Hi,
I work with MDCKs. Typically, we put Pen/Strep into our media. Recently, one of my transfections has been determined by the lab to be contaminated. This has made me worried. How do you tell if you have contamination? Obviously, I plan to do a Mycoplasma test on my cells since those are basically invisible. But what other ways can I tell?
The media does turn yellow after the cells have been in it for about two days (assuming I do not change it). But I'm pretty sure that's just exhaustion of media. I haven't observed any slowed growth. It takes about 2 days from thawing to confluence or from split to confluence. Their morphology hasn't altered, either. They look fibroblastic when growing and then nestle into nice little cobblestones when done.
I do see little black irregular dots oscillating at 40x. However, it seems like this is just Brownian motion as they don't really go in a specific direction. And there aren't many, even after two days with no media changes. There's probably one for every two cells? If I did see something swimming/darting around, I'd toss everything immediately. The lab says it isn't contamination.
Throwing the cells into wells has them all turn the same color at the same time at the same rate, too!
Hi Colin,
Contamination is the most challenging and commonly faced problem for all cell culturists. For identification of contamination, i.e. to be sure if at all there is something contaminating your cell culture you may try these:
1. Try not to discard the spent media but keep it in a plate (may be in a 60mm one), if the media turns yellow or cloudy even then, you know it is contamination! To determine what the contaminating organism is, microscopy is the best way.
2. Fix the cells and stain with DAPI. If you see DAPI staining in small dots outside nucleus, you know that it is staining the DNA of your contaminant.
3. Trypan Blue staining showing small dots with dark outline and lighter inner space indicates presence of contaminants.
4. For simple light microscopy, under 40X magnification, if you see black dots stuck on the plate which doesn't seem to go even if you change plate daily, and also has some kind of brownian motion, its bacterial cells that you are looking at.
To prevent contamination-
1. If you have already identified it to be mycoplasma, then there are many preventive measures which might help you. Like you can use 0.1 micron filter to filter sterilize your media and other stuffs of cell culture.
2. I heard Plasmocin from Invivogen is very effective to combat mycoplasma infection. You can follow the link for further information.
3. Also, even though your cells look healthy, their morphology or growth rate doesn't change, appearance of black dots indicate that you need to check for contamination.
4. Colour change of phenol red is not sufficient enough to determine the presence of contaminant, as rate of colour change depends on the confluency and number of cells seeded.
Hope it helps!
http://www.invivogen.com/plasmocin
https://www.researchgate.net/post/Contamination_problems_in_293_T_cell_culture_Any_help?_tpcectx=qa_overview_following&_trid=LIisdzQcAoQrYwx3WLZaYoqv_
Sorry Colin, that's contamination. Medium shouldn't deplete so quickly. The phenol red is in medium for exactly that reason - pH change ('premature depletion') indicates a problem. MDCK cells don't oscillate (at least mine don't). And bacteria don't normally synchronize their activities. You are unlikely to see mycoplasma without a stain. Fungus (yeast) is slow growing. Quick way to test for most bacterial contamination (which is what you probably have) is to toss a few microliters into a couple mLs of L broth and culture overnight on a shaker. Cloudy = problems.
Depending on a cell type and how well the medium is buffered, you can see a change in colour of the medium in two days without indicating an infection. It would also depend on the CO2 concentration in the incubator. The phenol red is a pH indicator and won't tell you if the medium is 'depleted'. If you have problems maintaining pH then you can think about using a medium with HEPES or sodium bicarbonate added.
The best indication of infection is cloudy media, or as Craig suggests transfer some to LB or other and see if it blooms. Bugs don't always swim around, so you might just see them wobble a bit with Brownian motion. If you're looking down the scope, try to find a patch with no cells to look at. Dark specks in cells are common, but an infection is unlikely to be purely inside your cells.
If you can't tell in the flask, put a ml in an eppendorf with fresh medium as a control and see if one is cloudier than the other.
It wasn't bacteria. This was confirmed by nearly everyone in the lab.
But it was Mycoplasma. Doesn't seem to affect the cell growth rate, but still, need to go about getting rid of it now.
Edit: The vibrating dots aren't bacterial in nature. Mycoplasma is present, but because it's sub-micron and cryptic, it's invisible under most types of microscope.
Nearly everyone in your lab said it wasn't bacteria? But mycoplasma is a genus of bacteria...
So now you can try and use mycoplasma-specific antibiotic therapy if you cant discard those cultures. It takes about 1 month and from that point on you become super cautious of mycoplasma in this culture, as it's a hard player to kill out completely in a host population.
Hi Colin,
Contamination is the most challenging and commonly faced problem for all cell culturists. For identification of contamination, i.e. to be sure if at all there is something contaminating your cell culture you may try these:
1. Try not to discard the spent media but keep it in a plate (may be in a 60mm one), if the media turns yellow or cloudy even then, you know it is contamination! To determine what the contaminating organism is, microscopy is the best way.
2. Fix the cells and stain with DAPI. If you see DAPI staining in small dots outside nucleus, you know that it is staining the DNA of your contaminant.
3. Trypan Blue staining showing small dots with dark outline and lighter inner space indicates presence of contaminants.
4. For simple light microscopy, under 40X magnification, if you see black dots stuck on the plate which doesn't seem to go even if you change plate daily, and also has some kind of brownian motion, its bacterial cells that you are looking at.
To prevent contamination-
1. If you have already identified it to be mycoplasma, then there are many preventive measures which might help you. Like you can use 0.1 micron filter to filter sterilize your media and other stuffs of cell culture.
2. I heard Plasmocin from Invivogen is very effective to combat mycoplasma infection. You can follow the link for further information.
3. Also, even though your cells look healthy, their morphology or growth rate doesn't change, appearance of black dots indicate that you need to check for contamination.
4. Colour change of phenol red is not sufficient enough to determine the presence of contaminant, as rate of colour change depends on the confluency and number of cells seeded.
Hope it helps!
http://www.invivogen.com/plasmocin
https://www.researchgate.net/post/Contamination_problems_in_293_T_cell_culture_Any_help?_tpcectx=qa_overview_following&_trid=LIisdzQcAoQrYwx3WLZaYoqv_
All above is good advise. however, co2-regulation mismatch gives the same results. check your incubator co2-level, it could be well over 5%.
Hi,
I am working with anaerobic bacteria. I kept my sterile media in the anaerobic chamber. It usually have 1-8% oxygen in it. Today I saw white spots on the surface of my media and on the walls of the bottle. I have no idea what it is. it did not penetrate into my whole media. How can i know what it is? how should I make sure it is not pathogenic?
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