Constant Infections(?) - I am in need of some serious help (Jan/20/2011 )

Hello. I am new to cell culture and research work in general (less than 1 year) and recently have been having a serious problem with my cell cultures. The cells I'm working with are A549, Rabbit fibroblasts, and RG-2. This post will pertain only to what has happened with the A549 cells, however, this problem has occured in all 3 cell lines. The media is DMEM with 10% FBS.

It started about a month ago, when I opened a new bottle of media and plated cells for an MTT. Overnight the media became turbid and it was filled with what looked like under the microscope to be small clear circles. I changed the media out, and waited to see what would happen the next day (since at this time I wasn't sure as to what the problem was, all I knew was that my cells died). The next day it was a dark purple, and very basic. I assumed this was the result of a bacterial infection since everything else was ok and since I don't use any antibiotics in my media (and the other cells next to this flask in the incubator were also doing fine). So I thawed out a new vial and plated. They looked perfectly fine for 4 days, then the same turbid/purple media resulted on the 5th day.

So I opened a new pack of flasks, new bottle of media,added pen/strep to 2%, made double sure to clean the hood out (I always clean before and after with 70% ethanol as it is but this time I did it 3 times before and after, spraying it very liberally and left the UV light on overnight and any time I wasn't in the hood), cleaned the microscope stage with ethanol, as well as wiped the incubator down with the same ethanol. Thawed out another vial, which looked good for 1 day then became turbid and all the cells died.

At this point I was totally at a loss as to what the problem was, but since I need cells to do experiments I tried it again. This time I opened fresh media, used fresh FBS, added 2.5mL of gentamycin (10mg/mL)to 500mL of media (the recommended concentration is to add 5mL per L) as well as the pen/strep this time at 4% and used a different incubator. This resulted in the cells being good for 2 days, then becoming cloudy and dead.

Now I'm at a complete loss and then some! So after some more research it looks like the turbid media is a result of a yeast infection. So now with yet another bottle of fresh media, I added 10mL/L worth of Antibiotic-Antimycotic (10,000 units penicillin, 10mg streptomycin, and 25ug amphotericin B per mL). I opened a freshly sterilized new container of pipette tips, flasks, 15mL tubes, and everything else I use. Everything was brand new and sterile. Now I used this media with a fresh vial of cells obtained from ATCC. They lasted 2 days (and looked healthy even in the presence of the anti-anti) before their media became turbid.

I tested out all of the new media solutions as well as the orignal as well by putting 5mL of each into separate flasks and incubating overnight. They all developed the same turbid look.

So please any help you can give me as to what I am doing wrong and what I could try in order to fix it is so greatly appreciated I'd kiss you if it works! I'd like to culture the turbid media and see if I can ID the (what looks like) yeast but do not have the means to be able to test it (or atleast I'm not aware of any way to ID it other than to pay $$$ for someone else to test, which isn't really an option just yet).

Sorry for the long post and thank you soooooooooooooo much for ANY help/suggestions/ideas you can give me!

Hi

How do you prepare your medium? Do you filter it?
If not, use a vacuum filtration system.
Something like this:
http://www.membrane-solutions.com/disposable_vacuum_unit.htm

Should get rid of any yeast spores.


A simple way to ID yeast would be to take some supernatant of your infected culture and put it on a potato dextrose agar.
http://en.wikipedia.org/wiki/Potato_dextrose_agar

Its a special agar for yeasts and molds.

if this happen to me, I probably think the cell is contaminated before cryopreserved.
I think you are doing fine with cell culture technique.

if you have any other cell line (other than what you are testing, and not contaminated before cyropreserved) stored in N2 tank in your lab,
why dont you thaw one vial and culture. If you are not doing anything wrong, you should not see any contamination in those cell culture.

I thought that it might be the cryoperserved vials that contained the contamination, however, when I ran out of cryovials I purchased new cells from ATCC and got the same infection 2 days after culturing the vial. Likewise, this same situation cropped up once with a different cell line (rabbit fibroblasts, which since I can't readily buy more of these, I stopped culturing them until this problem is resolved).

However, earlier today I took 200uL of RG-2 cells, one of the other cell lines I'm currently culturing, and plated it into a new flask. I intend to continue to culture this cell line to see if the yeast infection develops here as well.

I know I keep referring to this as a "yeast infection" but I'm not 100% sure that is what I'm dealing with. To my untrained and inexperienced eye it LOOKS like a yeast infection- basic media, what appear to be budding yeast under the microscope, the turbid appearance of the media - but I'm not an expert, not even close. Are there any tell-tale signs I can use to positively ID this as yeast? I'm going to gather more info, but as of right now I'm leaning towards the potato dextrose agar as previously suggested.

Thank you very much for the help so far!

Oh, and I almost forgot to mention - No, I don't sterile filter my media prior to use. I purchase it sterile filtered, and until recently, have never had a problem with it. I will however look into sterile filtering it - provided it won't be outside of my budget. Are there any other methods to sterile media and the like? I assume autoclaving it wouldn't work for a lot of reasons...

Thanks again!


EDIT: I do have access to 0.22 micrometer filtration equipment that I use to purify and degas solvents for the HPLC. Would I be able to sterilize this apparatus (I can autoclave it) and then use the 0.22 micrometer filter paper and vacuum pump that we already have?

you can use 0.22um filter that you can attached to the syringe to filter small amount of medium like 10-50ml,
but the 0.22um filter that I use come as sterile (individually packaged) so no need to autoclave them.
in fact, if you do autoclave those filter, you most likely damage the membrane and wont function.

if you want to test the filter system for the large quantity medium sterilization, I think you should go to a neighbor lab and ask one, then it is free (most of cell culture lab have them)

Also, I never had yeast contamination, but if you want to verify, just take picture and post here. Someone usually will identify them pretty soon.

I think your aseptic technique must be a problem if you have managed to contaminate all of your media solutions. Even if you are working with a contaminated cell line, this shouldn't get into your media bottle if you use correct technique. Is there anyone who is experienced with tissue culture in your lab or a neighbouring lab who could watch you work and see if there is anything you are forgetting/doing wrong?

Also, filtering your media will not help. It is already sterile so this would be a waste of time and money.

Another possibility is that you have contamination in one of your pipettes or pipette aids- could you use somebody elses, or give yours a thorough clean and then try again (maybe just with media so you don't waste any more cells?).

Just my thoughts...

I appreciate the responses. While I haven't had anyone watch me while I work to observe my aseptic technique, I religiously follow all procedures I have ever seen, heard, and read regarding aseptic technique. I'm not sure if the media itself is contaminated, or if the contamination is occurring at another step.

The pipettes and pipette aids that I use are used by several other people, and while it is possible they are contaminated, this yeast infections are not showing up in anyone elses cultures. Regardless, I will clean them and culture media alone and see if that helps.

I don't know how likely this is, but is it possible that the gloves that I have been wearing could be the source? The have been sitting in the lab for quiet some time now. However, I always spray my hands/gloves down with ethanol before touching anything inside the hood or anything going into the hood as well as before opening the incubators. What do you think?


Thanks again for the suggestions. keep them coming!

if there is some other people who is doing good cell culture, it is actually good to ask them to check what/how you do. or you can watch them how they do.
I do understand you read/follow protocol very carefully, but sometime that wont tell everything that you need to know.

I never heard contamination from glove although I cannot say it never happen. Also, I do not understand that you are using glove which was sitting in your lab for a while. Are you reusing glove or something? I am constant glove changer and use at least 10 pair or so a day since cell culture is not only the work I do.

as the other guy say if the medium that you purchase came as sterile, it is waste if you filter them. but some additive such as FBS might be contaminated, so I think it is good idea to filter complete medium (after you add all the additive) if you have filter unit.

But I still think if there is someone who is doing cell culture in good condition without contamination as for help to them, I am pretty sure you will get more useful info than asking here.

good luck

Hi there,

Sorry to hear about your contamination issues! What a nightmare

Just a little question to add: do you have any exposed skin near your gloves? There's a guy at work that was always getting contamination - until he starting wearing these kind of "sleeve covers" - disposable plastic things to cover the area between his lab coat/gloves.

Also, do you brew beer at home?

Clare