How can i decontaminated my cell culture ? - (Oct/17/2006 )

Hi..every body

I did tissue culture for ( MCF-7, Hela & HepG2),

I used DMEM with


......hight glucose+ L-glutamine+ pyridoxine hydrochloride+ 110mg/L sodium pyruvate+ without sodium bicarbonate}

.....Fetal Bovin Serum
....hyQ pencillin- Streptomycin solution 10,000 units/ml
....sodium bicarbonate..

i used 2% FBS as (mainatnce media) and 10% FBS as (growth media)

for 10% FBS i was prepared it with formula/ 500ml ..

NaCo3 5 ml
Pencillin Streptomycin 2.5 ml
Amphostate 2.5 ml
Serum 50 ml

for 2% FBS i prepared it with formula/ 500ml

NaCo3 0.75 Ml (microlitre)
Pencillin Streptomycin 0.5 ml
Amphostate 0.5 ml
Serum 20 ml

Equipments.....

..... Incubator at 37C & 5%co2 ....
......Orange cap flask for culture..

when i checked the cell within 24hours after cultured, the cells were ok with little bit turbidity in the media...so after 24hours i got several problems like.....

1st problem.. The color of the media was changed into yellow with something like milky turbidity floated inside the media

2nd problem..I found black dots over the cells with a change in the media color

3rd problem..sometimes the cells were detached from the surface and dead with a change in media color


4th problem.. the cells it take long time to grow


My Question:-

1-check if the formula that i am using for DMEM is right or not??

2- Identify my problem and give me solutions for it ???

3-How can i rapid the cell grow ??


thanx..Loai

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1. I don't know...never make DMEM from powder.

2. May be your tissue culture techniques...are you new to tissue culture?

3. MCF7 is slow growing cell line...it needs cell contact to grow rapidly...just increase the cell density.
I don't know about Hela & HepG2.

Hope this may help.

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Sounds like a bacterial infection.

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sounds like infection to me too, you shouldn't really see anything milky in the media at all.

one way to check if your media is actually contaminated is to put some (with all the supplements, but without the cells) into a 37 degree incubator and see if anything grows.

Alternatively, your flow cupboard could be contaminated, so I would thoroughly wash down with bleach and 70% etoh (or detergent) as well.

i wouldn't try and cure the cells from infection if you have back-up vials stored.

also, why are you using lower antibiotic concentration for the 2% FBS media?? seems strange to me

generally 10% FBS should be good for rapid cell growth, but like minnie mouse said, increase the seeding density should help.
you could also use conditioned media to help your cells grow better.

hope this helps

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Hi,

Careful with the waterbath. That's the main source of contamination. I've learnt it the hard way and now, I always fill in distilled H2O into a large beaker, place it into waterbath, and thaw or equilibrate my medium, FBS, antibiotics, supplements in it without touching the water surrounding the beaker.

No contam. now... hopefully that'll last. I agree with everyone that it looks like contamination. Try looking under a microscope to see if there's anything weird or not. If it's very messy, just throw away.

If they're patient specific cell lines, try decontamination by diluting the culprit and tolerate a little contam. It seems to me that these bugs are very difficult to kill, esp the black motile dots. My colleagues even convinced me that they're actually cell debris that is moving Brownian way, which I tried believing and cared less now. Ha!

Best of lucks

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