help! cell culture contamination too! - (Sep/13/2007 )
Hi-
I've been culturing some mouse lung fibroblasts that I received from another lab for my research and have been culturing these cells w/o problems for some time. However, I made a new batch of medium/trypsin and since then all the cells I subcultured seem to be dying- not sticking to the petri dish and not flattening out. I also see a lot of cell debris. I made an even newer batch of medium to no avail and today I found that a surviving plate had a "flower" thing growing in it with tiny little round stuff in it. I decided to throw all of my cells away and clean the incubator to prevent spreading. I'd autoclaved my incubator racks ( transported between building though, while wrapped in aluminum), wiped down with bac-down solution, DI water, and sprayed down with 70% and allow that to evaporate several times. I'd also cleaned the inside of the incubator using the same process. Is there anything else I could do? Our incubator doesn't have 95degree celsius decontamination capabilities.
-Ender-
Wow, you are very thorough!!! I would say that you have done everything that I could think of. The only other suggestion that I have is in future, keep cells given to you from someone else in quaratine (in a seperate incubator) until you can be sure that they are not contaminated (and are mycoplasma free also!!!).
-lauralee-
QUOTE (lauralee @ Sep 13 2007, 06:16 PM)
Wow, you are very thorough!!! I would say that you have done everything that I could think of. The only other suggestion that I have is in future, keep cells given to you from someone else in quaratine (in a seperate incubator) until you can be sure that they are not contaminated (and are mycoplasma free also!!!).
Couple of other things:
1. Freeze down cells at an early passage number so that you have cells to go back to if contamination strikes
2. Incubators these days have decontamination/high temperature cycles. If you have a chance in the future buy one of these i.e. Binder (180oC cycle), RS Biotech (121oC cycle), Heracell (90oC Cycle), Nuaire (95 oC wet cycle/140oC dry Cycle).
3. Change waterbath and incubator water regularly. Spray everything with copious amounts of 70% IMS. Use a seperate lab coat for culturing. Regularly clean the class II cabinet, top surface and underneath the stainless top.
4. REGULARLY MYCOPLASMA TEST YOUR CELLS........ both cell lines and primaries.
-Rhombus-
QUOTE (Ender @ Sep 13 2007, 06:41 PM)
Hi-
I've been culturing some mouse lung fibroblasts that I received from another lab for my research and have been culturing these cells w/o problems for some time. However, I made a new batch of medium/trypsin and since then all the cells I subcultured seem to be dying- not sticking to the petri dish and not flattening out. I also see a lot of cell debris. I made an even newer batch of medium to no avail and today I found that a surviving plate had a "flower" thing growing in it with tiny little round stuff in it. I decided to throw all of my cells away and clean the incubator to prevent spreading. I'd autoclaved my incubator racks ( transported between building though, while wrapped in aluminum), wiped down with bac-down solution, DI water, and sprayed down with 70% and allow that to evaporate several times. I'd also cleaned the inside of the incubator using the same process. Is there anything else I could do? Our incubator doesn't have 95degree celsius decontamination capabilities.
I've been culturing some mouse lung fibroblasts that I received from another lab for my research and have been culturing these cells w/o problems for some time. However, I made a new batch of medium/trypsin and since then all the cells I subcultured seem to be dying- not sticking to the petri dish and not flattening out. I also see a lot of cell debris. I made an even newer batch of medium to no avail and today I found that a surviving plate had a "flower" thing growing in it with tiny little round stuff in it. I decided to throw all of my cells away and clean the incubator to prevent spreading. I'd autoclaved my incubator racks ( transported between building though, while wrapped in aluminum), wiped down with bac-down solution, DI water, and sprayed down with 70% and allow that to evaporate several times. I'd also cleaned the inside of the incubator using the same process. Is there anything else I could do? Our incubator doesn't have 95degree celsius decontamination capabilities.
Sounds like you have a fungus or mold. There are anti-mycotic reagents you can add to your media if the problem persists. Otherwise, the only other thing I would recommend is changing the incubator filter. This may have your mold/fungus in it and now can spread spores. I would also recommend you test your incubator before re-starting cell culture by placing a plate of media in the incubator and leaving it for at least 24 hours, 48 would be better. This way you won't thaw cells only to have them become contamimated.
-rkay447-
QUOTE (Rhombus @ Sep 14 2007, 08:50 AM)
QUOTE (lauralee @ Sep 13 2007, 06:16 PM)
Wow, you are very thorough!!! I would say that you have done everything that I could think of. The only other suggestion that I have is in future, keep cells given to you from someone else in quaratine (in a seperate incubator) until you can be sure that they are not contaminated (and are mycoplasma free also!!!).
Couple of other things:
1. Freeze down cells at an early passage number so that you have cells to go back to if contamination strikes
2. Incubators these days have decontamination/high temperature cycles. If you have a chance in the future buy one of these i.e. Binder (180oC cycle), RS Biotech (121oC cycle), Heracell (90oC Cycle), Nuaire (95 oC wet cycle/140oC dry Cycle).
3. Change waterbath and incubator water regularly. Spray everything with copious amounts of 70% IMS. Use a seperate lab coat for culturing. Regularly clean the class II cabinet, top surface and underneath the stainless top.
4. REGULARLY MYCOPLASMA TEST YOUR CELLS........ both cell lines and primaries.
rhombus, what does "IMS" mean?
in another contribution, you mention "mycoplasm off"; what did you mean?
-The Bearer-
QUOTE (The Bearer @ Sep 15 2007, 04:07 AM)
QUOTE (Rhombus @ Sep 14 2007, 08:50 AM)
QUOTE (lauralee @ Sep 13 2007, 06:16 PM)
Wow, you are very thorough!!! I would say that you have done everything that I could think of. The only other suggestion that I have is in future, keep cells given to you from someone else in quaratine (in a seperate incubator) until you can be sure that they are not contaminated (and are mycoplasma free also!!!).
Couple of other things:
1. Freeze down cells at an early passage number so that you have cells to go back to if contamination strikes
2. Incubators these days have decontamination/high temperature cycles. If you have a chance in the future buy one of these i.e. Binder (180oC cycle), RS Biotech (121oC cycle), Heracell (90oC Cycle), Nuaire (95 oC wet cycle/140oC dry Cycle).
3. Change waterbath and incubator water regularly. Spray everything with copious amounts of 70% IMS. Use a seperate lab coat for culturing. Regularly clean the class II cabinet, top surface and underneath the stainless top.
4. REGULARLY MYCOPLASMA TEST YOUR CELLS........ both cell lines and primaries.
rhombus, what does "IMS" mean?
in another contribution, you mention "mycoplasm off"; what did you mean?
Dear Bearer
IMS.....is Industrial Methylated Spirit and should be diluted down to 70%
As for "mycoplasma off" this is a product I know nothing about. It was however mentioned by somebody else in the "mycoplasma thread " recently.
-Rhombus-