Choice of brightness/aperture and acquisition time

Getting the optimum brightness is a result of choosing good aperture and acquisition time pairs. To maximise sharpness of the image independent of the number of sharp objects the auto focus could use, aperture should have values higher than 4 best would be 8. As colonies generally do not move acquisition times up to 0.5 seconds could be accepted before there is a need to open the
aperture.

But what is the right brightness to choose? There are two general rules to optimise the images:

First the LT counter software has no problems to analyse images, which are a little bit darker than the value the human eye would like to be optimum for manual count.
As a consequence underexposure is no problem for image processing while overexposure is one. If the background of a petridish is clear white it does not contain any more information. It cannot be assured that e.g. small colonies with bad contrast are not more to be seen on the image and therefore do not exist for the machine.
To assure visibility of all colonies the background even of the whitest or clearest plate in a measurement series should have a light colour.
This can be easily achieved for backlight if the area around the plate has a slight grey colour.
If colonies tend to be very bright in top light avoiding overexposed i.e. maximum white colonies if needed if colonies should be separated properly.
Avoid any overexposured bright white area within the whole area of interest is best to assure best image processing possible.
Secondly further optimisation of image processing will be reached by maximising contrasts between colonies and background and borders between colonies according to visual impression. For this reason try if backlight ot toplight gives better contrasts for all colonies.