Quantification of Colony-Forming Units of Gut-Associated Bacteria in Drosophila Flies

Begin with a microplate containing homogenates prepared from Drosophila flies. These flies harbored a controlled population of a specific bacterium in their gut.

The homogenates contain lysed fly tissues and released gut bacteria.

Aspirate the suspended bacteria and serially dilute across multiple microplates.

Spot small volumes from each dilution onto agar plates, ensuring that the droplets remain separated.

Incubate to allow bacteria to proliferate into visible colonies.

Capture images of the plates to quantify colony-forming units (CFUs), which represent the number of viable bacterial cells capable of forming individual colonies.

Using image analysis software, align each image using a reference line, define a boundary, and apply a uniform grid to segment it into counting zones, each corresponding to a single spot.

Adjust the brightness threshold to distinguish individual colonies from the background, then run the analysis to count colonies in each spot and quantify the CFUs.

Place three rectangular MRS agar growth plates with their lids open in the biosafety cabinet to dry for at least 10 minutes. Prepare two dilution plates by adding 100 microliters of PBS to each well of a sterile 96-well plate using a 96-channel pipetter. Load a rack of P-20 tips onto the 96-channel pipetter.

Make a 1:10 dilution by aspirating 11.1 microliters of homogenate from the sample plate. Now, dispense it into the first dilution plate containing 100 microliters of sterile PBS per well. Keep the dilution plate on the plate shaker for 10 seconds at 600 RPM.

Mix again by pipetting up and down at least 10 times. Transfer 11.1 microliters from the first dilution plate to the second dilution plate and repeat the mixing steps for any further dilutions. For spotting plates, load two microliters from each well into the 96-channel pipetter.

Lower the pipetter head slowly onto the plate, avoiding stabbing into the agar, and ensure all the spots have been dispensed. Then, check that the liquid spots quickly soak into the agar and do not run together. Repeat the plating process for the remaining dilution plates as described in the manuscript.

Once the liquid has been completely absorbed into the agar, invert the plates and incubate them until the colonies have reached optimal size. Organize the plates logically and keep them in a specific order while photographing. Orient them so A1 is in the upper left corner for all the plates.

After removing the plate lid, place the plate on the stage with A1 oriented in the correct corner. Image the plates. Transfer the images to a computer and rename them, including the experiment name, the media type, the dilution factor, and other pertinent details.

Organize the images to be processed for quantification into a folder. The file names that distinguish the plates become column titles for each set of counts in the output spreadsheet. Make subfolders named Cropped and Receipts in the folder.

These folders will receive the output files. Now launch the Croptacular plugin from ImageJ and click OK to start cropping. A dialogue box to choose the source folder will pop up.

Click OK. Select the source folder and click Open. Then, click OK on the dialogue box for choosing the destination directory. Select the destination folder and press Open.

If the image is already straight, simply press Space. Otherwise, straighten the image by drawing a line along an edge that should become horizontal. Redraw the line as many times as needed if the image does not appear straight.

Next, draw a boundary box for the counting area and adjust its size and proportion until all the spots are within their cells. Drag the cursor outside the boundary box to refresh the grid. When the grid looks good, press Space.

Since the plugin assumes all the photos to be aligned identically, ensure that the next image automatically rotates to the same angle as the first one. If this is accurate, press Space to continue. Otherwise, straighten the image as before.

The grid also recalls the same position as the previous image. Adjust if necessary and press Space. Launch Count-On-It and select Gridiron.

Set the threshold colony intensity based on an upper and a lower pixel intensity value. Make the threshold as stringent as possible, while still selecting all the colonies. Click OK on the Action Required dialogue box when the threshold is satisfactory, then click OK to continue.

On the first image, the option is given to proceed or return to the setup menu. To proceed with the batch process, click OK. Then, select a folder to keep the receipts and results table. Use the Receipts folder created earlier.

After the first image, the following images will default to the same threshold as the previous settings. Click OK to use these settings or adjust the settings.