EPS Isolation from Pseudomonas aeruginosa

Contact: Melissa Starkey

Preparation

• Cut cellophane sheets to fit plates. Place in a container with water and autoclave.
• Autoclave centrifuge tubes ahead of time.
• Make ample LB plates.
• Prepare buffers and solutions

1. Place individual cellophane circles onto 50 LB plates, allow to dry.
2. Inoculate 5 mL of LB with frozen stock of bacteria (PAO1 is wild type). Incubate overnight (O/N) at 37^oC in shaker.
3. Plate 100 μL onto each plate. Allow to dry and incubate plates O/N at 37^o
4. Scrape biomass from cellophane (I use a bent pasteur pipette), and resuspend in 250 mL of 0.9% NaCl in a centrifuge tube. Vortex very thoroughly or use a homogenizer (5 min).
5. Spin cells at 20,000g (ideally, but our centrifuge does not go that fast, so use 13,000 rpm) for 1 hour at 4^o
6. Pour supernatant into a 1-L bottle, and add 750 mL of 100% ethanol. Put in refrigerator O/N.
7. Divide into 4 centrifuge tubes and spin as before (1 hr, 4^oC). Discard supernatant and add 190 mL 95% ethanol total to each tube. Spin for 30 to 45 min. The pellet is often very flaky and loose, so remove supernatant immediately. If the pellet is too flaky, try spinning again for a longer period of time. Finally, wash with 190 mL of 100% ethanol.
8. Allow the pellets to dry briefly, and dissolve in 30 mL H₂O. Freeze and lyophilize.

For Nucleic Acid-/Protein-free EPS

1. Resuspend lyophilized EPS in minimum amount of buffer (10mM Tris pH 7, 5mM MgCl₂)
2. Add DNAse and RNAse to 0.1 mg/mL. Incubate 37^oC, 5 hrs.
3. Add Proteinase K to 0.1 mg/mL.  Incubate 37^oC O/N.
4. Phenol extract 2X (Phenol with 10mM Tris, pH 6.7 (I use pH 8.0), 1mM EDTA), followed by 1 chloroform extraction.
5. Precipitate EPS with 3 volumes of ethanol, centrifuge, wash as before.
6. Resuspend pellet in minimum amount of H₂O, lyophilize.