal is to fill the chamber with steam. Sterilizing air is pointless since the second you open the chamber the air would be contaminated already.
 
I think in this case the sterilization factor is simply the high temperature. The steam makes sterilization quicker but is not necessary from a theoretical perspective.

steam sterilizes much more efficiently too. i think this is what the OP meant.

"When the goal of autoclaving is to achieve sterility, it is very important to ensure that all of the trapped air is removed. The reason for this is that hot air is very poor at achieving sterility. Steam at 134 °C can achieve in 3 minutes the same sterility that hot air at 160 °C takes two hours to achieve."

http://en.wikipedia.org/wiki/Autoclave
 
hmm... I don't think its the "steam getting inside the box" that kills bacteria persay, but rather the high temps/pressure. Regardless if the steam gets in, the high temps/pressure will purge those bacteria.

Here's a little thing you can do though. Take a piece of autoclave tape (normally no black lines; turns into black stripes at high temp/pressure) and put it inside your tip box. After that, autoclave your tips, then bring your tips somewhere sterile like a culture hood I guess and check if that tape changed color to soothe your worries. If it did, your samples are sterile. Congratulations!

I'm no expert on this so someone correct me before poor OP has crap growing in his cultures. 🙂

edit: OP if your lab has preracked tips those are sold sterile (RNase/DNase free) and will remain so if you are careful and use them under sterile conditions.

You are 100% right temperature and pressure make the kill!
 
Be like me and leave your pipette tip boxes slightly open with autoclave tape. Contaminated air can't land on them since you snap the boxes closed when you take them out and steam can penetrate more easily while they're in the cooker.
 
The autoclave operates under extreme pressure in order to make the sterilizing solution reach a temperature above its boiling point (otherwise, for instance the water would only get to 100C). That pressure drives it into that closed box even though it does not seem highly permeable to you (you will note it is not actually air tight). Under regular atmospheric pressure, the box is comparatively airtight and comparatively contaminant free after sterilization.

The sterilization is not provided by the temperature alone, but by the properties of damp heat. Steam is far more sterilizing at a lower temperature (and the requisite high pressure to make it) than dry air at a higher temperature--that is why autoclaves are used widely instead of high temperature ovens. The organisms you wish to eradicate will absorb heat energy (and die) more efficiently from vaporized solution with a higher thermal conductivity than from dry air.

As has already been mentioned:
To achieve the same sterility as an autoclave at the standard 134C in 3 minutes, dry air must be at 160C for TWO HOURS.
 
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