I am using human cancer lines for my studies. Everything was working fine for last 6 months and suddenly there comes contamination, only the culture flasks are contaminated and not the media bottles or FCS did contra cleaning of CO2 incubator fumigation of lab. Laminar hood is working fine, almost paranoid with handling, still culture flasks. The media turns turbid in 3-4 days or after the first or second passage of the cell line cells start detaching media gets used up.
Your cell line is contaminated or your flasks.. Have you tried flasks with media only, at least overnight? Have you tried a fresh vial of cells?
Yes..I have tried flasks with media only.... it is not contaminated for 3 days now... I have revived 4 different stocks of various cell lines... everything is fine for a week... and then in the first or second passage slowly the contamination starts and... then it takes its pace... n contamination is in every flask... within a day or two...
If I have an infection, I throw away all bottles I am currently using- PBS, trypsin, FBS, media, flasks that are from the same open bag (sometimes a batch of flasks are not sterilised correctly) and thaw a fresh vial of cells from as early passage as I have. I don't try to analyse or investigate the source- its more important to get the infection out quickly, so everything goes and start from scratch. Obviously clean hoods and incubators again before you thaw new cells. Anyone else sharing your hood and incubator having infections?
My approach is the same as Dr. O'Donnell. Toss everything. The fact you have this recurring issue is very damning of some key step or element that you've changed and overlooked. It's hard to dissect it out and usually it's faster to just start totally fresh.
If the problem doesn't show up till you passage that strongly implicates your trypsin, wash buffer, and plastic ware (centrifuge tubes, pipettes, etc.). Make sure you toss all those things.
Are you preparing anything yourself, for example autoclaving aspiration pipettes? That could be a source if something is being mishandled.
Are you using the same frozen stocks you used before the contamination? If I were you, I'd also order some fresh cells, since there is a non-zero chance you have some contamination in the frozen stocks, which you are reviving along with your cells.
I suggest you to filter sterile the media and FBS before use. Make sure that media is not contaminated by spreading 200-500 micro liter of growth media on Muller-Hinton agar plates and incubate at 37 degree Centigrade overnight. If the media contains any contamination, then you can observe colonies on the agar plate. Finally use appropriate antibiotics( I use Antibiotic-Antimycotic from Invitrogen. It really works well). Still you find contamination, I think its handling problem.
Good luck
@ Koula Gayathri: I always filter sterilize media and FBS and all other buffers used... All of them are stored in the same CO2 incubator... There is no contamination observed in only media or only FBS....
Thank you for the response...
Hi Shruti! If the culture media is contamination-free and the incubator and hood are also not a problem, then the culprit can be either PBS or trypsin. I had faced a similar problem earlier. You can try doing this: in a 24 well plate add PBS+media in one well and Trypsin+media in another well. Keep the plate in the incubator for 3-4 days and check if either is contaminated.
In my case trypsin was contaminated.
Good Luck!
@Amanda O'Donnell and @Peter Sobolewski: Thank you for the response...
Actually 3 of us in my lab are using same hood and incubator....We prepared fresh media.. FBS... buffers... sterilized everything... freshly autoclaved all plasticwares...CO2 incubator was also cleaned .. still problem persists...
About stocks...We also doubted the stocks at the end..but we have revived the stocks that we have prepared and some stocks prepared by our seniors... Can all stocks prepared at different point of time be contaminated?? we found it very unlikey...
About flasks: We use corning T25 flasks... we used a fresh stock of flasks which were not opened before....We also used a fresh stock of T75 flasks as well... contamination is there in both
@Amanda O'Donnell yeah... you r rit... one needs to get rid of contamination as quickly as possible.... But all 3 of us are facing the same contamination....
I dont understand this... I mean how to get rid of this!!!
@Poornima Paramasivan: Thanks you for the response... and Yess we use antibiotics in the media...
@Shrikant Pradhan : thank you for the response....
As such the bottle containing trypsin and PBS is there for a week in the incubator... there is no contamination in them...
Shruthi, I think your cells are not contaminated, it is just dying after 2-3 days. I had the same problem and I did all the suggestions mentioned above without getting any success. Finally I found the culprit after 4 months, it was the CO2 cylinder. When I changed the CO2 cylinder, everything back to normal, I did not have any problem after that. You can also try it.
Have you change your decontaminant spray ? Recently, I found a kind of biofilm in a decontaminant spray... Some batches can be 'dubious'...
Have you checked whether it's yeast or mycoplasma. I would recommend checking for mycoplasma contamination. Also, do you filter your FBS? FBS is a major culprit when it comes to media contamination. Hope your problem is resolved soon. Cheers!
@Amrish Sharma.... We have plated the supernatant of conatminated culture (the contaminated media).. on blood agar plates.... its bacterial.....and yess we filter FBS..only FBS is fine....
And yess... thanks... hope so...
@Natacha Broucqsault: we did not use decontaminant spray as such.... we fumigated the culture room... we cleaned hood and incubator as well...
@Silvia Villar: We use ddH2o.. filter sterilized...and laminar... we kept a bloor agar plate open in laminar hood infront of the flow for 1/2hr... and incubated it... there was no growth,... so laminar hood must be fine ...and Mn content... we have not checked it...I will look into that matter... Thanks...
@Hoa Trummell: We autoclave glassware and plasticware simultaneously in the autoclave... glasswares used for storing culture media... and that is fine....So i believe autoclave is working fine... but as such we have not tested our autoclave.... I will look into this aspect as well... thank you...
@Boopathi Subramani: CO 2 cylinder??? is it so? we have not checked this aspect... infact never suspected this.... CO2 is supplied after passing through a filter rit??(before entering the incubator)... anyways..I will consider this... Thanks a lot...
It's really a horrible situation so first of all you should dispose of everything these cells have come in touch with; culture media, PBS, FBS, trypsin etc. And if whatever they are; bacteria or fungy are antibiotic resistant. That's definitely no good
Once you have disposed off everything you should decontaminate everything, hoods, cabinets, incubators, water baths. In matter of fact I would seal the room and fumagate it (put fumes of formaldehyde in the room overnight this will kill everything).
It's just worrying about the source of contamination. And you should be using 20% FBS with these cells.
If they were precious I would try other antibiotics. But you can buy them ready from various suppliers
Sounds like a nasty infection.
Get rid of everything, start fresh and examine your aseptic technique.
Use the 1:1 mixture of water and 35%formaladehyde.
But to do this you have to take utmost care and should not use the room for atleast three days completely and better try this in this weekend.
You have to completely seal the room so that its vapours will not come and you are leaving the doors of cabinets open during the process.
Strict precaustions should be followed as its vapours are dangerous and is a potential carcinogen at higher concentrations. You should put a big notice on the door so that no one enters the room during the process.
After three days leave open all the doors after closing the cabinet doors and leave the room open for ventillation for two to three hours and u can use it afterwards.
But be carefull with process and have a look at the precautions that you have to take while carrying out.
Good luck and it works good to avoid all the contaminations in the Microbial safety cabinets.
Try to clean the MSC filter before carrying out the above process, which will furhter helps you to reduce the contamination.
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