FUNGAL CONTAMINATION! - URGENT! (Jul/03/2006 )
Hi! All,
We are a lab working with breast cancer patient samples...trying to isolate & enrich the epithelial cells in suspension culture.
We had been doing pretty fine till the past 2-3 weeks, from when we have been facing regular fungal contamination problems. I have checked every reagent/media that we are using.
We also use Fungizone (from Sigma) at a concentration of 1 ug/ml. In the past, we have not faced such a major fungal problem & it seems to have suddenly come up.
Every tissue that we are handling comes up with those perfectly spherical, floating fungal colonies within 3 days of seeding in culture!
The fungizone, we had prepared about 1&1/2 years back & the aliquots are stored in -20oC...Can that be a problem? Does anyone think the concentration of fungizone that we are using could be a problem?
Collection method of the tissue in the hospital is unchanged from the past.
I have no clue where could the problem lie! COULD SOMEONE PLEASE HELP? I would be very, very grateful! 
Thanks!
DD
hi biologic ! fungizone is the trade name of Amphotericin B and is the most effective drug available in the market against fungal infectuions/ contaminations. however Amph B is stable for only three days at 37 C .another drug of choice against most fungal contaminations is Nystatin.
iam sending you a table of antibiotics and antiofungals as an attachment. hope this will help you
all the best
Dear Shiva and Biologic,
The contamination is coming from the environment and is always difficult to erradicate. However using Fungizone is definetely not the answer. Amphotericin B has many effects on cells and in the past we have learnt by our mistakes. In our early days in Nitric Oxide research we used Amph B to stop fungal contamination. However the primary cells we were using at the time stopped producing NO when under these conditions.
Routine Antibiotics in cell culture seems to be a common way for people to disguise poor technique. I teach Asceptic technique in our Institute and can never understand why people have so many problems. 30 years ago we were culturing cells on the bench with bunsen burners and glass flask's, if you were lucky you could use a laminar flow cabinet. Now everything is so much easier. I come from a production background where we made vaccines (Polio) in human cell lines and contamination was not an issue. The people doing the work did not have any academic qualifications but had FANTASTIC TECHNIQUE. When I first came into research I was appalled at the bad practice by academics who generally blame everything and everybody, apart of course themselves.
Please therefore be careful using these agents as they are not selective but are DIRTY COMPOUNDS and can have profound effects on the cells you want to use for your experimentation
dear Rhombus ! i appreciate your concern and the suggestion about using antibiotics and antifungals in cell culturing. infact i faced this problem of unwanted signaling that might be due to use of antifungal compund amphotericin. now i do it without antibiotic/ antifungal compounds.
unfortuantely not all people are perfect in technique. either they are learners or contaminations come due to some unavoidable circumstances. in such cases if you want to save your precious cell lines/ patient samples etc. then you can always use antibiotics / antifungals depending on the need. when i say depending on the need this means that as long as you are not having any problems with these extra additives (we routinely produce monoclonal antibodies and we use antibiotics and antifungal agents .......cant take chances you see). they are there for our convenience. other wise they are not required.
Hi Biologic
Assuming you use an open culture system my guess is that the source of your fungal contamination is your incubator(s).
Every time we've had problems with fungal infections it is because we've been slack with our incubator maintenance. If its occuring regularly you may be able to see a patch of white-ish haze somewhere on the wall(s) of the incubator. On one occasion I recall we kept getting sporadic infections even after several thorough cleans (this was only happenning in one incubator - all the others were fine). We eventually dismantled the inner casing of the incubator and discovered that the fan circulating the C02 was covered with fungus. My advice would be to thoroughly disinfect your incubator. Isolate (or discard) infected cultures as this sort of contamination spreads very easily.
We have found fungizone to be quite toxic at high concentrations but we use it prophylatically in our media at lower concentrations (can't recall the conc. at the moment - will check tomorrow). It should be fine stored at -20.
05/07/06
Final concentration is 0.5ug/ml. This keeps yeast at bay. We would treat infected cultures at a higher concentration if we experienced the fungal contamination you describe (and even then the aim is to do what we need to do with the culture and then get rid of it as quickly as possible). If it's not absolutely critical to have the culture we would ditch it straight away.
iam sending you a table of antibiotics and antiofungals as an attachment. hope this will help you
all the best
Hi! Keshava!
Thanksa lot for your suggestions & the attachment. I had actually gone thro it some time back & I have accordingly made a Fungizone stock of a higher concentration & have started using that. Today, we again see the same contam in all the plates (with the new stock).
I strongly feel the organism could be coming from the hospital from where we are getting the samples since all samples, & with every user, we are getting the same thing. What do you think on this?
Biologic
hi biologic ! karyotyper is right . if your incubator is the source of contamination then it keeps recurring.. contamination from the hospital is one possibility but always the same type of fungal growth????????? try cleaning your incubator . if possible fumaigate it with methanol or formaldehyde . make sure that no traces of methanol vapours or formaldehyde is left in the incubator while transfering your cell cultures to freshly fumigated incubator.
one more possibility is that your media is contaminated. add media suing a syringe filter and check for contamination. is the contamination persists then this possibility is ruled out
all the best