Strange contamination issue - (May/08/2018 )

I'm culturing cells in 6-well plates to expose them to a drug for 24h. 

They are grown in media with antibiotics and serum.

After they're reached around 80-90% confluency, I swap the media with serum-free media containing my drug solution. 

However, after 24h all the wells are cloudy. 

Since this is the second time this has happened, I was extra careful during the second run - consistently spraying the hood and even wiping the closed plate edges with ethanol. I changed pipette tips frequently. I follow strict aseptic technique, that also includes aliquotting of media, not moving arms above plates etc.

Under the microscope it looks like a layer of small dots. 

What could this be? 

I'm at a loss. 

It cannot be the media or cells since they grow fine in 96-well plates and work well doing the exposures. 

Could it be from touching the inside edge of a well when slowly adding the new media? 

Many thanks.

So the only difference in this is that you have added a drug (in a vehicle of some sort presumably)? 

Check the drug + medium to see if it happens in the absence of cells, confirm if it is bacteria/yeast (plate some out, or fix on a slide and stain). If it is bacteria or yeast, you may be able to sterile filter your drug solution.

It could also be that the drug is insoluble in water and is precipitating or is forming visible micells

bob1 on Tue May 8 16:07:22 2018 said:

So the only difference in this is that you have added a drug (in a vehicle of some sort presumably)? 

 

Check the drug + medium to see if it happens in the absence of cells, confirm if it is bacteria/yeast (plate some out, or fix on a slide and stain). If it is bacteria or yeast, you may be able to sterile filter your drug solution.

 

It could also be that the drug is insoluble in water and is precipitating or is forming visible micells

But the drug exposure works fine in 96-well plates. This is an issue with the 6-well plates somehow. But they come straight out of packaging only when under the hood.

I have already tested media by itself and that is fine. 

It's also only ever during the second step. When initially plated into 6-wells all cells are fine,they grow and do not show cloudyness. 

The drug is dispensed in the exact same serum-free media that is mixed with serum to initially grow the cells...

Whatever makes the media cloudy is also advancing rapidly. I changed the media in some affected plates and it was beginning to get cloudy again within 1 hour.

P.S. I should add that the dots in the media move with the media. And also the media is pink in colour, so no colour change to yellow.

OK - I would be very surprised if it is not also happening to your 96 well plates then. Microscopy in 96 well plates is difficult due to the light being distorted by the walls of the wells, so you may not be able to see it directly.

As you have stated that they start to get cloudy an hour after changing the medium and there is no change to the colour, I think you will find that this is neither bacteria nor yeast contamination, but is an effect of the drugs in medium. If it were bacteria, with that sort of growth, you should be easily able to put some on a slide and see it under high power (40x or 100x) on a microscope. It could be the cells dying by apoptosis, but you should be able to see that by changes in confluency and cell morphology.

Try putting the drugs in medium in a well by itself and see how it goes. Also try diluting in a different serum free medium (e.g. DMEM without serum) and see if anything changes. 

bob1 on Tue May 8 18:56:17 2018 said:

OK - I would be very surprised if it is not also happening to your 96 well plates then. Microscopy in 96 well plates is difficult due to the light being distorted by the walls of the wells, so you may not be able to see it directly.

 

As you have stated that they start to get cloudy an hour after changing the medium and there is no change to the colour, I think you will find that this is neither bacteria nor yeast contamination, but is an effect of the drugs in medium. If it were bacteria, with that sort of growth, you should be easily able to put some on a slide and see it under high power (40x or 100x) on a microscope. It could be the cells dying by apoptosis, but you should be able to see that by changes in confluency and cell morphology.

 

Try putting the drugs in medium in a well by itself and see how it goes. Also try diluting in a different serum free medium (e.g. DMEM without serum) and see if anything changes. 

Thank you.

It's definitely not happening in the 96-wells - it did happen before to only some of the wells in a 96er so I know what it would look like. 

Since my controls are also cloudy I assume it must be handling step and not a drug-media issue. 

I just can't seem to figure out what else I could do in terms of aseptic handling...I wasted half a bottle of 70% ethanol the other day just ensuring it wouldn't happen again (and it did anyway).