Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

CFU determination is necessary to measure the efficacy of the immune response against bacteria. However, it is very well throughput and becomes cumbersome and expensive when applied to pathogenic bacteria such as mycobacterium tuberculosis. This is a significant limitation when we need to test large panels with treatments.

The research focuses on tuberculosis and on the assessments of TB vaccines. It explores safety, immunogenicity, and efficacy of vaccines against mycobacterium tuberculosis. The study presents an optimized method, the micro CFU for enumerating bacterial colonies to assess vaccine efficacy in preclinical studies.

This protocol aims to optimize CFU determination by generating results from a small droplet, similar to those from a regular Petri dish. Quantifying bacteria viability from such a small sample allows us to analyze thousands of data points using a fraction of the materials and time necessary for the classical methods. Our research aims to dissect the antigen presentation machinery and to identify the most relevant antigens to optimize antigen presentation.

This will inform us to design and develop better vaccines to improve or replace the current TB vaccine, which is BCG. Begin by collecting the BCG culture at the mid or late log growth phase. Centrifuge it at 3000g for 10 minutes.

Then discard the supernatant. Next, add 10 milliliters of PBS to wash the bacteria. After centrifugalization, discard the supernatant.

Resuspend the pellet in five milliliters of infection media. Place the tube in an ultrasound bath for 15 minutes at full power with a frequency of 80 hertz. Now, centrifuge at 1000g for 10 minutes.

Collect the supernatant. And discard the pellet. Measure the optical density of the supernatant.

To perform serial dilution using a well plate, first, place mycobacterium suspensions on rows A and E of a 96-well plate. Add 180 microliters of deionized water into the remaining wells for the serial dilution process. Utilizing a 12-channel pipette, resuspend the suspensions in row A and transfer 20 microliters to row B.Repeat the suspension transfer for rows B and C until the last dilution is reached in row D.For micro-droplet plating, use a 0.5 to 10 microliter multichannel pipette with thin tips to transfer five microliters from each row of the 96-well plate to the solid medium square plate.

Allow the droplets to slightly touch the agar, ensuring proper transfer and minimizing liquid retention inside the tip. Allow the droplets to dry and close the agar plate. Incubate it at 37 degrees Celsius while monitoring bacterial growth.

Optionally, place the agar plates in a sealed plastic bag to prevent drying. After six to 10 days of incubation, check for individual colonies visible to the naked eye. Place the plate under an inverted microscope using the lowest magnification objective.

Alternatively, use a camera to take a picture of the droplet and manually count colonies on the computer using ImageJ for automated colony counting. The micro-colony forming unit or micro CFU assay was used to produce high amounts of data from a small amount of agar plates. Using liposomes for a saquinavir delivery increase the efficacy of the treatment against the mycobacterium strains with different drug resistances.

To execute the manual counting method, begin launching the Fiji software. Open the image containing the micro-colony forming units. Navigate to Plugins, then click on Analyze, followed by Cell Counter.

On the cell counter menu, initialize the program by clicking on Initialize and choose a counter. Proceed by clicking on each colony. If an accidental click occurs, click Delete to undo.

Note the value displayed on the counter. Press the Reset button to restart the count and open a new image to count additional samples. For automated counting, convert the image in Fiji to an 8-bit grayscale image by selecting Image, then Type and clicking on 8-bit.

Using the oval tool in the toolbar, draw an oval around the colonies, adjust the oval if necessary. Then select Edit, followed by Clear Outside to remove any interference from outside the area. Now, select Image, then press Adjust and choose Threshold to adjust the sliders in the threshold menu until the colonies appear in red with minimal background noise.

Click on Apply and exit the Threshold window generating a black and white image. Now, select Analyze. Then click on Analyze Particles and specify the range for colony area and circularity in the Analyze Particles window.

Choose Outlines in the show popup menu, enable Display Results for detailed measurements and check Clear Results to erase any previous measurements. Click the Summarize Box and initiate the analyzer by selecting Okay to display the summarized results of the measurements. Observe as a new window appears showcasing all outlined colonies detected and counted.

The details of each colony can be seen in the results window and the total colonies counted in the summarized results window.