What is the correct sequence of steps in a typical direct fluorescent antibody (FA) test on a patient-derived slide?

Culture the organism from the patient sample for 48 hours, fix the colonies to a slide, then add fluorescently labeled tertiary antibodies that bind to host complement proteins and image under electron microscope.

Add fluorescent secondary antibody to empty slide, air dry, incubate with patient serum, then visualize under brightfield microscopy after Gram staining.

Place patient sample on slide, add fluorescently labeled primary antibodies, wash, then visualize with fluorescence microscope.

Incubate patient serum with enzyme substrate, perform ELISA plate reading, transfer to slide, then confirm with immunoblot to visualize fluorescence.