Robustly find N circles with the same diameter: alternative to bruteforcing Hough transform threshold

Question

I am developing application to track small animals in Petri dishes (or other circular containers). Before any tracking takes place, the first few frames are used to define areas

Answer 1

You don't explain why you are using a black background. Unless you are using a telecentric lens (which seems unlikely, given the apparent field of view), and ignoring radial distortion for the moment, the images of the dishes will be ellipses, so estimating them as circles may lead to significant errors.

All and all, it doesn't seem to me that you are following a good approach. If the goals is simply to remove the background, so you can track the bugs inside the dishes, then your goal should be just that: find which pixels are background and mark them. The easiest way to do that is to take a picture of the background without dishes, under the same illumination and camera, and directly detect differences with the picture with the images. A colored background would be preferable to do that, with a color unlikely to appear in the dishes (e.g. green or blue velvet). So you'd have reduced the problem to bluescreening (or chroma keying), a classic technique in machine vision as applied to visual effects. Do a google search for "matte petro vlahos assumption" to find classic algorithms for solving this problem.