It doesn’t look like you changed much from the example pipeline for what you uploaded; I think it is worth you perhaps looking at the included PDF and/or the module notes if you want to get a better sense of what each individual module was supposed to be doing, and therefore where yours went wrong. The image in your screenshot that you showed doesn’t look like what it ideally should before going into IdentifyPrimaryObjects (a pretty dark background with your colony spots looking bright), so it’s not surprising that it isn’t necessarily doing a good job.
A simple pipeline on one of your images that worked well for me, without even needing a template, was this-
- ColorToGrey, to split out your channels
- ImageMath using the Invert option to invert your Blue channel, making the blue spots now bright-on-dark
- EnhanceOrSuppressFeatures with the Speckles option to bring out the spots and delete almost all of the background
- IdentifyPrimaryObjects to call the spots.
(Plus then you’ll want probably want some SaveImages/Export/etc modules to write out your results.
The exact tuning should be up to you, because I don’t have a good sense which of the borderline spots are real or not, but it looked pretty decent to me in the View Workspace mode. I can’t promise it will work for all images; you may want to use a plate template or something ala the original pipeline, but that should be enough to get you started.