CM Engine measurement modes. (a) Images of colonies in grayscale before and after application of a threshold filter. These images represent the three main ways cells are deposited onto agar plates in high-throughput growth experiments (discrete points, blunt tipped and liquid spotted). Three different CM Engine measurement modes (Standard, Summation and Background Subtracted) have been developed to handle each of these colony deposition methods. The grayed boxes in b, c and d indicate the preferred measurement mode. Prior to analysis, each image is symmetrically divided into partitions, one for each colony. (b) Standard mode is designed for robot pinned colonies that come from a discrete point. Images are subjected to a threshold filter and only the largest particle of each partition is considered. Anomalies are presented to the user and may be removed manually. All colonies in image I are measured accurately since the linear artifact in partition 2 was detected and removed. In image II, only colony 1 is correctly measured since Standard mode chooses the largest particle in each partition, incorrectly measuring the non-contiguous growth in partitions 2 and 3. None of the colonies in image III were measured accurately since the automatic threshold filter used in Standard mode is insensitive to density differences between partitions. (c) Summation mode is designed for colonies from blunt tipped pins. Images are subjected to a threshold filter and all particles within each partition are considered part of the colony. In this case, all colonies in image II are measured accurately. Colony 2 of image I was incorrectly measured due to the inclusion of the linear artifact in that partition. This mode also fails for image III since, like Standard mode, it is insensitive to density differences. (d) Background Subtracted mode is designed for cells spotted from a liquid culture. The background of the grayscale image is subtracted from each partition and the remaining pixel values are averaged to quantify cell density. In this mode, image III colonies are measured accurately, while image I was not due to the linear artifact in partition 2. Additionally, image II was not measured accurately due to the high background value of the grayscale image. For b, c and d, values are normalized to the largest colony size. Accuracy was determined by manual threshold and measurement as described in the documentation of ImageJ software.