Effective antigen transfer was based on three characteristics, the first being the ability to support the growth of E. coli O157:H7 to produce enough antigenic mass for visualization by the CLI, the second being the ability of the PVDF membranes to bind antigen from the agar surface without discoloration of the membranes, and the third being the ability of some bacterial cells to remain viable on the agar surface for further characterization.
Coloration of the PVDF membranes by anything other than the substrate reaction is undesirable, because contaminating substances may interfere with interpretation of the CLI results.