DISCUSSION
A CLI for the detection and quantification of E. coli O157:
H7 was successfully developed, optimized, and evaluated. Optimization
involved the determination of the appropriate membrane
and substrate types, antibody concentration, and exposure
time. PVDF membranes (Millipore) were selected for use
with the CLI, because of their durability, ease of use, and good
retention of the substrate color reaction. Furthermore, once
saturated in methanol, the hydrophobic PVDF material appeared
to have a higher affinity for the protein (antigen) attachment.
The antigen transfer step, however, had to be performed
while the PVDF membrane was still moist with 100%
methanol. Up to 20 membranes could be processed in a single
reaction mixture with little risk of antigen cross-contaminating
the membranes. The brief exposure of the colonies to methanol
on the agar surface did not adversely affect their viability,
which allowed subculturing for biochemical and serological
testing and storage.