Recently I tried to culture striatum and hippocampus neuron from mice pulps but I had seen fungal contamination. Although I tried every possible way to stop microbial contamination like using 70% ethanol to clean all apparatus, biosafety cabinet II but I failed.
Hi Emran Hossen ,
Probably your medium and cultures are contaminated too.
Dispose the contaminated cultures or all if you suspect them to be. Check the bath for contamination too. Clean the incubator with ethanol or hypochlorite solution. Leave it for a while and then start your work by adding a cocktail of appropriate type and concentration of antibiotics (such as gentamycin).
Are you seeing the contamination in all of your plates (wells, coverslips..) after a single isolation or only in some of them?
In my experience (I work with mouse postnatal neurons) contamination in only one well (out of many) is not a problem. Perhaps one contaminated dust particle got in accidentally...
If you are seeing contamination in all wells then most likely one of your reagents is contaminated. Which reagents were replaced recently? Which solutions were prepared recently, before the problem started? If you suspect one particular solution - replace it. Otherwise have to replace all of them and start from the beginning.
Needless to mention, all solutions must be sterile, never exposed to air outside of biosafety cabinet, all equipment must be wiped with ethanol, everything that comes in contact with cells must be sterile. Everything that ever comes in contact with something that comes in contact with cells later must be sterile, etc, etc.
Make sure you wash the pieces of tissue at least 3 times with dissociation medium before dissociation.
Good luck!
Misha Monakhov out of 24 wells I found most them were contaminated (21 wells). I will prepare everything new. Then see what will happen. Thank you..Also Thanks to @Bahman Sadeghi..
I dissect mouse fetal brain in a quiet room where there is no traffic. I do not do it in a hood, which I've not found to be necessary. Tools are autoclaved and the dissecting microscope is wiped with 70% ethanol before the dissection. The mother mouse's abdomen is doused with 70% ethanol and tools are dunked in a bath of ethanol if they get hair on them. We also pre-culture each new bottle of medium to make sure it is sterile.
We battle mold all the time. It is very humid and warm here and everything grows. Every month, we sterilize the incubator to keep the mold at bay. We autoclave shelves and supports from the incubator and clean it out with generous amounts of fresh 10% bleach applied with a sponge (including the seals around the door). Leave the bleach on for at least 10 minutes, then wash off with water. Usually if we do this monthly, we only get occasional wells contaminated with mold (fungus). Ethanol doesn't kill mold spores, but bleach does. So don't use ethanol to sterilize your incubator. It only kills bacterial spores.
Earl W Godfrey Thank you for your valuable comments..I solved the problem by changing everything new. The things that I added that was wearing mask during neuronal culture and boring the tips of tweezer which I used to take cover slide.
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