Digitally+Counting+Colonies

Basic process that seems to work on ImageJ
 * Adjust brightness and contrast
 * set image to an 8 bit image type
 * threshold so that only the colonies are left
 * Analyze particles and get a cell count

Counting Colonies in One Image Using ImageJ
 * Create image (take a picture of the plate with a phone, scan it with a scanner, or use a gel imaging instrument to take a picture of the plate (I do it this way))
 * Open the image file
 * Adjust the brightness and contrast as desired (Image->Adjust->Brightness/Contrast, adjust as necessary and then click apply)
 * Convert the image from a red, green blue image to a grayscale 8 bit image (Image->Type->8 bit)
 * Crop away unnecessary parts of the image by copying the part you want and creating a new image.
 * Select the oval tool. Highlight the region you are interested in. Copy this.
 * Make a new image (File->New->Internal Clipboard)
 * Apply a threshold to the image so that only the colony spots are remaining.
 * Image->Adjust->Threshold. Adjust the minimum and maximum threshold levels so that only the colonies of interest and possibly some of the cropped background remain. Apply this threshold
 * Count the particles
 * Analyze->Analyze Particles (may want to have the following settings applied: Size (pixel^2): 0-Infinity, Circularity: 0.00-1.00, Show: Outlines, display results, clear results, and summarize). Click OK

Automatically Counting Colonies in Many Images Using ImageJ >>> setMinAndMax(0, 157); >>> call("ij.ImagePlus.setDefault16bitRange", 0); >>> run("8-bit"); >>> makeOval(123, 45, 332, 330); >>> run("Copy"); >>> run("Internal Clipboard"); >>> setAutoThreshold("Default"); >>> //run("Threshold..."); >>> setThreshold(134, 255); >>> run("Convert to Mask"); >>> run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00 show=Outlines display clear summarize"); >>> close; >>> close; >>> close; >>> runMacro("L:\\Colonies on plate 2-16-12\\Macro.ijm"); >>> open("L:\\Colonies on plate 2-16-12\\2 B 10-2.jpg"); >>> runMacro("L:\\Colonies on plate 2-16-12\\Macro.ijm"); >>> etc. .. .
 * First create a macro
 * Plugins->Macro->Record
 * perform all of the steps above for "Counting Colonies in One Image Using ImageJ" (note that adjusting the brightness and contrast does not seem to work so you may want to do that separately)
 * When all of the steps have been performed, click Create then File->Save As and save the macro with the name you want in the place you want. Note that the macro can be edited in any standard text editor
 * Run the macro
 * Plugins->Macro->Run
 * Select your macro
 * Set things up to process many images at once
 * I like to create one macro that does all of the image processing, and one macro to open all of the images and call the image processing macro
 * Example Image Processing Macro
 * run("Brightness/Contrast...");
 * Note that in order to obtain the values for the brightness/contrast command that you want for the setMinAndMax command you can play with an example image first. Adjust the brightness and contrast the way you want. Make note of the min and max values, and then click reset. To generate the commands in the Macro you can then click set, and then set the min and max values to the numbers you noted previously.
 * Example file handling macro
 * open("L:\\Colonies on plate 2-16-12\\1 B 10-2.jpg");

Example Images from ImageJ Processing

Here's an article about counting colonies Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: Comparison with manual counting Read More: http://informahealthcare.com/doi/abs/10.3109/09553002.2011.622033