Begin by adding 200 microliters of bacterial inoculum into each well of a sterile 96-well plate prepared for microscopic analysis. Incubate the plate at 25 degrees Celsius for 24 hours before removing the supernatant with the pipette. Next, carefully add 300 microliters of filter-sterilized deionized water into each well.
Remove the supernatant using a pipette once again. In another tube, prepare a diluted mixture of SYTO 9 and propidium iodide and filter-sterilized deionized water to the respective desired final concentrations. Then add 200 microliters of the mixture into each well and incubate the plate for 20 to 30 minutes at room temperature in a dark setting.
Confocal laser scanning microscopy revealed that biofilm formation significantly varied depending on the strains. A substantial amount of biofilm was found in Acinetobacter junii, Acinetobacter baumannii, and Acinetobacter ursingii with distinct biofilm morphologies.
