ATCC also says to maintain cultures at a cell concentration between between 1 x 10exp5 and 1 X 10exp6 viable cells/mL. Yours were seeded at about 2.4 x 10exp 6 cells/ml and then were allowed to grow for 36 h. Your medium turned yellow because the high cell content caused the medium to become acidic. It was also probably depleted of nutrients. At this point, you most likely lost some cells. The problem is that you now have a population that went through a selection process. In short, you no longer know what you have. Based on this alone, I would toss them out and start over with a fresh vial.I noted a cloudy clot without bacterial or yeast appeareance
. Any kind of cloudy clot is suspicious of contamination, possibly fungal in this case. Another reason to start over with fresh cells.
As long as the cells look like they are supposed to (see picture
), they should be fine regardless of passage number. Here are some references that may help:
PNAS Beltan et al Dec. 19 2000, Vol 97, No.26, pp 14602-14607
PNAS Beltran et al June 25 2002, Vol 99, No.13, pp 8892-8897