The test microorganism is prepared, usually by growth in a liquid culture medium.
The suspension of test microorganism is standardized by dilution in a nutritive broth (this affords microorganisms the potential to grow during the test).
Control and test surfaces are inoculated with microorganisms, in triplicate, and then the microbial inoculum is covered with a thin, sterile film. Covering the inoculum spreads it, prevents it from evaporating, and ensures close contact with the antimicrobial surface.
Microbial concentrations are determined at "time zero" by elution followed by dilution and plating.
A control is run to verify that the neutralization/elution method effectively neutralizes the antimicrobial agent in the antimicrobial surface being tested.
Inoculated, covered control and antimicrobial test surfaces are allowed to incubate undisturbed in a humid environment for 24 hours.
After incubation, microbial concentrations are determined. The reduction of microorganisms relative to initial concentrations and the control surface is calculated.