Encyclopedia of Experiments: Immunology
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Transcript
Take a microtiter plate with a lid featuring cylindrical supports, or pegs, coated in bacterial biofilms.
Place the biofilm-coated peg-lid onto microtiter plate wells with buffer to eliminate loosely attached or planktonic bacteria, focusing on biofilm-attached measurements.
Transfer the rinsed biofilm-coated peg-lid to a challenge plate with increasing test antibacterial agent concentrations. Incubate without shaking.
Depending on the agent's concentration and antibacterial potential, it disrupts the bacterial metabolic activity and biofilm integrity.
Post-incubation, place the peg lid onto buffer-containing plate wells to remove planktonic bacteria.
Transfer it to microtiter plate wells with a biofilm recovery medium containing resazurin, a cell-permeable, weakly fluorescent dye. Incubate.
In metabolically active bacteria, dehydrogenase enzymes reduce blue-colored resazurin into pink-colored, fluorescent resorufin.
Lack of resazurin conversion indicates biofilm eradication, while conversion to resorufin suggests the presence of viable cells.
The lowest test antibacterial agent concentration yielding no resazurin conversion denotes the minimal biofilm-eradicating concentration.
