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 multi-channel 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 saquinavir delivery increased the efficacy of the treatment against the microbacterium strains with different drug resistances.
