For information about installation of the Image Analysis tool, access the “Simulator” tab.
Step 1: Follow the instructions from simulation tab to download and install ImageJ software.
Step 2: ImageJ will be open in a new window as shown below
Step 3: To analyze an Image into ImageJ, we have to import the image into ImageJ. To perform that, Go to files -> Open. A popup window will appear. Use Choose button to choose your respective images into ImageJ software
Step 4: The imported picture will show in a new window.
Step 5: Choose the line symbol from the shape buttons and draw a line that was placed in the image for scaling.
Step 6: Go to Analyze ->set scale and set the known value to the box and click “OK” .
Step 7: The image will be in the RGB format. Convert the RGB image into a gray-scale image: Image -> Type -> 8-bit
Step 8: To invert the image, Go to Edit ---> invert. This step is not necessary if the bacteria are already darker than the agar.
Step 9: Click on Image -> Adjust -> Threshold. The "thresholding" of the image is done by setting all pixels above certain intensity value to black and leaving background as white. This is called image segmentation.
Step 10: Adjust the threshold by using the sliders to highlight only the bacteria.
Step 11: Select the circle image from the tool bar. Draw a circle around the colonies where the imageJ will measure from.
Step 12: To count the objects, Open: Analyze -> Analyze particles.
Step 13: Adjust the size: “0- infinity”, show: “Outlines” and tick on all boxes except “Record Starts”. Click "OK"
Result interpretation:
The number of colonies present per selected area is 4. The area covered by colonies are 1487 which is 14.431% of the total area. From these 4 colonies, forth colony is large and third colony is small compared to others.