Colony counting on Omnitray media

I am very much new to CellProfiler and have been trying to adapt the ExampleYeastPatches pipeline to use for counting my plates. I am using a serial dilution and spotting technique to counting starting CFU/ml of the respective sample. Each row is a separate sample and gets diluted across the plate. The instrument then spots 5ul of a chosen dilution onto a rectangular agar plate.

I need an more automated / high-throughput means to count the resulting colonies, other than counting each by eye, as I am doing currently. Does anyone have input for me? I am going cross-eyed trying to determine which module to use and in what order to result in a countable image.

I have attached an image to use as an example of the type of format from which I need CellProfiler to count colonies.

I appreciate any assistance you can provide.

Picture1.tif (3.0 MB)

Hi @cory,

Welcome to the forum!! That is a perfect start with the EXampleYeastPatches pipeline!!
Modify this pipeline which should work for your image. You might have to remove modules like “ColorToGray” since your image is already gray image. You might hvae to play around with the illumination correction a bit to work for your image set and other parameters too according to your need. In case you have tried already in this & still not working, you might have to share the modified pipeline so that we could help you better.

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Join us at, https://www.slas2020.org

@lakshmi
I have been trying to adapt the pipeline but it still needs a lot of work, I think. I’d appreciate any input, as I am not familiar with many of the modules to know which to use to accomplish a given task.ExampleYeastPatches.cppipe (18.9 KB)

Hi @cory,
Its fine. To start with you could explore the tutorials available here.
I tried with your image. You need to playaround with the parameters slightly. From the example, pipeline I have added two modules extra “ImageMath” (This is to correct after illumination correction) & “EnhanceSupressFeatures” (To enhance your speckles structure by supressin the BG). For your image “Closing” is optional based on your need. In case you need a small speckle like structures you need not use this, whereas if you are looking for the whole colonies then you might have use the “closing” Module. Please find the screenshot of both,
Without closing,

With Closing,

Here is the pipeline,
ForumYeastPatches_notclosing.cpproj (694.8 KB)
Hope this helps.

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Join us at, https://www.slas2020.org

those look fantastic @lakshmi!
Thanks for the assistance. I’ll work with the pipeline and see if I can implement it.

1 Like

you are welcome!!

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Join us at, https://www.slas2020.org

@laksmi, the pipeline worked pretty well at locating the isolated colonies, but now i’m trying to determine how to use the output data to indicate colonies which will lead to a count within the grid that I have it drawing.

Hi @cory,

You are welcome. Since you had mentioned about the grid, I have a suggestion. CellProfiler has a “DefineGrid” & “IdentifyObjectsInGrid” module were you can define your own grid & identify the objects within it.

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Join us at, https://www.slas2020.org

Hi @lakshmi,

I appreciate your reply.

I have added DefineGrid to identify the grid, though I can’t seem to get it to automatically determine appropriate shape / location on the designated image. Beyond that module, I have also added the IdentifiyObjectsinGrid. I’m looking
for the means by which I can have the software tell me how many colonies are counted within a specific region, e.g. A6 or D7, on the grid. Thoughts?

Hi @cory,

The purpose of IdentifyObjectsGrid module is exactly what your asking for. In your “MeasureObjectSizeShape” module you might counting the colonies where you might have to add the result (I mean the Name) of of “IdentifyObjectsInGrid” module as another object. This would give the count of the objects in each of the grid.
Hope this helps!!

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For CellProfiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Read more on our site.
Yokogawa CV8000 - The Ultimate in Confocal HCS
https://www.wakoautomation.com/products/yokogawa-high-content-imaging

Join us at, https://www.slas2020.org

@lakshmi, thanks a bunch for your help so far. I am quite close to getting it to the point of employing it on a large number of images.

I was curious if you know how to possibly assign a name to a location, so that when exported the associated counts are linked to a well location, rather than objectnumber.

Hi @cory,
@bcimini
You are welcome. In case of extracting the measurements for the result of IndentifyObjectsinGrid, the object number is the location of the grid in that particular csv file corresponds to the well based on how you have defined the grid.
But I am not sure if you can location as A01, B01,etc…

Regards,
Lakshmi