Hello everyone! I am differentiating NSC to neurons for my new project that I need to expose with 3 different drugs!

Everything looks perfect until I split the cells to 96 well plates and after 1-2 days I see fungi hyphae in just some wells! not all of them! it happened to both drug treated and un- treated plates.

I cleaned the hood, the incubator, the pipettes (we use filter tips) and the vaccum suction which is in metal and we use for 96 plates with Contec Prochlor and ethanol and UV light! I changed all the chemicals but only one medium I didn't which I tested with my NSC in 4 well plates and nothing grew. I threw all the tips and took new one! I got cells from my coworker who is working in the same lab and had seen no infection with her culture!

I started again! everything looks good until I start with 96 well plates (they are sterile and single packed) again there is exact same things in just few plates!

I lost 2-3 months without no result!

I couldn't find the source and I have to start again! the last thing I did was seeding the cells in the exact same medium in another type of 96 well plates, changed the coating and didn't use vacuum! I did expose them with the drugs! It was just the test no experiment!

I seeded 10 wells per plate (3 in total )! Well there is no infection!

I have to start my experiment from the very beginning and I am extremely worried!

could it be the plates? could it be the coating or the vacuum chamber. I tested them separately! I put medium in one 96 well plates, I used vacuum in another plate and just put some medium without cells and I put the coating in a separate well in the incubator with some medium! well nothing grew!

I am really dreading to start...

I welcome any suggestion and thank you in advance