Begin by adding one milliliter of the bacterial inoculum to each well of a sterile polystyrene 12 well plate. Incubate the plate at 25 degrees Celsius for 24 hours. Then, remove the supernatant using a pipette.
Carefully add 1.5 milliliters of sterile phosphate buffered saline or PBS to each well, and again, remove the supernatant with the pipette. After adding one milliliter of sterile PBS to each well once more, use fitted cell scrapers to scrape off the bottom and wall surfaces of the wells. Transfer the harvested cell suspension to a sterile plastic tube marked as 10 to the negative one containing nine milliliters of saline and 10 to 20 glass beads.
Vortex the tube for 60 seconds at maximum speed. Next, perform a tenfold serial dilution by transferring one milliliter of the cell suspension from the previous tube to another sterile tube marked 10 to the negative two containing nine milliliters of saline solution. Continue this process of serial dilution up to tube 10 to the negative seven.
Spread 100 microliters from each dilution onto separate acinetobacter agar plates. Incubate the agar plates at 30 degrees Celsius for 24 to 42 hours. Then count the number of typical red colonies on each plate manually, considering those within the range between 10 and 250.
All the acinetobacter isolates except for A.bouvetii had cells equivalent to seven to eight log CFU per well in their biofilms. While A.bouvetii had a much lower cell number at 4.4 Log CFU.
