7 replies to this topic

[Sugan]

I am working in Adipogenesis using 3T3 L1 cells. Recently I have got some problem in the culture. 2-3 days after resuscitation of the cells from liquid nitrogen storage, there is a cloudy and milky appearence in the culture. The closer picture (attached) shows dots moving randomly here and there. Is it a bacterial contamination or any other thing? Is it is a contamination, how to overcome this problem? Is it possible to save my present culture by addition of antibiotics? Normally, I dont use antibiotics in my media. I cleaned my CO2 incubator with 70% alcohol and change the water weekly once. Please help me curb this problem.

[leelee]

Yes it is contamination, no it is not possible to save your culture. Throw them out and start again.

It is unlikely that the source is your CO2 incubator.

[Sugan]

leelee, on 28 September 2011 - 10:54 PM, said:

Yes it is contamination, no it is not possible to save your culture. Throw them out and start again.

It is unlikely that the source is your CO2 incubator.

Leelee

Thank you for your reply. This is third time I have faced the problem. Each time I started with a new ampule from Liq. N2. But I need to stop this problem. Please tell any solutions.

Sugan

[leelee]

Are they all vials from the same frozen stock batch? Could be that particular stock was contaminated prior to freezing.

If not, then the problem is occurring during thawing and plating. So I would do the following:

- thoroughly clean your t/c hood and all of your equipment (pipettes etc) with 70% EtOH
- Start with a brand new, unused bottle of media and fresh supplements
- have somebody with a lot of t/c experience watch you to see if you are making any mistakes you aren't aware of


Is anyone else in the lab having the same contamination issue?

Edited by leelee, 28 September 2011 - 11:20 PM.

[Rnotk]

why you are not adding antibiotic in medium?
any particular reason?

[Sugan]

leelee, on 28 September 2011 - 11:19 PM, said:

Are they all vials from the same frozen stock batch? Could be that particular stock was contaminated prior to freezing.

If not, then the problem is occurring during thawing and plating. So I would do the following:

- thoroughly clean your t/c hood and all of your equipment (pipettes etc) with 70% EtOH
- Start with a brand new, unused bottle of media and fresh supplements
- have somebody with a lot of t/c experience watch you to see if you are making any mistakes you aren't aware of


Is anyone else in the lab having the same contamination issue?

I also suspect that the vials could have been contaminated before freezing. I did all the measures you have suggested. Presently, I am the only guy working in cell culture...I have asked my colleague who was working earlier to repeat few of his experiments to check for contamination in his cultures.

[Sugan]

Rnotk, on 29 September 2011 - 10:52 AM, said:

why you are not adding antibiotic in medium?
any particular reason?

The reason is my boss told me that adding antibiotics may affect proliferation and differentiation.

[leelee]

Sugan, on 29 September 2011 - 04:54 PM, said:

Rnotk, on 29 September 2011 - 10:52 AM, said:

why you are not adding antibiotic in medium?
any particular reason?

The reason is my boss told me that adding antibiotics may affect proliferation and differentiation.

It is best practice to avoid the use of antibiotics in tissue culture, as in addition to possibly having an effect on your cells (as Sugan's boss says) it can also mask low level contamination.