ChemBioMatrix
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Sterilizing unsterile syringe and membrane filters
Hello, so I have to sterilize my syringe filters, and membrane filters and they claim to be autoclavable at 121*C. How do you guys recommend I go
about placing them, should I simply set them in the autoclave? Or pouch them up? wrap them loosely in foil perhaps? and the membrane filters im more
concerned about, if anyone has any tips or methods on placing these into an autoclave, or more so just the entire process to achieving sterile filters
after the process i'd greatly appreciate it!
[Edited on 28-4-2015 by ChemBioMatrix]
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Tsjerk
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Make sure they're not submerged in water. Put them in a plastic or glass container or something with some aluminium foil over it. Some water will
always find its way in the container, but you can evaporate that overnight at 60 degrees or RT for a couple of days, leaving the foil in place.
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Kitsune1
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Hi ChemBioMetrix,
I would always aim to place any instruments I am sterilising inside of a autoclave pouch or bag with an indicator tap, if these aren't available then
loosely wrapped in foil. I am guessing you are using a pressure cooker as an autoclave? if so then don't place the items directly on the bottom, use
the rack/trivet that would have come with it and I prefer to use a thick cloth underneath which will insulate the items being sterilised from the
extreme hot of the bottom as much as possible because I have had Polypropylene melt in the past even at 121C 15psi. Keep items away from the sides of
the vessel which can also get very hot.
Allow the autoclave to cool down on its own, to at least 50C, even better to leave it over night. Wrapping things up in foil as well as keeping excess
water off (doesn't work too well, things almost always still end up wet) gives a barrier against contaminants for when you finally open the autoclave
lid, I prefer to only open the lid once I am ready to use the equipment, then transfer it straight to the glovebox, no waiting around. Remember to
wipe down everything on the outside of the autoclave before opening with ethyl or isopropyl alcohol and leave to evaporate before actually opening it
and wear sterile gloves/ or alcohol sanitised gloves when you transfer your foil/autoclave bags. There are bacteria and fungi spores everywhere which
will contaminate your project.
Kit
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ChemBioMatrix
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Wow okay great! Thank you both very much for answering my question so thoroughly! I am so glad I asked prior to doing so!
I am using an automated autoclave actually, not a pressure cooker style autoclave, and I was going to actually ask this question depending on the
feedback I received, since I have an autoclave with different automated per-programmed settings choices, (unwrapped, wrapped, pouches, liquids) I have
a choice of a drying cycle after the autoclaving is finished, should I utilize the option for drying? Again thanks so much for replying and also for
such detail, I greatly appreciate it!
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Tsjerk
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I can't say anything about the different programs as I don't really know them. But if I where you I would go for dry/wrapped, with the additional
drying step. When you wrap it in aluminium foil and dry it before opening you should have minimal change of contamination. That is at least how I
always do it.
I don't know how much experience you have with autoclaves, or if there is someone else who does, but keep in mind that even during the "dry" program
you need water in the autoclave. Dry or wet just reverse to the sample you put in. Otherwise you will probably either get an error or you destroy the
machine.
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Kitsune1
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It really depends upon the capabilities of the machine you are using, programs can vary between different models; check out the information that came
with the equipment you are planning to sterilise, I have found that things from Fisher tend to come with rather good guidelines for use and if they
don't, speaking to your account manager usually does it.
For single use items like syringe filters and membranes, I would always aim for pre-packed, gamma radiation sterilised disposables with a batch number
and a guarantee of sterilisation; I tend only to sterilise reusable equipment like forceps and scalpels. This saves all the rigmarole of
sterilisation/tyndallisation and risk of something going wrong like a component melting (HDPE, LDPE or PETG will melt and can sometimes be used in
syringe filters) or incomplete sterilisation. Be careful with dry cycles as these can get hotter than the usual wet run and can (depending on the
machine) but your equipment should say whether it is suitable for a dry cycle or give you the optimum autoclave cycle.
If in doubt, try it out on the small scale first; as you are using a professional, automatic autoclave; aim to use an autoclave basket (a perforated
PP tray to sit all equipment in) it makes things far easier to clean up for when things go wrong and is good practice plus far easier to handle if
removing from a hot autoclave.
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ChemBioMatrix
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Great yea actually that is the cycle I chose, I can choose between unwrapped instruments which is preset at 135* C but can adjust exposure times
starting as low as just 10 minutes, which also has a adjustable dry cycle with door closed, then of course the additional open door dry cycle once
it's finished. To high of temp. According to the whatman PTFE and Nylon membrane filters and syringe filters. Well actually all they suggest is that
they are autoclavable at 121*C for 15 minutes... Well here's my issue, the two cycles that are preset at 121*C which one is a "liquids" cycle which
doesn't have the optional post dry cycle, so the only one left that I am able to use that's 121*C with a drying cycle is the "Packs" cycle which in
that case it'd my desired temperature, as well as the dry cycle after, but! The minimum exposure time is 30 minutes. I'm assuming they suggest 121*C
for 15 minutes as that's sufficient enough exposure time to indeed sterilize the filters. I just hope the addidition 15 minutes is indeed safe to
continue. When I put my PTFE membrane filters in to sterilize they came out a little warped looking, which I believe was caused by the drying to be
honest. The Nylons on the other hand come out looking perfect. Anyhow thanks a bunch guys, any suggestions or comments are always appreciated! Have a
wonderful day.
By the way my autoclave is a Harvey MC-10, almost new condition! Had 24 cycles when I got it! Works amazingly, more then what I can ask for really.
[Edited on 29-4-2015 by ChemBioMatrix]
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ChemBioMatrix
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Well I went ahead and simply placed a few unsterilized syringe filters into a beaker, covered the breaker with foil loosely, and put them through a 30
minute cycle, with a 15 min dry cycle, opened the door slightly and there was still some condensation in the beaker so with sterile gloves on I
reached in and made sure the foil was much more tightly conformed to the beaker not allowing air in, then closed the door so it was only about half an
inch open and ran an open door dry cycle that cleared it all out. and just to be safe I ended up doing one more entire cycle, lets hope these indeed
are sterile! I guess I could just filter some sterile water through them and incubate the filtrate for colonies couldnt I?
[Edited on 1-5-2015 by ChemBioMatrix]
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Kitsune1
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Quote: Originally posted by ChemBioMatrix  | Well I went ahead and simply placed a few unsterilized syringe filters into a beaker, covered the breaker with foil loosely, and put them through a 30
minute cycle, with a 15 min dry cycle, opened the door slightly and there was still some condensation in the beaker so with sterile gloves on I
reached in and made sure the foil was much more tightly conformed to the beaker not allowing air in, then closed the door so it was only about half an
inch open and ran an open door dry cycle that cleared it all out. and just to be safe I ended up doing one more entire cycle, lets hope these indeed
are sterile! I guess I could just filter some sterile water through them and incubate the filtrate for colonies couldnt I?
[Edited on 1-5-2015 by ChemBioMatrix] |
Yes that could be a good idea although I would suspect that you have already succeeded at reaching sterile conditions (a few times over); be aware
that excessive sterilisation can cause some equipment to prematurely weaken or fail).
A streak plate would show up any contamination after a couple days with very obvious results (colonies); but this should be totally unnecessary as
your typical 30 minute cycle at 15 psi (121C) will be enough to kill off any contaminants you may have. These filters are now likely to be the
cleanest things in your lab/cleanroom.
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