Mammalian colony counting


Dear all,
I would like to use CellProfiler to count mammalian cell colonies (stained in blue) on 6-well plates.
I have tried to adapt the yeast colonie counting pipeline, but I was not so successful. Does anyone tried something similar?
Here is an example of pictures:
I’ve tried to ‘filter’ the picture using the blue layer (where the colonies are invisible) to remove the plate itself and the background but I didn’t manage to adjust the intensities before doing that, even using the AdjustIntensity module.
Any ideas are welcome… :smile:


We have a pipeline that will be helpful for you. Please download the “Colonies” example (identifies and measures yeast colonies growing on agar plates). You will need to follow a similar strategy as the example. (In the example, we are identifying, measuring, and classifying red colonies.) For your images, you will need to adjust it to use the blue channel, as well as change the size requirements for identifying and cropping the outside well area. Additionally, we have a ‘plate template’ of the well–you will need to make a similar template for your specific size wells.

Hope this helps!


If anybody wants a mammalian colony counting pipeline, I have one I am happy to email you if you PM me. The main difference from the yeast colony example provided is that it contains a module to invert the image so that colonies stained blue/purple and scanned on a white background appear light on dark and can be counted by the software.



Thanks for the offer! Just to let everyone know, we will be publishing a chapter in the upcoming edition of Current Protocols for Molecular Biology using the Colonies pipeline. If you would like a preprint, please contact me directly at



I new in the field, do you have any pipeline for automated counting of mamalian colonies on 6 or 12 wells` plates with the size determination?




We don’t have a pipeline for 6 or 12 well plates. However, you can try to modify either the ‘Classify Colonies’ pipeline or the ‘Grid’ pipeline.


Dear Martha,

I would like to ask if if you could help with the pipeline you offered? Just let you know a bit about my work: doing agar assay to test cell transformation and count the number of colonies formed on the agar assay (on images taken). I am new to it and do not know how to use it, but got know through a paper recently in Biotechniques and your paper on Curr. Protoc. Mol. Biol. 82:14.17.1-14.17.12. 2008.
However, when I tried to load pipeline (ExampleYeastColonies_BT_Images-2), it appeared that the load image module could not be found or erred. I have tried many times without succeeding, would appreciate it if you could send me the pipeline and instructions if possible. My e,mail:
(I tried your email but couldnot reach you, do not know why?!)
Many thanks and look forward to hearing from you.


Hi all,
I encountered problems when using CellProfiler. After installing the software, I tried to load the example pipeline (downloaded from the web, unzip and place all files in one folder) but without succeeding in loading the pipeline, it always appeared when I tried loading the pipeline: "The loadimages couldn’t be found in the directory specified or an error occurred while extracting its variable settings.This is uncommon, the module may be corrupt…"
I have tried exactly what suggested in the help and even reinstalling the CellProfiler many times, whithout being able to load the pipeline for my colony counting assay (from images taken). I would appreciate it very much if someone could offer help on this problem, please email me at
Thank you.

Hung, Duke-NUS.



This error usually occurs when your preferences haven’t been set correctly. Try going to File>Set Preferences, and make sure the directory where Modules are stored is in fact the one where your ‘Modules’ folder was downloaded to.



Thanks Kate,

I have tried that and still got the same problem in my Mac. I just tried on my friend PC, a Windows and it worked well. The software works really well for my purpose but it is a bit pity that I have a Mac , not a PC. I am not sure if any one got this problems before.
I have tried many times and captured all the windows showing errors, but cannot post here, maybe I will send you by email?

Thank you for your help.



It’s important to download the correct version for you Mac. Go to Apple>About this mac and look under ‘Processor’. That will tell you if you have an Intel or PPC processor, which dictates which Mac version you should download. Try checking this and make sure you have downloaded the correct version from (If you have a PPC, we have not gotten the latest version compiled on PPC yet, but the 5811bugfix version works fine).



Just to update, if anybody is still interested in my pipeline for counting mammalian colonies using CellProfiler 1.0, feel free to email me at
conradvink1 gmail com