We are running two bacterial killing assays in our lab, both require colony counting. One of those assays requires counting colonies of Streptococcus pneumoniae (pneumococcus) grown on a Todd Hewitt agar. The pneumococcal colonies are a reddish brown (after TTC staining) and the agar is a wheat colored light agar. Tif grayscale images of the plate are captured on an Alpha Innotech FluorChem using white light to illuminate the plate. Colonies are easily counted using colony counting software called NICE developed at the National Institute of Standards and Technology. This works OK.
The second assay is the reason I have been interested in using CellProfiler. The assay seeks to measure the ability of a patient’s serum (antibody) to kill Haemophilus influenzae (H. flu). The readout is, again, tons of colony counts on serially diluted serum samples to which H. flu has been added. The problem here however is that the agar used to cultivate H. flu is chocolate agar (named for the fact that it looks like chocolate, that is, it is brown and opaque). The colonies of H. flu are off-white; I have attached a typical image named 1-1.tif. NICE is unable to “see” these colonies.
I’ve been through the Current Protocols article by Vokes and Carpenter that describes a pipeline designed for yeast colony. That pipeline is not designed to begin with grayscale images and so I have tried to modify (also attached) it with mixed results.
So finally a question or two.
First, do you think a pipeline can be constructed that will enable CellProfiler to count these H. flu colonies on chocolate agar and still discriminate colonies that are touching one another?
Second, The 1-1.tif image shows a single plate with 8 different samples. Is there a way to partition the plate into separate regions that could be counted independently.
Each plate can be counted manually using the FlurChem but I have ~300 of these plate images that need processing. I’m hoping that CellProfiler can come to the rescue.
Infectious Disease Unit
Bob’s pipeline 21 Apr.cp (11.2 KB)