Does anyone know what could this dark sticky material growing in my 4T1 cell culture flasks be?

Hi,

Is this a fluorescent microscope image? why is it green? does medium color turn to yellow quickly? It seems to be bacteria. To check, drop some sample on a clean glass slide and fix it by heating the slide on a 50 C hot-block until the liquid is evaporated. Then stain the sample by DAPI. After washing check the slide under a fluorescent microscope. If you see something like attached your problem is bacteria.

Vikrant Mehta

1) Like Babak suggested i guess u shud start by taking 1-2 drops of the media in which ur cells r growing and fix them and observe under microscope. Additionally, take ur cell suspension media (about 20 ul) and spread them on a Nutrient agar media or Potato dextrose agar. How about a DAPI stain for mycoplasma as well?

2) Next thing that can be done is the daily washing of the cells with PBS but of a little higher pH (maximum 7.8 or 7.9 pH). Be careful about the concept of osmolality here cuz your cells might start to show vaculation or granulation which is a sign of unhealthy cells.

3) How about your cryopreservant.? If u r using expired glycerol or DMSO then that can also lead to toxicity in the cells and they might come up with some unusual appearance although i would like to see some study in the context of this statement.

4) Another thing that can be reviewed upon is the no. of passages your cell line has gone through and whether it is growing as per the expected rate because if u r supplementing them with adequate growth conditions and they r not growing then they might be heading for senescence.

5) Try and do daily viability checks (Usual trypan blue etc)

6) Last but not the least review your aseptic technique and check for any contamination in LAF hood or incubator . U should then go for fumigation of the culture room and thaw an entirely new batch

If i have something new in this direction i'd definitely let u know.

Good Luck

Waseem Akram Malla

Thank you Dr Babak and Dr Vikrant for your suggestions. The above pictures have been taken in green filter of an inverted microscope (non-fluorescent). Some other details that may help:

Passage Number: This time, I had started with cells at P11 that had been frozen previously (by someone else as I've joined the lab recently). Earlier, I had tried P2 cells as well. In both the cases, after thawing and re-establishment of culture, the cells grow well for 1 or 2 more passages. However, afterwards, P13 this time, the growth almost comes to a halt, cells start detaching from the flask and enter a kind of steady-state where the number of adherent cells remains constant whereas those floating around in the media increase in number. It's almost around the same time that the black background appears and spreads around.

Color change in media: I don't think the media changes color quicker than it used to in the earlier passages. Instead, the color change seems to become slower, as I said the growth is reduced significantly.

Testing for Mycoplasma: Yesterday, I performed Mycoplasma testing on one of the flasks (using EZdetectTM Hoechst Stain Kit for Mycoplasma Detection). Since the monolayer had not grown significantly, I was able to see just a few nuclei on fluorescent microscopy, which were large in size but not that much bright. I concluded that the flask is Mycoplasma negative as I could not find any pin-point foci as shown here: http://himedialabs.com/TD/CCK008.pdf .

Regarding DAPI staining: Prepared slides today but was unable to observe them under microscope. Will inform you as soon as the results come.

Fumigation: We go for it often. The last one was done just 4 days ago.

Achromobacter contamination: I'll go through the paper and compare their results with what I see.

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