Question

Hey guys, I am having trouble creating a sterile nutrient agar for growing human skin cells. I bought an agar online which is instructed to mix with chicken broth. The samples stay clean for a few days, exhibiting enormous cell growth, but after just a few days, the samples may turn greenish, and the agar develops colored circles all around (mold?)
Any ideas on how to avoid this??

Answer 1

and you autoclaved everything? also, are you pouring agar plates or doing something else?

Answer 2

hmm good questions. Unfortunately, I do not have the supplies necessary to successfully autoclave the petridishes, however I attempted to sterilize them beforehand by soaking them in bleach/water mixture. They were then placed upside down on a towel to aerate.

I am also pouring the plates myself. It is a mixture of water and chicken broth heated on the stovetop and then the powdered agar added in. It is poured and cooled until solid. Fromthe bleaching step-on, i attempt to keep all insides as clean as possible.

I understand my procedures are not great and not very sterile but I am doing the best I can. I even built a Sterile Transfer Box with rubber gloves so the petri dishes are even cleaner.

hope this helps you help me.

Answer 3

ok, after consulting the guy who grows all our cells for us (were spoiled), he told me you want to use something called dodeca modified eco media. I googled it and couldn't find much, so ill double check that spelling tomorrow, maybe i just messed it up. Anyway you have a couple major problems

First, without a more sterile environment, contamination is really just a matter of time. You could try using a low dose of an antibiotic or anti-fungal since you are growing animal cells instead of bacteria. If you can get your hands on gentamicin, i know that that antibiotic cannot penetrate animal cells. You could wash your cells with it to help keep the cells free of contamination.

Second, the skin cells on the surface are not able to reproduce. you must be sure to have the underlying layer of monopotent stem cells that are constantly making new skin. These are able to grow into more cells. They will want some sort of membrane to grow on, since they are used to growing (i think) on a layer of collagen or similar ECM.

Third, animal cells almost always require growth factors to signal them to divide. If you can get ahold of some fetal bovine serum, that should work well. The chicken broth is probably cooked, so the growth factors are most likely degraded or just not similar enough to human.

So basically, i would use a good plastic reaction plate if you can get them and use a liquid media instead of an agar. Animal cells are usually pretty good at attaching to surfaces, so if you can get them to form a nice layer they should grow nicely. Hope all this helps and i will get back to you with more if i can. Its never simple with biology!

Answer 4

WOW brendan! those are some great tips! It is amazing how difficult it is to grow these cells, but i suppose thats why it is just now becoming popular in lab settings. Also, I do make sure I have the right skin cells, which is clear by the growth that does occur, apprently I am just not making sure it is sterible enough.

Again, thanks a lot for the help. Keep in touch as more information comes into play, I would very much appreciate it!