Counting colonies of clonogenic assays by using densitometric software

Everyone who has already counted colonies from a clonogenic assay knows that this is hard work which takes a lot of time. Many groups have thought about an improvement of the counting system [1-8] and the cited publications certainly won't cover all attempts, but no method has achieved a widespread use at all.One may ask what the reasons are. Two major reasons are certainly the following: all presented methods had problems when faced with clustered colonies. Some systems have managed to avoid this problem by constructing ingenious scanning systems, but these systems are only commercially available.For this purpose we developed a new software that is able to evaluate the clonogenic assays by using densitometric methods and other special parameters. This revealed highly fitting results which were compared to manual countings with the microscope.The newly designed software requires normally scanned six-well-plates or flasks (200 dpi is optimal for excellent results) which should be scanned as grey-scaled photos and saved as jpeg-file. Even better is a reflection light scanner but this is no condition for good results. It is anyhow recommended to avoid (as much as possible) disturbing shades which are caused by scanners, although the software can usually recognize and neglect these shades.Flasks can be scanned and evaluated without editing the scanned pictures. In our institute it is a routine activity to archive the clonogenic assays by scanning them so that this is no additional work.When launching the program (a more detailed description is given in the manual of the program, see 1) you are asked to select a data file. After selecting a file the program will start (see fig. 1). The area to be counted can be selected and copied to the working area. By counting the first time you usually won't get the result you expected. So you have to adjust the available parameters. This presumes that you have counted one well of the plate (usually the one that contains the minimum