Image analysis and mathematical correlation between cell number and diameter of colonies in soft agar as a...

Automated image analysis was used to measure the colony diameter and estimate the cell number in colonies under conditions for anchorage-independent growth (AIG). The calculated values for the number of AIG cells in a colony of a diameter greater than or equal to 60 microns were decidedly different from the actual values obtained by either automated or manual cell counting. Like animal cells that exhibit AIG, these colonies demonstrating anchorage independence can be enumerated reproducibly. However, the assumption is incorrect that the number of cells (N) in a colony expressing AIG varies directly with a change in the diameter of a colony (d). Whether one counts the number of cells manually in a colony of a definite diameter or arbitrarily uses a diameter function of greater than or equal to 60 microns as a linear distance that indicates the presence of 50 or more cells in a colony, each procedure has its own built-in bias. In procedures that require quantitative data, the most reliable procedure is to standardize the values for diameter of colonies with cell numbers in colonies.