Fungal contamination in 1 well of 96 well plate?

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Ihatefungi's picture
Fungal contamination in 1 well of 96 well plate?

Hi all,

I'm dumbfounded.  I culture human nasal cells in a transwell plate and every 12h take apical samples for 4 days.  Then, I take those apical supernatant samples, serially dilute them 2-8 times (e.g. A1-H1 in a plate) and run a TCID50 with 6 duplicates (e.g. A1 is put into A1-A6 in a new plate) on MDCK cells to measure viral replication kinetics over time.

About a month ago, I noticed that out of say, 50 plates, 4 to 5 plates would have fungus growing.  It looked white and puffy so I'm guessing yeast.  Surprisingly, the contamination only starts in 1 well around 5 days at 32 to 37C. If I leave it for 7 days, maybe the adjacent wells will have growth- maybe. 

We cleaned and decontaminated the hoods and incubators, filtered the reagents with a small enough filter, and ran a test with the old and newly filtered reagents on MDCK cells for 2 weeks at 37C. No growths. We were ecstatic.

I ran TCID50s with my supernatants that came from the human cell cultures and I have 2 plates, each with a single well contaminated. 1 has fungus growth, the other plate has media discoloration that suggests the presence of fungus. These plates were from two different patients as well. The wells were also in the middle of the replicates and the serially dilutions, so we would think that if the original sups are contaminated, more wells above and around them would be contaminated.

Our last thought is to fumigate the hood because maybe A spore landed in these two wells?!

Anyone have any other advice?