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.