counting in soft agar - (Nov/01/2005 )
When I read articles about the soft agar assay, it seems like it is so easy to count the colonies. When I look at my cells the colonies consist of tiny tiny cells and I cant see how anyone can count them and see if there are more or less than a certqain number of cells.
How does the crystal violet/iodonitrtetrazolium violet work? How do they help the counting?
How does the crystal violet/iodonitrtetrazolium violet work? How do they help the counting?
Are you sure that you are looking at colonies and not at the original cells?
When I read articles about the soft agar assay, it seems like it is so easy to count the colonies. When I look at my cells the colonies consist of tiny tiny cells and I cant see how anyone can count them and see if there are more or less than a certqain number of cells.
How does the crystal violet/iodonitrtetrazolium violet work? How do they help the counting?
Are you sure that you are looking at colonies and not at the original cells?
No I am not sure.
At first I was wondering if what I see is apoptotic cells or something, but they seem to grow allt he time. The cells are small from the beginning. I have never done this before and no one around here seems to have done it either...
Depending on the cell type it takes 2-3 weeks to see colonies in the soft agar. At 2 weeks these are pretty small, think pin head size. You should not need a microscope to see these.
I take the plates out of the incubator and let them sit on the bench top for about an hour until the phenol red in the media has turned a nice purple as easier to see the colonies.
You can count the colonies directly from the plates, but I have found scanning them in with our gel visualization system then counting off the jpg is much easier and saves on eyes and neck.
If you don't think you are getting colonies I would try plating ~0.5E4 cells in a 150mm plate without selection and see if colonies form that way.
I take the plates out of the incubator and let them sit on the bench top for about an hour until the phenol red in the media has turned a nice purple as easier to see the colonies.
You can count the colonies directly from the plates, but I have found scanning them in with our gel visualization system then counting off the jpg is much easier and saves on eyes and neck.
If you don't think you are getting colonies I would try plating ~0.5E4 cells in a 150mm plate without selection and see if colonies form that way.
I have gotten something that I think are colonies, but the cells seem so tiny. But it seems strange if all my "colonies" would be apoptotic cells or something like that. This is what the colonies looked like without selection. They often have a bigger cell in the middle and then more tiny cells around that.
Ok, while not the same expt you are doing, here is what a general colony assay looks like with the naked eye. These are colonies after three weeks of growth in soft agar on a 150mm plate.
What controls have you dont to ensure you are doing the colony assay correctly?
And when you say a "bigger cell" surrounded by "more tiny ones." Do you really mean individual cells which you need to look at with a microscope, or is it something you are seeing with your naked eye?
What controls have you dont to ensure you are doing the colony assay correctly?
And when you say a "bigger cell" surrounded by "more tiny ones." Do you really mean individual cells which you need to look at with a microscope, or is it something you are seeing with your naked eye?
I can not see anything with the naked eye, but I have not used any dye to visualize them either yet. So the cell structures I see are only seen in a microscope.
Reference with the plate photograph. So what i see is dark spots on a transparent background. I presume that each dark spot is a colony. Has staining been done to these cells. if yes, would like to know how to do it. Also, would like to know if this will work with mammalian epithelial cells. Is this photograph taken with the help of a microscope or these colonies are visible to you with the naked eye after it is stained.
I am doing soft agar cloning too and also found difficulties to count the cell. I have also try to using Image Analyzer to capture the picture. But the machine still cannot do a proper counting. Any one can help? Thank you. 
What kind of image analyzer are you using? The best way is to do it manually with a microscope going through the different planes of the surface one at a time. Unless, of course, if you can invest in a colony counter (Gelcount).