Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

WHen you autoclave


  • Please log in to reply
16 replies to this topic

#1 c0ok1e

c0ok1e

    member

  • Active Members
  • Pip
  • 22 posts
4
Neutral

Posted 16 April 2009 - 12:16 AM

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

#2 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 16 April 2009 - 12:24 AM

when I autoclave a forceps, I put it in aluminium foil to keep it sterile out of the autclave. I don't put aluminium foil around the box containing the tips as the box is closed, but some people are more than careful, and put also aluminium foil.
The same when I was using cotton wool to stuff, I never added aluminium foil, and the medium has never been contaminated.
I think it's useless, and not good for our environment

#3 GeorgeWolff

GeorgeWolff

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 294 posts
0
Neutral

Posted 16 April 2009 - 01:40 AM

It may be unnecessary - try it without the foil and see.

But forget the enviro silliness - it makes no difference in that regard.

Edited by GeorgeWolff, 16 April 2009 - 01:41 AM.


#4 Astilius

Astilius

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 50 posts
4
Neutral

Posted 16 April 2009 - 01:51 AM

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?


The aluminium foil will help keep the cotton bung dry. It won't interfere with autoclaving.
You don't want the bung to get wet. It's purely a practical step.

The same principle applies to other items. Wrapping in aluminium foil can keep the items dry-ish. It means that drying off tips after autoclaving will take a short while instead of a long while.

It's purely a practical step.
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#5 klinmed

klinmed

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
1
Neutral

Posted 16 April 2009 - 11:17 AM

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

There seems to be some miss-understandings of the principles behind steam-sterilization.

Non heat-conductive items like cotton wool, plastic pipette tip boxes (and the tips!!) etc MUST be in contact with steam throughout the sterilization process. If aluminium foil keeps the items "dry" (prevents access of the steam) they will not be sterilized with the times/temperatures used for autoclaving.

Autoclaving is usually undertaken at 121 oC for ca 30 mins or at higher temps for shorter periods. In contrast, dry heat (oven) sterilization requires much higher temperatures/times ca 160 oC for 2-3 hours. Thus, in the absence of steam contact, autoclaved pipette boxes etc will not get hot enough, for long enough, to assure sterility.

The principle behind this is simple: Steam is many times more effective at conveying thermal energy to the item than hot (dry) air. For example, in the kitchen, potatoes can be cooked in a few minutes in a steam pressure cooker (115 - 121 oC) while cooking at THE SAME TEMPERATURE would take many hours in a hot-air oven.
In addition, steam "wets" the bugs and thus facilitates lethal heat-induced protein coagulation.

Small items like forceps can be autoclaved when wrapped tightly but only because they conduct heat rapidly.

In critical applications (hospitals for example) special indicators are placed in the center of dry loads to check for adequate steam penetration (eg Bowie-Dick test).

Thus, if you must wrap dry items with foil before autoclaving, do it loosely. However, it is better (and required for GMP, ISO..) to use steam-permeable bags specially made for the purpose.

Remember that standard autoclave tape ONLY shows that the article has been exposed to steam. Not that it has been treated long enough for sterility. Appropriate autoclave test strips that integrate time and temperature can be purchased from a number of sources.

Sorry to go on, but think this is important.

Edited by klinmed, 16 April 2009 - 11:43 AM.


#6 c0ok1e

c0ok1e

    member

  • Active Members
  • Pip
  • 22 posts
4
Neutral

Posted 16 April 2009 - 06:33 PM

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

There seems to be some miss-understandings of the principles behind steam-sterilization.

Non heat-conductive items like cotton wool, plastic pipette tip boxes (and the tips!!) etc MUST be in contact with steam throughout the sterilization process. If aluminium foil keeps the items "dry" (prevents access of the steam) they will not be sterilized with the times/temperatures used for autoclaving.

Autoclaving is usually undertaken at 121 oC for ca 30 mins or at higher temps for shorter periods. In contrast, dry heat (oven) sterilization requires much higher temperatures/times ca 160 oC for 2-3 hours. Thus, in the absence of steam contact, autoclaved pipette boxes etc will not get hot enough, for long enough, to assure sterility.

The principle behind this is simple: Steam is many times more effective at conveying thermal energy to the item than hot (dry) air. For example, in the kitchen, potatoes can be cooked in a few minutes in a steam pressure cooker (115 - 121 oC) while cooking at THE SAME TEMPERATURE would take many hours in a hot-air oven.
In addition, steam "wets" the bugs and thus facilitates lethal heat-induced protein coagulation.

Small items like forceps can be autoclaved when wrapped tightly but only because they conduct heat rapidly.

In critical applications (hospitals for example) special indicators are placed in the center of dry loads to check for adequate steam penetration (eg Bowie-Dick test).

Thus, if you must wrap dry items with foil before autoclaving, do it loosely. However, it is better (and required for GMP, ISO..) to use steam-permeable bags specially made for the purpose.

Remember that standard autoclave tape ONLY shows that the article has been exposed to steam. Not that it has been treated long enough for sterility. Appropriate autoclave test strips that integrate time and temperature can be purchased from a number of sources.

Sorry to go on, but think this is important.


I like your explanation. So does it mean that usng the aluminium foil to cover our non heat condusive items are wrong that we should not use the foil?

#7 klinmed

klinmed

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
1
Neutral

Posted 16 April 2009 - 11:34 PM

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

There seems to be some miss-understandings of the principles behind steam-sterilization.

Non heat-conductive items like cotton wool, plastic pipette tip boxes (and the tips!!) etc MUST be in contact with steam throughout the sterilization process. If aluminium foil keeps the items "dry" (prevents access of the steam) they will not be sterilized with the times/temperatures used for autoclaving.

Autoclaving is usually undertaken at 121 oC for ca 30 mins or at higher temps for shorter periods. In contrast, dry heat (oven) sterilization requires much higher temperatures/times ca 160 oC for 2-3 hours. Thus, in the absence of steam contact, autoclaved pipette boxes etc will not get hot enough, for long enough, to assure sterility.

The principle behind this is simple: Steam is many times more effective at conveying thermal energy to the item than hot (dry) air. For example, in the kitchen, potatoes can be cooked in a few minutes in a steam pressure cooker (115 - 121 oC) while cooking at THE SAME TEMPERATURE would take many hours in a hot-air oven.
In addition, steam "wets" the bugs and thus facilitates lethal heat-induced protein coagulation.

Small items like forceps can be autoclaved when wrapped tightly but only because they conduct heat rapidly.

In critical applications (hospitals for example) special indicators are placed in the center of dry loads to check for adequate steam penetration (eg Bowie-Dick test).

Thus, if you must wrap dry items with foil before autoclaving, do it loosely. However, it is better (and required for GMP, ISO..) to use steam-permeable bags specially made for the purpose.

Remember that standard autoclave tape ONLY shows that the article has been exposed to steam. Not that it has been treated long enough for sterility. Appropriate autoclave test strips that integrate time and temperature can be purchased from a number of sources.

Sorry to go on, but think this is important.


I like your explanation. So does it mean that usng the aluminium foil to cover our non heat condusive items are wrong that we should not use the foil?

Things to remember when you steam sterilise:

For dry goods: Wrap in a way that does not hinder steam penetration. A loose covering of aluminium foil MAY be ok but it is not good practice. As mentioned previously, steam-permeable autoclave bags are preferable. For small instruments, like forceps, large test-tubes plugged with cotton wool can be useful.

With tip boxes adequate steam exposure is problematic especially if you tightly close the lid. If you don't believe me try autoclaving a sterilization indicator strip (one that tests both adequate temperature AND time) in a closed box and under a full layer of tips. I did this many years ago using a well maintained high pre-vacuum autoclave. Sterilization takes considerably longer than 20 mins! I now autoclave slightly opened tip boxes that are packed in autoclave bags. After cooling, I close them without opening the bag.

Remember that sterilized packed goods must be fully dry before placing them on a non-sterile surface!

With wet goods: Keep bottle caps loose or use cotton wool plugs (always use the non water-absorbent type of wool. Remember that sterilization time depends on volume (2 liters of fluid takes considerably longer to sterilize than 10 ml).

It is good laboratory practice to test at least once a month how well your autoclave functions. We do this by 1)autoclaving a sealed glass ampule of G. stearothermophilus spores (available commercially) in 1 liter of water (for wet cycle). 2) Autoclaving a non-biological indicator (intergrating!) packed at the bottom of a 1 liter beaker full of microfuge tubes (dry cycle).

Better safe than sorry...

Edited by klinmed, 16 April 2009 - 11:36 PM.


#8 Astilius

Astilius

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 50 posts
4
Neutral

Posted 17 April 2009 - 02:22 AM

klinmed is right.
Ignore what I said about tips (it's not a common practice to wrap them in aluminium foil anyway - some folk autoclave already 'sterile' tips and wrap the box tightly in aluminium foil as a kind of wrapping. The autoclaving, seems to be, a paranoid last line of defense) but you will want to protect your cotton bungs from getting wet. The cotton bungs should be sterile to start with and thus full autoclaving shouldn't be necessary*. You really don't want them getting sodden. But when you wrap the aluminium foil around the bung don't do it very tight, just wrap it round and secure with a squeeze of your hand.

But aye, I wasn't very exact in what I said. klinmed's description is far better than mine.





*Actually, now that I've typed this I hate it. It's standard practice in a few places I've been but it does seem not to be best practice.
To the last, I grapple with thee; from Hell's heart, I stab at thee; for hate's sake, I spit my last breath at thee.

#9 c0ok1e

c0ok1e

    member

  • Active Members
  • Pip
  • 22 posts
4
Neutral

Posted 17 April 2009 - 04:54 AM

klinmed is right.
Ignore what I said about tips (it's not a common practice to wrap them in aluminium foil anyway - some folk autoclave already 'sterile' tips and wrap the box tightly in aluminium foil as a kind of wrapping. The autoclaving, seems to be, a paranoid last line of defense) but you will want to protect your cotton bungs from getting wet. The cotton bungs should be sterile to start with and thus full autoclaving shouldn't be necessary*. You really don't want them getting sodden. But when you wrap the aluminium foil around the bung don't do it very tight, just wrap it round and secure with a squeeze of your hand.

But aye, I wasn't very exact in what I said. klinmed's description is far better than mine.





*Actually, now that I've typed this I hate it. It's standard practice in a few places I've been but it does seem not to be best practice.


Its good that we brought up this discussion.. so now we kow the standard practice in most of our labs are unnecessary.. so the next time when i autoclave i wont use aluminium foil to cover unneccessary items like medium and pipette tips...its just wasting resources...

#10 josse

josse

    Student

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 17 April 2009 - 07:17 AM

klinmed,

when we use the autoclave we have the pipettips in a plastic box and around the lid of the box (the gap you see) we put some indicator tape (completely around the box)
Now reading your posts here I wonder whether the tips are sterile like this, because they are in a box and sealed.

And what with other objects like a little jar or tweezers. We wrap aluminum foil around it, put in the autoclave and then we take it out and put it in the cabinet and we assume it stays sterile. But is this even ok?


and another question:

Remember that sterilized packed goods must be fully dry before placing them on a non-sterile surface!


Why?
and when the autoclaving is done, you simply let the products in the autoclave to dry or?
I mean: when I finished autoclaving the products are always wet but we take them out and put them inside the cabinet.
(keep in mind we do use the simplest autoclave possible: a stovetop autoclave)

Edited by josse, 17 April 2009 - 07:29 AM.


#11 klinmed

klinmed

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
1
Neutral

Posted 17 April 2009 - 12:32 PM

klinmed,

when we use the autoclave we have the pipettips in a plastic box and around the lid of the box (the gap you see) we put some indicator tape (completely around the box)
Now reading your posts here I wonder whether the tips are sterile like this, because they are in a box and sealed.

And what with other objects like a little jar or tweezers. We wrap aluminum foil around it, put in the autoclave and then we take it out and put it in the cabinet and we assume it stays sterile. But is this even ok?


and another question:

Remember that sterilized packed goods must be fully dry before placing them on a non-sterile surface!


Why?
and when the autoclaving is done, you simply let the products in the autoclave to dry or?
I mean: when I finished autoclaving the products are always wet but we take them out and put them inside the cabinet.
(keep in mind we do use the simplest autoclave possible: a stovetop autoclave)


Often we follow a procedure because it is the "way things have always been done in this lab". More often than not the procedure will work fine. Most of the time.... But why risk a cloning experiment or a primary cell culture because of the poor preparation of sterile starting materials? We have enough to worry about with our experiments than being concerned about something being "sterile enough".

A bit of equipment being "sterile enough" is like a woman being "almost pregnant"! It is or it is not.

As mentioned above, if you hamper steam penetration (eg by sealing tip boxes with autoclave tape), you risk inadequate sterilization. Always wrap dry goods in steam-permeable packs if they are to be autoclaved. Special autoclave bags are best, but filter paper is also adequate.

I always use a machine that pulls a high vacuum after sterilization in order to dry the goods. If your stovetop autoclave cannot dry well it is probably a good idea to place the sterilised material on a ethanol-cleaned LAF bench until dry.

By the way, I found out how to sterilize items correctly AFTER losing $ 13, 000 (yes three zeros) worth of immunomagnetic beads because of an inadequately autoclaved bottle. Once done, never forgotten.

#12 josse

josse

    Student

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 18 April 2009 - 05:54 AM

I see, thanks klinmed.

I do know that the boxes we use always seem to be wet inside. Maybe its some sort of plastic that lets steam pass ? Dont you think that the people that make those pipetboxes build them so that steam can penetrate?



however still another question: why does it need to be dry when storing it? does a wet object attrackt more micro-organisms?

Edited by josse, 18 April 2009 - 05:55 AM.


#13 c0ok1e

c0ok1e

    member

  • Active Members
  • Pip
  • 22 posts
4
Neutral

Posted 19 April 2009 - 06:42 PM

I see, thanks klinmed.

I do know that the boxes we use always seem to be wet inside. Maybe its some sort of plastic that lets steam pass ? Dont you think that the people that make those pipetboxes build them so that steam can penetrate?



however still another question: why does it need to be dry when storing it? does a wet object attrackt more micro-organisms?


I think its because wet area, microorganisms can grow on it so it will not be sterile anymore. There are microorganism in the air also so if its wet then it will be contaminated very fast.

#14 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 20 April 2009 - 12:14 AM

I would like to add some comments :
first sterility is not black or white. It's just a way to reduce the risk of a contamination to the lowest point, however the probability of a contamination will never be zero.
Second, the tip boxes are not so well closed that they do not let the steam enter. So, your tips are sterilised. If you open the autoclave to early, there will be water inside the box, it's the condensation of the steam. We let all the material dry in a Pasteur oven, to be sure there will be no water inside the boxes.

#15 fitriyusof

fitriyusof

    member

  • Members
  • Pip
  • 5 posts
0
Neutral

Posted 09 May 2009 - 04:12 AM

guys, i want to ask additional question, my colleague used autoclave machine to sterile his cloning plate containing media for bacterial culture before he discard it.
any suggestion or advice to avoid leaking, or negative effect? since last time, i asked him is it safe to autoclave media containing plate becos i afraid it will leak and clog the autoclave drainage system.

tq




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.