Eliminating fungal contamination - (Apr/13/2012 )

Hi,

I'm a second year PhD student and I'm having serious fungal contamination problems . I've been doing cell culture since last summer and everything seemed to be going fine and then my fibroblasts got fungal contamination! I've taken numerous steps to prevent this i.e. changed all my stocks, I always throw out my media if I've had contamination, I changed the type of tubes I use to put my pen-strep and L-glutamine in, I've started pre-screening my media before use, I ethanol everything to death before I use it i.e. equipment (which I am sure some of you will tell me will not help with fungal contamination), I change lab coats, tie my hair up, wear gloves, UV the hood, check the hood for spilled media - summing up I've tried everything. I'm pretty sure its not my technique either as I clean up media, I try not to touch the tops of tubes etc, I use equipment (tips and pipettes) that haven't been opened but I can't seem to convince my supervisor that the problem is environmental unless everyone else has a problem. Other people i've asked all say that what I'm doing is overkill but seen as its just me being affected I don't know what else to do. I asked about the maintenence routine for our incubators and they get cleaned every 6 months and topped up with distilled water when needed. We have Hereus fan assisted incubators. Because of the problems our tech was kind enough to clean the incubators but I'm not sure what this entailed and whether it was good enough as there was white stuff growing in it which I have read is likely fungus, plus this week I was pre-screening some media as I'm getting some fresh fibroblasts next week and that had contamination! I'm convinced its the incubator as the culture flask I was using didn't have a filter top it had a lid where you just have to leave it loose as we'd run out of the other kind plus I had some Raji cells growing and they haven't got contamination (touch wood). I've even taken to eyeballing other people's culture flasks to see if they are contaminated and its just not getting reported. This has been a problem for about 6 months now and its really starting to stress me out as I've completely lost confidence in my ability.

Any suggestions would be much appreciated as I just don't know how to continue if its down to me as my peers tell my my practice is fine. Thanks.

C

There are many many potential sources of fungi in most labs, the two most common are incubators (as you mentioned) and waterbaths. Idealy these should be cleaned routinely about once a month, or more often if necessary. The cleaning of an incubator should involve removal of all prts that can be removed, scrubbing these with detergent, then autoclaving. The main body of the incubator should be washed down with an anti-fungal (e.g. barrycidal) and then washed with water (to remove detergents) and 70% ethanol. Parts should then be replaced after spraying with 70% EtOH.

Waterbaths should be washed down with anti-fungals as well, then rinsed and re-filled with distilled H2O.

It sounds like your techniques are probably OK, though you may want to get someone else to view how you work and see if there is something you are forgetting (e.g. wiping flasks down before putting in the hood).

Note that you could have contamination in frozen stocks of the fibroblasts. If they are particularly valuable for some reason, the you can try treating with anti-fungals, but they do strange things to cells, so it is usually best and cheapest to throw them out and get/make some more.

Perhaps the filters in your hood are contaminated.

EvilTwin on Tue Apr 17 09:15:49 2012 said:

NO NO NO NO NO NO NO

99.9% of all fungal contamination in CO2 incubators is caused by THE PEOPLE WHO USE THE TC FACILITY USING IMPROPER ASEPTIC TECHNIQUE

i.e

NOT changing the water in the incubator on a weekly basis
NOT wiping their TC flask's/dishes in and out of the incubator
NOT using gloves/coats/other PPE when doing their work
NOT changing the water in the waterbath on a weekly basis

OLD WIVES TALES:-
USE UV IN CABINETS........USELESS
USE ANTIFUNGALS........USELESS AND BAD SCIENCE BECAUSE THEY ARE EXTREMELY DIRTY COMPOUNDS

In the 1970's we were growing cells on the open bench with glass flasks that could be flamed, glass pipettes that could be flamed, using pleny of 70% alcohol AND FAN ASSISTED CO2 INCUBATORS........talk about against the odds....but we got CLEAN cells.


Listen to bob1 as well, he has obviously been trained at the same "school of TC" as me.

I do hope some of this is useful

Kindest regards

Uncle Rhombus

By hood i meant "biochemical safety cabinet" = the place where you work with cells.
Incubator is the place where they live, right?

EvilTwin on Tue Apr 17 13:06:44 2012 said:

Dear EvilTwin,

If you mean the biological class II safety cabinet and the 0.22uM HEPA FILTER that is fitted to "sterilise" the circulated air in the hood ...then that in my experience is NEVER the source of the fungus. The "hoods" should be tested annually for :-

HEPA filter integrity
Operator protection
Product protection

The CO2 Incubator is "where they live" and in simple terms that is correct.......BUT ......checks are required on all incubators:-

Is the temperature correct
Is the CO2 working concentration correct
Is the incubator humidified
IS THERE ANY CONTAMINATION PRESENT
Is the incuabator correctly situated in the TC room
ARE COMPETENT STAFF USING IT!!!!!

Kindest regards as always

Uncle Rhombus

So we had a miracle in our lab. Don't shout at me or I'll contaminate your filter too

Hi curlychick,

Do you use filter tips? And filtered stripettes? If not, consider using those and otherwise clean out the inside of your pipettes/change the filter in your pipetboy. Using clean new tips is of no use if your pipette blows spores through them.

I always package my primary cell cultures with plastic wrap, it allows free exchange of CO₂, but blocks any contamination, has worked for antibiotics free cultures in what turned out to be contaminated incubators (or I was just lucky).

You said you used new tubes for pen/strep l-glut, but did you use new stocks as well for those? Antibiotics can be infected with fungi. Same goes for trypsin

Avoid using the waterbath. Most cells are fine with room temp media for short periods, or warm your aliquots in a heat block.

Go even more mental and sterifilter everything into autoclaved bottles. I do when other people have infections in their cultures.

You could buy plastic sleeves that cover your arm up to your elbow.
It could just be that you are unlucky, I knew a senior post-doc who always had fungi and yeast infections whatever he tried, and had the culturing work done by others. (no bread and bananas before you start culturing)

Hope you solve it,

S