5 replies to this topic

[madelingirly]

Dear Guys ,
I have been facing some infection during the last couple of weeks.
So I decided to massively clean every thing before I restart.
especially my incubator which has this strange smell.
Any way, I switch off my incubator, then dismantle all parts.
wash inside incubator with hypochlorite solution
Irrigate with water
Then wipe it with alcohol , No smell of hypocholrite is observed.
Then clean all shelves and water tray  with enathnol and added new sterile water.
Switch on my incubator,
Wait tell temperature become 35 then pump the Co2
After that, I waited around 1 hrs.
then added 1% antibiotic to my culture medium (previously I have not used antibiotics) then cultured my cells from stock.
Today (almost 12 hrs later ) cells are floatings, not adhere at all
I dont know if there is remaining of hypochlorite or chlorine gas could this destroy my cells (even there is no smell).
If Something accidentally happened to the Co2 sensor during cleaning, and it was not working (although it shows me 5% Co2 level) does uncontrolled Co2 level could cause this massive cell destruction.
my stock was fine I have used it for several times before and it was fine and viability around (60 % ) after thawing.
Does any one has previous experience could share it with me.
  Thanks guys in advance

[bob1]

If the CO2 is too low, the medium (assuming it contains phenol-red) will turn purple.  Too high and it will turn yellow/orange quickly.  This could affect your cells, but it is unlikely if they are commonly used cell culture lines.

Ideally to clean the shelves and water tray in your incubator, you can scrub them with ordinary detergent and then autoclave them to ensure that they are sterile.  Wiping with ethanol before they are returned to the incubator is also a good plan.

[madelingirly]

Dear

bob1

I am very grateful for your priceless help

the color of medium is fine.
I wonder what made my cells dead.
I will try again. culture new cells, try to determine the viability after thawing in my new plate.
and put them again in the incubator and see what is going on ???

[madelingirly]

I have cultured a new batch with new medium
Test viability after thawing it was 92%
and lets see, is the problem really in the incubator!!!!!

[madelingirly]

New culture cells are adherent, however, around 50-60% of my cells are floating in the medium???

previously I was using different FBS (CCB) http://www.nichirei....Bioscience.html
Then I bought biowest FBS ( and the cells showed a slower rate of growth compared to the other FBS.
http://www.biowest.n...php?pg=Products

then I have stopped my cell culture.
and return back after around 2-3 weeks from my stock.
then when I recultured my cells from stock, a lot of cells (40%) was not adherent too. however, I didn't thought it is a big deal because there are a lot of adherent cells too.
After I cleaned my incubator,  and culturing from stock around 95% of my cells was not adhere and floating I thought the incubator may be the problem.
So I tried subculturing yesterday
However. today my plate looked like my previous plate before cleaning the incubator.
The viability test using trypan blue was 92% after thawing.
the old plate showed 95% viability during sub culturing and cells was fine but slow growing compared by other FBS + medium.

Can FBS cause these massive damage during culturing from stock.
Anyone has similar experience and ready to share with us .

Best Regards

[bob1]

Short answer: yes. there are lots of threads on here about testing FBS and ensuring that is performs with your cells in the same manner as your old batch.