I'm working with MDCK cells, but unfortunately there's a contamination appear on my cell culture with the some characteristics as follow; 1) the colour of the medium is clear yellow, not turbid; 2) some cells are still attached to the cell culture flask surface but it ain't growing anymore; 3) there's such a thin-circular formation on the monolayer cell culture appear as in the picture below. Is it mycoplasma or anyone have an idea?
Hello
The culture medium has turned acidic because of which the color has turned yellow. You need to add fresh medium to the culture. The cells are not looking healthy which could be because of the acidic medium.
There seems to be no contamination. But if you are suspecting mycoplasma you can always test it by using mycoplasma detection kit. You can also test for bacterial contamination by streaking a few micro litres of this medium on agar plate and incubating at 37 degree C overnight. Check for bacterial colonies the next day.
Best,
The cells do not look healthy at all. You should check for contamination. In case you change media regularly and still see this, start with a new media bottle.
A few options here:
1. Revive new cells and remake the culture media. Check the concentration of all components and the pH of the media.
2. Check the incubator. CO2 level, temperature variations, light can affect nutrient breakdown and stress the cells out.
3. Use a mycoplasma detection kit. Here's a paper talking about some of the most popular detection kits and how they work. CDC uses ATCC and abm's detection kits, with abm's having the best value. If you don't already have one, might be a good idea to invest in one for future use.
https://www.atcc.org/products/30-1012k
https://www.abmgood.com/pcr-mycoplasma-detection-kit-g238.html#mycoplasmapcr
Article Mycoplasma detection in a historical arbovirus repository: C...
thank's a lot for the suggestion! Malcolm Nobre Nancy Zenan Li Utsa Roy Md Mezbah Uddin
It is not contamination but maybe the cell cytotoxicity so check the media pH before use for subculturing and also check the type of flask used for culturing. The cell also shows such characteristics when reviving from the cell storage, so follow the subculturing protocol by pre-warm the growth media containing fetal bovine serum and tryptose phosphate broth (TPB) before use.
I used to revive my cell culture using a growth media containing 10% of fetal bovine serum, may I know the function of the addition of tryptose phosphate broth (TPB)? because I'm not familiar with it before, thank u Yeneneh Tesfaye Alemu
Dear Dasilva Nuswantari, you may formulate the growth base media 80%, TPB 10%, and Newborn calf serum 10% to make complete cell growth media. Adjust the pH media one pH below the cell growth media pH because when you filter (0.2 µm pore size), the media pH increase with one pH after filtration. Regarding your question about tryptose phosphate broth (TPB): It is valuable in tissue culture procedures where the peptone content is considered to be a stimulating factor for cells. The tryptose component is a peptone (mixture of amino acids and short peptides) derived by the mixed enzymatic hydrolysis (pancreatic enzymes) of the milk protein casein. Tryptose Phosphate Broth (TPB) comprises pyrimidine-based components and is crucial for pyrimidine synthesis and electron transport chain in cells. Sodium chloride maintains the osmotic and ionic equilibrium and disodium phosphate provides the basic buffering capacity. In chick embryo fibroblast, it alleviates chloramphenicol inhibition. Tryptose Phosphate Broth (TPB) is composed of four components: Tryptose (20g/L); Dextrose (2g/L); NaCl (5g/L) and Disodium Phosphate (2.5g/L) typically adjusted to pH 7.3.
Regards,
Yeneneh
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