How to remove lid and read absorbance on a portion of a 96 well without contami - (Jun/11/2014 )

I seeded cells into a single 96 well plate for an MTS assay at different time points. I want to read the first few rows at 16 hours, next few rows at 24 hrs and remaining rows at 48hrs. I was thinking I could just add MTS to the wells for that time point, read the absorbance and continue culturing the plate for the other time points. But then after seeding the cells I remembered that I need to take the lid of the plate to read and I face a contamination issue. Has anyone used the approach and how did they prevent contamination? Parafilm the wells that you want to keep covered? Or is there some other simple solution?  Or opening the plate for such a brief period of time does not cause a problem because the cells will not be exposed that long nor will they be cultured much longer. 

Thanks!

Solution:

1 - Simple solution, plan the experiment better and do not seed cells for different time points in the same plate.

2 - Read the plate with the lid on at each time point and subtract the background using the blank wells.

Just curious, did you face contamination problems ? I thought taking the lid off for a brief time when doing it in the middle of the room where there is lots of air currents should normally not be a problem, although it is not the safe way.

Do you have antibiotics in your cell culture ?

Tabaluga on Wed Jun 25 15:19:37 2014 said:

Just curious, did you face contamination problems ? I thought taking the lid off for a brief time when doing it in the middle of the room where there is lots of air currents should normally not be a problem, although it is not the safe way.

Do you have antibiotics in your cell culture ?

I decided to read the plate with the cover on. I am repeating the experiment and will plan it better ie different plate for each time point. I do add pen-step to my media but I did not want to risk taking the cover off to read the plate considering I would be culturing the cells for another 36 hrs after that point. I thought the disadvantage of  the inflated absorbance from reading with the cover on outweighed the contamination risk and possibly not being able to read the plate at my later time points. I got a good viability curve over a 48hr period after subtracting out the background and I saw difference between my control and experimental cells. 

I had the same concern about contamination when I started growth curves, but others in my lab assured me that a brief exposure would not lead to contamination. I took the lid off immediately prior to placing in the plate reader and replaced it immediately after. Even after reading the plate daily for 3-4 days, no contamination has occured.