Process of single cell isolation using LCM (Step 4). (a) Zeiss Laser Capture Microdissection (P.A.L.M.) system used. (b, c) As an example of single bacterial cell isolation from a clinical sample, sputum from a 42 year-old CF patient was treated with sputolysin, proteinase K as previsouly described11, followed by step 3A in this protocol (Live/Dead® BacLight stain, paraformaldehyde fixation, and spreading on to membrane slide). Fluorescent bacteria were observed under 1000x oil immersion objective lens. DIC and fluorescence image overlays before (b) and after (c) single-cell laser micro-dissection and catapulting. Black bars equal 10 μm.