Categorized: Microbiological Techniques
- Grow bacteria to an OD620 of 0.8.
- Wash 3x in PBS containing 2% FCS.
- Resuspend to the original volume with 1mM MgCl2. Dispense 0.8 ml into Eppendorf tubes.
- Add monoclonal or polyclonal serum and vortex (3 on Vortex Genie Fisher)
- Incubate for 45 min. at 37ºC with agitation.
- Wash 3x with PBS containing 1% FCS, 1 mM MgCl2. (Microfuge for 1 min)
- Incubate at 37ºC for 45 min in PBS containing 2% FCS and a 100-1000 fold dilution of rabbit anti-mouse IgG.
- Repeat step 6 and then step 7 this time with goat anti-rabbit Ab coupled to FITC.
- Wash with PBS.
- Add 10 µl to a slide. Add a cover slip and with a fluorescence microscope measure the fluorescence emitted at 525 nm.
Another method uses bacteria fixed on slides. For each incubation step place the slides in a humid chamber for 10 min. at 37C. The slides are washed by dipping in PBS.