Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa

Materials and Reagents

1. Bacterial culture [Pseudomonas aeruginosa PA14]. This method has also been performed for Escherichia coli (BW25113) and Staphylococcus aureus (ATCC, catalog number: 6538)]
   
2. LB broth Lennox (BD, catalog number: 240230 )
   
3. Ciprofloxacin hydrochloride (Corning Incorporated, catalog number: 61-277-RF )
   
4. Agar (BD, catalog number: 214040 )
   
5. MgCl₂^.7H₂O (VWR International, BDH^®, catalog number: 0288-VBD )
   
6. DI water (distilled water)
   
7. Sodium chloride (VWR International, BDH^®, catalog number: 0241-VBD )
8. Spooled Masterflex peroxide-cured silicone tubing, L/S 14, 250 ft. (Cole-Parmer, catalog number: UX-96407-14 )
9. Masterflex Norprene tubing (A60 G), L/S 13, 50 ft. (Cole-Parmer, catalog number: UX-06404-13 )
10. PVDF barbed Y connector, 3/8" ID, 1/4", 2-3/8", 1-3/4"; Pack Of 10 (Cole-Parmer, catalog number: WU-30703-93 )
11. Barbed fittings, Straight Connector, Clear PP,1/16" ID, 1/32", 25/32", 1/4" (Cole-Parmer, catalog number: WU-30506-00 )
12. Barbed fittings, Reducing Connector, Clear PP,1/8" x 1/16" ID, 3/32", 13/16", 1/8" (Cole-Parmer, catalog number: WU-30506-06 )
13. Barbed fittings, T connector, Kynar, 1/4" ID, 1/8", 1-15/16", 1-5/16"; 10/pack (Cole-Parmer, catalog number: WU-30703-75 )
14. Sterile Petri dishes
15. Microfuge tubes
16. Syringe 60 ml
17. Syringe needle (Gauge 21G)
18. Micropipettes (P1000, P200, P20)
19. Aluminum Foil
    
20. Saline (see Recipes)
    
21. Saline with Mg₂Cl₄^.7H₂O (see Recipes)
    
22. LB agar with Mg₂Cl₄^.7H₂O (see Recipes)
    

Equipment

1. Peristaltic pump (Cole-Parmer, catalog number: EW-07553-80 ) with an 8 channel, 6 rollers, 3-stop Ismatec minicartridge pump head (Cole-Parmer, catalog number: EW-78002-50 )
   
2. Inoculation ports (VWR^® Sleeve Stoppers) (VWR International, catalog number: 89097-534 )
   
3. GeneMate Incubated Shakers (BioExpress, catalog number: H-2000-M )
   
4. General Purpose Laboratory Incubators, SHEL LAB (BioExpress, catalog number: G-1400-1 )
   
5. Nalgene^® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
   
6. Nalgene^® Top Works^TM Aseptic Closure System, Silicone, for Bottles and Carboys, Thermo Scientific (VWR International, catalog number: 2135-8303 )
   
7. Acro^® 50 Vent Filters, Pall Laboratory (VWR International, catalog number: 28143-616 )
   
8. Erlenmeyer Flasks (150 ml)
   
9. Homogenizer (PRO SCIENTIFIC, model: Bio-Gen Pro200 )
   
10. Table top refrigerated Centrifuge (Eppendorf, catalog number: 5418R )
    
11. Spectrophotometer (BD, catalog number: DU700 series )
    

Procedure