How to clean the incubator after bacteria infection? Virkon? - (Apr/19/2008 )

Hi!

We recently had a contamination in one of our incubators, it seems like it was rod-like bacteria. I trashed all the cells and I want to give the incubator a good cleanig, but I am wondering what would be the best way to do it.

I read some previous posts suggesting autoclaving the removable parts of the incubator, but for that I would have to take the parts out into the open air when they leave the autoclave and go back to the tissue culture room. Is autoclaving really essencial, and would it be any good?

The other options I thought of was spraying the incubator extensively with IMS or washing it with Virkon. I suppose Virkon would be more efficient, but at the same time, would't it leave stains on the metal?

And just one more question: after doing the cleaning, how long should I wait to grow my cells again, to be sure the contamination is gone and that there is no residual strong smell left, that could harm my cells?

I would really appreciate if you could give me some tips…

Thank you very much!

Julianne.

PS. Medium and everything else are fine.

I think formaldehyde vapor is sufficienrt to kill all living things. Just guessing, I never try it.

There are a variety of methods of cleaning incubators. The most common is to remove all the bits that can be removed, wash thoroughly in detergent and water then autoclave. Meanwhile, wipe the inside and non-autoclavable bits with virkon or other bactericidal/fungicidal detergent, rinse with water to remove residues and then spray with ethanol and allow to dry. Then replace the autoclaved bits, spraying with ethanol as you do this to prevent re-infection. This is pretty good at keeping the incubator infection free provided it is done about once every couple of months. depending on usage.

Other options include formaldehyde disinfection, which while being fantastic at decontamination has a real problem getting rid/stopping leaks of the formaldehyde and with the amount of down time for the incubators (usually overnight). I would suggest getting a trained professional to do this option, the people who check and maintain your class II hood should be able to do it.

Self cleaning incubators are OK too, but usually have problems with not actually removing persistent infections, especially of the fungal variety.

Having said all this, in my experience, bacterial infections in culture usually result from the waterbath for heating media and/or bad technique, they are typically easily prevented by spraying everything well with ethanol. Fungus is more commonly from incubators and is hard to remove as the spores are resistant to ethanol

I add Incuwater from Applichem whenever I add water to the incubater water tank, and I autoclave the water before adding it. Applichem also sells a disinfectant spray for the incubator, also.

- Eli

bobs right - unless you are smearing your cells on the incubator wall - the problem probably doesnt lie there.
sorry to say - a lot of people do use this as an excuse when the real problem is they keep touching the rim of a bottle with gloved hands (or something similar) - check your technique with a pro first

as for the clean - quick scrub with virkon and a spray of 70% ethanol should do the trick

dom

When we clean the incubator, our lab will swab the racks with 10% bleach; then spray with 70% ethanol.

we'll then leave the incubator on overnight before placing any cells in it.

Hope your contamination issues are resolved.

Hi! First of all, I'm so sorry for the double post, for some reason I didn't find my previous one, so I thought I didn't get to submit it or something! Sorry about it!

Thank you so very much for all your help, it was a great support!

About the cleaning, I did what you guys suggested, except for the autoclaving (our autoclave is very far from the culture room, so I was afraid of getting the racks even more contaminated in doing so)

So the story is basically this: these cells have been fine for months, until one day I arrived and they were totally dead, rounded, floating. I don't recall seeing any signs of contamination back then, and it happened in only 24h! I trashed everything (medium/cells/trypsine etc) and got a new aliquot. It got contaminated after 2 passages, and this time you could see clearly the bacteria wriggling around, turbid medium etc.

That's when I came for your help.

So now I've sprayed the whole incubator with IMS, 2 days later wiped everything with Virkon and washed with IMS. Added new autoclaved water in the pan, prepared new media and everything else. I also tested the old medium and PBS and there was no signs of contamination. Got new aliquot. Contamination returned 24h later.

If it was something that would happen everynow and then, I agree it could be some fault in my technique, but now it seems like a recurrent problem, the same contamination in the same cells everytime, which makes me suspect of the incubator (since everything else is new). My other cell lines, in other incubators, are just fine too, which made me desconsider the hypothesis of our equipment being contaminated. Other ppl's cells are fine as far as I know, so it can't be hood/waterbath.
Since many ppl once in a while do mess up and get contamination, I don't dare saying my technique is absolutely perfect. But what I don't get is how the contamination keeps spreading to new stocks, if I am using everything new!

I am pretty positive stocks are fine, because 1) they are from very different dates, almost 1 year apart, and I don't recall them ever having this sort of contamination, and 2) I used these stocks before and they were fine.

There are no other cells in the incubator except mine, so I can't know for sure.
Right now, I am the only one using this incubator, with only one cell line, and this is the one that is contaminated. I have another cell line in another incubator which is just fine, so I believe it is not related to any of our equipments.
As far as I know, no one else in the room had this problem, but then again, they don't use this incubator. So I thought it couldn't come from hood or waterbath.

The cells are grown in plates, and we do use pen/strep and another antibiotic to keep the selection of the cell line (although the cells didn't have the other antibiotic when they first got infected, since they were only in passage number 1 and we wait til passage 2 to add it, so it's less harmful).

The HEPA filter has been changed about 2 weeks ago, although the contamination was already there, so it might have gotten contaminated too, maybe…

So I am wondering if this may be some really resistant kind of bacteria that might leave their spores in the incubator or something, and even after IMS/Virkon they would stay there… so that's why I thought of the stericycle, although I am not sure if that would be feasible, or even effective…

Any other alternatives?

I will certainly check for the formaldehyde with the maintenance guy.

Thank you so much for your help!!

All the best.

Just a quick note with regards autoclaving the removeable parts - if you place the parts in an autoclave bag and seal it with autoclave tape then you can carry the parts too and from the autoclave and they wont be exposed to the general air. Our autoclave is 4 floors below & thats what we do. When you bring the parts back up from the autoclave and into your cell culture room spray them liberally with 70% ethanol as you are putting them back into the incubator to keep them as clean as possible!

Julianne W,

My cell rounded up too but no bacterial nor fungal growth could be obeseved in the flasks even after observing it everyday for 2 weeks.

If you think Virkon is not working, use multiwell plates (such as 48 wells or 24 wells) add in some VIRKON
example: well 1 0.5ml complete media+ 1ul VIRKON
do some dilutions on the VIRKON add it in and aliquot in some of your contaminated samples (for example 1~10ul/well). Of course starting bacterial titers will affect the results but we are just here to see wheter the little fellows will survive in VIRKON. Or add in some VIRKON, do some washings on the well and add in the culture medium with some of your contaminated samples. This will sort of imitate the washing conditions which you carried out on your trays.

Or if you have access to bacterial culture plates, you can grow your contaminated samples (spread them out with hockey stick) and lay round filter paper discs containing different concentration of antiseptics or antibiotics. This will give you their MICs.

And if you want to find out which spot is the source of contamination, leave multiwell plates filled with your complete media, expose it in your incubator/ hood for a couple of minutes, close them and incubate them. This will determine wheter spores are present in your incubator.

And try to sample some water from the water bath as well. "Other people" might be doing "little things" to avoid contamination, which could be unnoticible. This will tell you wheter your water bath is alright or not.

I had tried doing it with contaminated cultures in the past to determine the titer in which cells could survive and bacteria would die. I once obtainted a contamination which was penicillinG (benzylpenicillin)/streptomycin sulphate resistant but was found to be suseptible to low concentrations of ampicillin. The controls (without antibiotics) grew very well of course. You can also see morphological changes in the bacteria when you add in antibiotics. If they are wriggly rod like creatures, they will start to twist and burst.

There are 2 groups in our lab, one which swears on not using antibiotics at all and the other one is constantly using it. I am in the one which does not use antibiotics but we keep our flasks in the same incubator.

I guess continuos use of antibiotics will select your cells AS WELL AS the bacteria to be resistant towards the particular antibiotic.

Everything was fine for me till recently when all my cells die within 6 hours without reason. I guess technique is really important.

Everything in science has a reason. And we here to get it**

**No matter how absurd it might sound to others.

Dear All,

Thank you so very much for your answers, I will definetely try it all. I'll make sure we get the autoclave bags in case I need to do the whole cleaning, and I wil test the virkon, it is a great idea.

The test the open plates is great too! Thank you so much for sharing all the tips.

I did a test these days using the medium of my other cell line, which is in another incubator and is not contaminated. I left 10cm dishes in the incubador I think is contaminated with this medium, one was left open and the other closed. They have been sitting there for 3 days now and so far none of them got the contamination.

If they indeed don't get contaminated, I'll be quite puzzled.
Could it be that the bacteria likes one kind of medium and not the other, just because of their different composition? If the incubator is indeed the source of the contamination, shouldn't every medium I place in there get contaminated?

Thank you so much, I'll let you know how the next tests went!

All the best,

Julianne.