I am facing repeated contamination while culturing SHSY5Y cells. I am attaching representative images for different phases for reference. Pic no. 1 - Initial stage. Pic no 2. Contaminated plate (Discarded the plate later). Pic no. 3 - Plate with a new stock of cells I had. I guess very little number of cells could adhere and they are stressed and stretched. Pic no 4.- New stock of cells (got from my colleague). I am also adding few pictures of SiHa cells.
I have done contra con. Changed the PBS, FBS, DMEM media stock as well. Cleaning the hood with EtOH properly. Sometimes it is better, but in most of the time this contamination is coming.
Please help me with the situation. What can be done? What is the best way to discriminate different types of contamination & how to get rid of them? When to stop trying with the lot & just discard the cells/stock? Thanks in advance.
The first thing to look at is the cell medium, visually inspect for cloudiness. Cloudy medium means bacterial contamination.
Fungal contaminations do not make the medium cloudy but you can still see growth for some yeasts. However, the yeasts are very clearly visible in microscope as they are quite large. In your pictures i unfortunately could not tell if it is fungal contamination, but yeasts should be round and maybe 1/10th diameter of the cells or larger.
Finally you could have mycoplasma contaminations but they are best detected with kits.
The first moment you see contamination it is best to discard the cells. Since you are working with cell line it should be okay. If you really need to save the cells you should take some antibiotics or antifungals, but i have never personally used them.
When we have contamination we also clean everything in the laboratory. There is no way to tell and contamination could come from many sources. Clean and sterilize the hood and CO2 incubator, and see if the filters are working properly. Also clean the water bath regularly. check cell stocks because they could also be contaminated, make sure you thaw cells that you are sure are clean.
Cell media, Trypsin, PBS, FBS and all liquids can be either filter-sterilized or autoclaved. Then, you can place a dish with different combinations into the incubator and let grow for some days to see if there is any contamination.
Finally, aseptic technique, good use of the hood is important. The hood is UV sterilized for 15 minutes before each use. We use gloves and coat when working in the hood; spray everything that goes into the hood with 70% ethanol and make sure we do not pass anything over open bottles or boxes while in the hood. Also we close windows or air flows that could break the laminar flow in the hood.
However careful you are there is still some chances of getting contamination but with time and practice you can minimize the chances.
It looks like you might have a mycoplasma contamination. I would recommend to use a PCR based detection of mycoplasma. If the mycoplasma is confirmed then add anti-mycotic along with PENSTREP.
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