|We are having major difficulty keeping our mouse and human stem cells from becoming contaminated. We clean our incubators every two weeks and the hood every time we use it. Our media consists of:|
Nueral Basal media + glutamine+P/S(for ms.)
EGF and FGF
*this is all filtered*
We use a 70% ethanol solution to sterilize all tubes, etc. What could we do differently?
|Start by identifying what the contamination is - is it bacterial, fungal, mycoplasma .. We had mycoplasma once and it took us shutting down the culture lab entirely cleaning the whole room from top to bottom (twice - the first time didn't get rid of it) and then strongly enforcing the proper technique to rid ourselves of the problem.|
I would also check my hood - do you use UV light to keep it sterile - if so are their things you leave in the hood while doing so - racks, pipettors etc.
One of the major sources of contamination is from the operators - so have someone who is reliable watch everyone else to see if they can spot technique problems.
Also I personally like to use an alcohol foam to "wash" my hands (or gloves). It is easier than soap and water and doesn't dry out the skin and according to the medical literature is more effective at reducing contamination on the hands.
|Thank you very much for your reply seanpeel. The contamination is bacterial. We do leave items in the hood, but have done so for years and never had the problems we are having now. We use a UV lamp to sterilize the hood when it is not in use, and our hood was serviced two weeks ago and everything came out okay. We even spray the hood with a bacteriocidal compound, still we get contamination. If there is anything else you can think of, please let me know. |
|According to the safety guys that I have worked with in the past....alcohol makes microscopic holes in latex gloves so you don't want to "wash" your gloved hands with alcohol. If you are concerned about your gloves being contaminated you should wear sterile surgical gloves or double glove.|
|In addition to what Sean has said, make sure your "source" or master bank is not contaminated as well. When do you see contamination after thawing a frozen vial? Is the contaamination slow growing? i.e. it shows up after your first or second passage of cells after thawing? To rule out that your source is not contaminated as well as a slow growing contamination, do not discard excess cells after passaging and see if all of them get contaminated or not.|
Hope this helps,
|During the cell passage we collect cell conglomerates that are too big to filter through the 70 micron filter and always discard them. I do not know how to scrap the cells off this filter in order to use them again in culture. Any suggestions?|
Secondly, what types of bacterial infections are common in human stem cell contamination? Are any of these bacteria capable of eliciting a harmful response in humans? If so, what form of protection is necessary to prevent such occurences? Currently I just wear latex gloves solomente.
Thank you again for all of your help. We really appreciate it.
After reading all of the messages I have some questions that you should ask to try to figure out where you can go from here...
1. Do you know if the same organism is contaminating the culture each time? (You should run a gram stain and try to culture the bacteria on a rich plate, such as a Blood Agar Plate, to determine if the colonies look uniform)
2. Are you discarding EVERY open media component bottle that is associated with the contamination?
3. Are you tracking lot numbers of the components to try to associate the contamination with a component?
4. Are you warming media in a water bath and wiping it down with ETOH? ETOH (or any other disinfectant)does not work immediately so the items should be allowed to sit for a few minutes before they are wiped off.
5. Are you using disposable pipets that are well wrapped? I have found that there can be holes in the wrapper that can cause conatminations if they are used. I think that the holes often come from placing the pipets in canisters or holders in the hood.
6. Are you using a suction device to remove media in any part of the manipulation? If the tubing to the flask is contaminated it can migrate into the cells without much trouble.
If you can demonstrate the the contaminations are alike than you can, without much risk, assume that you are looking for a common source rather than multiple sources.
Not to discourage you...but in the 20 odd years that I have been doing cell culture I have found the biggest source of contamination to be the operator. You might want to slowly walk through the manipulations that you perform and identify areas that you think could cause a contamination and make sure that the contamination isn't operator related.
Good luck and keep us posted how your are progressing,
|It has been 23 days and we have not had any contamination. Thank you for all of your help.|
|Thanks for visiting.|
- To ask or answer a question, you need to register here.
- You can browse by clicking on a message subject to see the complete message.
- If you have previously registered, you can log in below.