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Autoclaving is my problem

autoclave decontamination

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32 replies to this topic

#16 science noob

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Posted 02 January 2012 - 06:15 AM

Dear science noob, I have a simple question regarding taking wet object out of autoclave into oven , shall I wear gloves while doing so, or I can carry them with my bare hands?????

What ever it is autoclaved or not, and autoclaving only kills cells not chemicals. It is good practice to use Always Gloves

Dear science noob, I have a simple question regarding taking wet object out of autoclave into oven , shall I wear gloves while doing so, or I can carry them with my bare hands?????


Most of the time, we use the inside of autoclaved bottles to fill up with media for cell culture. So, technically you can do whatever you want to the outside JUST follow the golden rule - sterilise everything that goes into the cell culture hood with ethanol.

but similar to Biouday's point, I would automatically put on a pair of gloves whenever I step foot into a lab regardless if I'm doing cell culture or not. It's your hands and you don't know what chemicals have been around it. Gloves are cheap but you only have 2x hands and can't afford to stuff up your experiments.

#17 leelee

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Posted 02 January 2012 - 05:50 PM

When taking objects out of the autoclave, you should absolutely be wearing gloves- thick, heat resistant ones! The inside walls of the autoclave remain hot for some time and you really don't want to burn yourself on that. All it takes is a slight touch, and I've seen the results. Not pretty.

#18 madelingirly

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Posted 02 January 2012 - 07:28 PM

Dear
science noob,

I have followed ur advice in putting an empty petridish with medium only in my incubator to check if my incubator is contaminated or not, I can see the circular shapes in it, from 3 days of incubation, it is much less than the plates which contains the cells but it is there, I have added a photo with my 10X lens1.jpg ,
however, this result may suggest too that my medium may be the source of contamination, because it is the same one which I use in my plates, I dont know, but it is much less compared to original plates with cells

#19 madelingirly

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Posted 03 January 2012 - 10:31 PM

Hi Guys,
I have upload some pictures of my plate, I will be deeply grateful if any one can give me more opinions and comments about these pictures and spherical shapes.
Best Regards
plate-1.jpg
plate-8.jpg
plate-7.jpg

#20 bob1

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Posted 04 January 2012 - 12:27 PM

I have followed ur advice in putting an empty petridish with medium only in my incubator to check if my incubator is contaminated or not, I can see the circular shapes in it, from 3 days of incubation, it is much less than the plates which contains the cells but it is there, I have added a photo with my 10X lens1.jpg ,
however, this result may suggest too that my medium may be the source of contamination, because it is the same one which I use in my plates, I dont know, but it is much less compared to original plates with cells

It is very common to find precipitates in FBS. The photo you have provided is not clear enough to be able to tell if these are precipitates or some other contamination.

Hi Guys,
I have upload some pictures of my plate, I will be deeply grateful if any one can give me more opinions and comments about these pictures and spherical shapes.
Best Regards
plate-1.jpg
plate-8.jpg
plate-7.jpg

Plate 1 shows a clump of cells that either hasn't attached when you last split the cells, or has detached from the plate.
Plate 8 shows what looks like a very confluent plate with the cells looking a little worse for wear (either need to be split or medium changed or they are just getting old). The objects you have put arrows to are all either cell debris (red arrows) or intracellular structures such as vacuoles (black arrows).
Plate 7 the arrows point to floating (and probably dead) cells. It is very normal to see dead/floating cells in any population of cells you are growing, indeed some cell types such as SH-5YSY have a proportion of their normal living population that floats as suspension cells.

#21 philman

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Posted 05 January 2012 - 04:00 AM

Regarding autoclaved glass and plastics, as long as the lid is on and they are sealed then the inside should be sterile wherever you take them, whether is 6m to the oven or to the other side of the city! (obviously the lid should be loose and not tight while it is in the autoclave, so that the sterilising steam can get inside the bottle)

The shapes in your photos look like mammalian cells, not fungi or bacteria, but the cells don't look particularly healthy. I am concerned if you are the only person in your lab doing cell culture work at all, if your equipment is all suitable for biological work. Your incubator is a 37 degree + 5% CO2 incubator right? And you do all work inside a hood with filtered air flow? Sorry for the simple questions but if no-one working with you does cell culture then you might not be aware of the obvious things!

Everyone has problems with sterile technique when they fist start doing cell culture, with no-one physically there to train you it must be even harder!

#22 EvilTwin

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Posted 05 January 2012 - 07:24 AM

Do you use a hood with horizontal or circular air flow? I mean, is the air blowing in your face when you work or is it circulating, and there is a glass screen in front of your face?
Case 1) Do not speak, cough, sing or whatever into the hood. Turn your head away even if you have to sigh (and I suspect we all sigh very often)
Case 2) Do you clean the hood underneath the metal plates that make up the bottom surface as well?

-> Autoclaving: if the container you autoclave is closed tight, theoreticcaly you can do whatever you want with the outer surface and the inside stays sterile. If you are not shure if the container is closed tight or not, you may try covering it with aluminium foil.

How is the air in your incubator moisturized? If there is simply a water container at the bottom, it also has to be refilled with clean water. We also use copper sawdust, which acts antiseptically, but i don't know if this is a common practice and does any good. It's kinda like magic: everyone does this and noone dares to check whether an incubator without copper sawdust would be contaminated with bacteria.

Edited by EvilTwin, 05 January 2012 - 07:29 AM.


#23 bob1

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Posted 05 January 2012 - 11:53 AM

We also use copper sawdust, which acts antiseptically, but i don't know if this is a common practice and does any good. It's kinda like magic: everyone does this and noone dares to check whether an incubator without copper sawdust would be contaminated with bacteria.

Copper does work to some extent, but only in the water tray, it will not prevent growth of fungi and/or bacteria on other surfaces, unless they are also made of copper. Incubators without copper definitely do become contaminated!

#24 madelingirly

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Posted 05 January 2012 - 04:58 PM



Dear

philman


EvilTwin,

SO I shall put my bottles in the autoclave and close them when I take them out, (they will contain water drops as my autoclave has no drying function, then put them in the oven and re-open them there ( to allow them to be completely dry).




Another question, does Aluminium foil is enough to protect sterility, I put my pasteur pippittes in a beaker, then put them in the autoclave, and take them out of it (wet) and put them in the oven to dry, and after drying I cover the top of my beaker with Aluminium foil.
when I use pasteur pipptes in the clean Benche ( I have a horizonatl clean bench as I feel the air blow in my face), I open the aluminium foil take one, and leave it open in the hood and take another one if I need.
Then After I finish, I use the flame to heat part of the beaker which I may accidently touch, then close it with the aluminium foil cover again.

Does this a good aseptic practice?????
Please I will wait ur generous comments.

Secondly

bob1

I never putted any copper in my water tray (as I read it will damage my stainless steel tray )

however I read in ( Culture of animal cells a manual of basic technique and specialized application R.Ian Fresheny)

that I may use 1% Copper sulphate in my water tray, which is better???

can I use copper on my water bath too, or it only works in the presence of Co2??

Thanks Guys for your comments and helpful discussion


#25 philman

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Posted 06 January 2012 - 06:58 AM

Hmm sounds like your problem there is the lack of sterility taking things out of the autoclave. Anything that you are going to use to touch the media or cells in any way, whether bottles, pipettes, other things, must not be exposed to the air! The air in the lab, the dry oven or anywhere is full of fungal spores, bacteria, and all sorts. So when you uncover your pasteurs to dry them they are becoming contaminated straight away! The same with your bottles, as soon as you take the lid off outside the hood they become contaminated. The only places where the air is clean is in the hood where the air is filtered.

With the bottles you should autoclave them with the lids on, but loose so that the steam can get in. then when you take them out you can leave the lids loose for a little bit when you dry them. Personally I don't think a little bit of water is a problem as it will still be sterilised water, it was 135C steam not so long ago!

With the pipettes you should have them in a glass beaker or something with foil over the top, but not sealed, just fold it over the top and the foil should keep the shape of the beaker, keep the foil over the top the whole time they are in the autoclave and also the whole time after you take them out! Only take the foil off when they are inside the sterile hood!
Also you can get paper bags that are designed for autoclaving collections of small items such as pasteurs or eppendorf tubes, the top folds over and is sealed by some autoclave tape, when you autoclave the bag a dark black spot appears so you know they are sterile. But of course as with anything, only ever open then in the hood!


And the copper sulphate in the water bath or water tray is fine, stops anything growing in the water bath and is commonly used.

#26 madelingirly

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Posted 06 January 2012 - 10:26 PM

Dear

philman

But I read in a book ( I will try to put its name for you).
That wet object will lose its sterility
so I should not wrap anything autoclave if it is wet from inside.
these drops of water may affect my medium conectration, ny trypsin and my reagents too.
if in a bottle is 2 drops, and in another bottles is 50 drops I dont know.
so I want to ask you, if u r using autoclave like mine, with no drying function???, so items come out with water drops from it????

#27 EvilTwin

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Posted 09 January 2012 - 12:20 AM

Hi!

Philman is right, anything that is sterile, stays sterile only if you don't open it outside the hood. If your gloves are clean, there is no need to burn the edge of the beaker. Just try not to touch the remaining pipettes. The best way is to tilt the beaker and try to shake it gently untill one pipette slides out.

I know this question was not for me, but I can't remember any moment, when I put medium in a bottle, that was previously autoclaved. When do you do so?

We use sterile bottles, in which the media come, and single-use falcon tubes to prepare any solutions. As far as I remember, they are gamma-sterilised and are sterile inside untill you open them. The only thing that we autoclave before working with cell cultures are all kind of pipetes/pipet tips (they are single-use as well, but not sterile). Even if there is some water in the box containing pipette tips, it doesn't touch the tips, there is no way it can affect the concentration of reagents You measure. Only if some bacteria get into the box, they will grow better if there is water inside.

Bob1, thank you. I have always wondered if it's really so important.

Edited by EvilTwin, 09 January 2012 - 01:24 AM.


#28 philman

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Posted 09 January 2012 - 02:49 AM

The water inside an autoclaved bottle will still be sterile as long as it is not opened outside of a hood. In our lab, and I expect many labs, we autoclave entire 2L bottles of milli-Q water to ensure it is sterile, and it is sterile, up until it is opened outside the hood.

the single worst thing you can do to contaminate anything is to expose it to the air outside of a hood.
(well apart from something silly like stirring it with an ungloved finger or something)

Edited by philman, 09 January 2012 - 02:49 AM.


#29 leelee

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Posted 09 January 2012 - 10:53 PM

But I read in a book ( I will try to put its name for you).
That wet object will lose its sterility


If the source of the wetness is not sterile. Sterile water (which is what the condensation inside your bottles is) will not be the source of contamination. And a few drops will by no means dramatically alter the concentration of your reagents.

At any rate, I agree with EvilTwin on the use of disposables for tissue culture wherever possible. What are you using the bottles for?

#30 science noob

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Posted 10 January 2012 - 12:41 AM

leelee and EvilTwin just doubly reiterated the use of commercial pre-sterilised, single use tubes for cell culture which I have been emphasising previously. You can't afford to add another potential source of contamination to your cell culture and the medium is what you use most and can't afford to contaminate with an unclean glass bottle. With the commercial tubes, you can be guaranteed that your media is kept sterile (unless you dip your dirty gloves in it or somehow technically contaminate the media).





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