How to avoid contamination in tissue culture? - (Aug/29/2006 )

As the topic puts it my querry is which are the ways in which the fungal and bacterial contamination can be avoided in animal tissue culture. We get tissues from the hospitals which are collected in media cotaining pen,strep, genta and fungizone. The tissue is washed with PBS containing 2X of same antibiotics. Still the problem of contamination (mainly fungal) is not completely eliminated. It feels really annoying when you see fungal colonies growing in the media where you exprect your cells to grow. (Inspite of somebody telling:" your tissue culture is not contaminated, it is just sharing its living quarters!!" I request all you people to post all suggestions you have regarding this. Thanks.

it seems to be largely an issue of collection practice? unless you can pinpoint the source to your lab

A recent discussion abt cell culture has some interesting suggestions by Rhombus and others with regard to good cell culture practices. just search for it in the forum.

This may simply be my misunderstanding of your post, but I would suggest keeping fungizone and antibiotics in the media that the primary cells are growing in. If I am understanding correctly, you are only treating the tissue with the antibiotic/fungizone? You are most likely introducing pathogens durring the isolation of primary cells.

From my own experience with macerated human tissues I would say the fungus is either present at tissue collection or is living in your incubator and is secondary to the tissue collection procedure (check for a patch of haze on the incubator walls).

Firstly I would ensure your incubator is clean (shut it down and completely decontaminate it; if you can access the fan double check that it doesn't show evidence of fungus on the surface as I've also seen this before).

Assuming your tissues are the problem you will need to keep an antifungul in your media as cwarnes has suggested. Filamentous type fungi can be extremely dangerous in a humidified incubator - the spores spread easily and once it's in your incubator it can be extremely difficult to get rid of. We have a policy of immediately discarding cultures with this type of fungus. It's too risky to keep them in an open culture system CO2 incubator with non-infected cultures.

After surgery the tissue is carried in a sterile zip lock bag to the pathology lab .The pathology lab where the tissue is is not that sterile though. So most probably the source of contaminaitn could be from hospital and the problem is the strain looks resistant.
In the Binder incubator whihc we have there is no visisble growth but definitely heat stelrilization at 180C might help.
Do anyone use sigmaclean or aquaclean?

Dear Calvin,

Just a few of my thoughts on the subject, which maybe duplicated from a previous post :

I have a group in my department who have had loads of fungal contamination recently. The group consists mainly of Post Doc's. They were all convinced that they were using best practice and that the contamination was coming from the Class II cabinets in their TC Room which we maintain and test. I was convinced that it was their bad practice that was to blame and that the fungal contamination was coming from their CO2 incubators and NOT the cabinet or TC room they are using. I decided to pour some SETTLE plates (W/O antibiotics) and locate them in sensitive areas i.e Cabinets, TC room and CO2 Incubators......... 2 hour exposure, then incubate at 37oC for 2 days.

Results : 2 x Heto Holten Cabinets...................No visible contamination

TC Room ( by the sink, on top of a tall cupboard and on a shelf unit......No visible contamination.

CO2 Incubators : Massive fungal contamination in both Incubators.


So this proved that in fact the general TC room was a clean environment and was much cleaner than their CO2 incubators.


As stated previously :

Use best practice and swab cultures (70% IMS) in and out of Incubators.

Do not try to cover up contamination with anti-fungals as these potentially have massive effects on the cells themselves.

Try and buy NON FAN ASSISTED CO2 Incubators as they only make the situation worse. We use non fan assisted incubators that have a high temperature decontamination cycle.

Try and protect the cells from the Incubator environment i.e. use TC flasks with 0.22uM Filters, rather than TC plates.

Monitor cultures regularly, I agree with Karyotyper, chuck away any contaminated cells ASAP to prevent further problems.

Have a TC rota in place i.e. spread the general duties around the staff who use the TC facility

i.e Change water in water bath weekly
Change incubator water weekly
Clean the floors weekly
Have seperate lab coats for TC work
Make up fresh IMS and Virkon weekly
Fyrite your incubators weekly
Clear TC bins when full
Stock TC cupboards to make sure that you are not moving around to much from area to area.
REGULARLY CHANGE YOUR 0.22uM FILTER IN YOUR PIPETTE GUN.... WEEKLY or if you contaminate it with media
Aliquote all TC stocks i.e. Glutamine, FCS, Pen/strep... this reduces contamination risk.
HAVE ONE PERSON RESPONSIBLE FOR THE ROTA AND TO MONITOR PEOPLE DOING THEIR JOBS.

In combination all the above will reduce the risk of contamination. Sorry for the length of this post

Good luck

Extremely good common sense advice Rhombus. All budding tissue culturists should print that post and stick it on their incubator.

Given Calvin*'s samples are collected aseptically it suggests the contamination is unrelated to the specimen (assuming the tissue being biopsied is also sterile). I'd be putting my money on the incubator.

You mentioned the strain appeared resistant. The problem with this type of fungus is that you have to hit it with such a massive dose of fungizone (or equivalent) that you practically kill your cultures in the process. If you must keep your cultures I can advise of ways to help retrieve them but I think you would be better served by throwing them out.

We need to grow most of our primary samples on coverslips in petri dishes so unfortunately can't take advantage of filter capped TC flasks. However, even these won't help in cases of poor aseptic technique.

Just out of curiosity Rhombus, what brand and model incubators are you using? I agree about the problem with fans, although they're a lot better than they used to be.

Dear Karyotyper,

Thanks for the vote of confidence. All the measures mentioned have been taught over 30 years in Tissue culture. I used to in fact go to abattoirs to collect tissue for the primaries I was using and my professor at the time did'nt want to hear about contamination problems.....just RESULTS. I am sure that operating theatres are much cleaner environments that abattoirs?

In 1999 we had to purchase approximately 35 CO2 Incubators for our new Institute. Companies were falling over backwards to lend us their incubator for upwards of a year before hand to test them out. We again had the choice of Fan or non assisited fan incubators. Also new on the market were incubators with high decontamination cycles.
Potential problems and questions to be asked

i) Do CO2 and temperature levels vary widely in non fan Incubators.

ii) Do the high temperature cycles have a negative effect on the incubator components i.e. PCB and IR CO2 sensors.

Having tested out the following models..... RS Biotech, Hereaus, Sanyo, Heto Holten, Forma, Triple Red, and others I have forgotten, we eventually went with RS Biotech. I have to say they have been excellent and the backup from the company quite outstanding. The events as described in the "Settle plate" incident were actually from a Fan assisted incubator that was brought into the Institute from Cambridge University, where this group moved from. It only reinforces the fact that in my opinion Fans make the situation only worse.
We still have contaminations in our non fan assisted incubators but they are very much easily controlled. All the incubator trays and there supports can be removed and autoclaved, as well as having the 120oC high temp. decontamination cycle. After 7 years in situ, the Incubators have been as reliable as any I have used before.

We are now possibly looking to buy another 50 Incubators for a new Institute and are again looking at the market. Binder have been mentioned by somebody in this thread, they have a 180oC decontamination cycle, but are Fan assisted.
Does anyone have a unit they use? and when using the cycle, do you have to remove the CO2 sensor or any other components? Can anybody recommend them or others we should be looking at ?

Thanks in anticipation

Rhombus

Thanks for the quick reply Rhombus

We have always used Hereaus and Forma. The older models had open fans that could cause all sorts of problems when they started collecting bugs. We had sporadic contamination with fungus on and off for months and it ended up being in the fan casing. Every time we cleaned the incubator the fan just respread the fungus until we actually identified the source!

Our Institute TC lab has about 8 or 10 Binder incubators. They were bought because of their heat sterilization function if I remember correctly. I don't know too much about them but I'll ask how the TC staff find them and report back tomorrow.

Cheers.