Procedure for Rhamnolipids Quantification Using Methylene-blue

Materials and Reagents

1. Borate Buffer Packs BupH^TM (Thermo Fisher scientific, catalog number: 28384 )
   
2. Centrifuge round bottom reaction tubes (30 ml) (Krackeler Scientific, COREX^®, catalog number: 1-8445-30 )
   
3. Greiner CELLSTAR^® 96 well plates (clear polystyrene wells flat bottom) (Sigma-Aldrich, catalog number: M3687-60 EA )
   
4. Microcentrifuge tube [2 ml, transparent polypropylene (PP) with Lid] (Sigma-Aldrich, Brand^®, catalog number: Z628034-500 EA )
   
5. Sterile culture tube (PP) (14 ml) (The Lab Depot Inc., catalog number: TLDT8235 )
   
6. Centrifugal Filter (0.1 µm pore size, Non-Sterile) with Durapore^® PVDF Membrane UFC40VV25 Ultrafree^® (2 ml) (Merck Millipore Corporation, catalog number: UFC40VV25 )
   
7. Aluminium foil
   
8. Micropipettor (10 µl,100 µl and 1,000 µl) tips
   
9. Pseudomonas aeruginosa (P. aeruginosa) PAO1 Wild-type (strain PAO0001) (http://www.pseudomonas.med.ecu.edu/)
   
10. Chloroform (≥ 99% ) (Carl Roth GmbH & Co. KG, catalog number: 3313.1 )
    
11. Ethyl acetate (> 99.5%) (Merck Millipore Corporation, EMSURE^®, catalog number: 1096231000 )
    
12. Hydrochloric acid (HCl) standard (1 N solution) (Sigma-Aldrich, Fluka analytical, catalog number: 318949-500 ml )
    
13. HCl standard (0.2 N solution in water) (Sigma-Aldrich, Fluka analytical, catalog number: 343102-500 ml )
    
14. Luria Bertani (LB) Broth (Lennox) Powder microbial growth medium (Sigma-Aldrich, catalog number: L3022-250 G )
    
15. Methylene blue solution [1.4% (w/v) in 95% ethanol] (Sigma-Aldrich, catalog number: 1808-50 ml )
    
16. 3-(N-morpholino) propanesulfonic acid (MOPS) (99.5%) (Sigma-Aldrich, catalog number: M1254-100 G )
    
17. Rhamnolipids (90%, Standard) (Sigma-Aldrich, catalog number: R90 )
    
18. Sodium hydroxide (NaOH) solution
    
19. LB-MOPS broth (see Recipes)
    
20. Borax buffer (50 mM) (see Recipes)
    
21. Methylene blue aqueous solution (see Recipes)
    

Equipment

1. Micropipettor (100 µl-1,000 µl)
   
2. Micropipettor (10 µl-100 µl)
   
3. Autoclave
   
4. Stopwatch
   
5. Measuring Pipette (5 ml) (Jaytec Glass Limited, catalog number: WJ.485 )
   
6. 96-well microplate spectrophotometer (e.g. Molecular Devices, model: SpectraMax M2 device )
   
7. Vacuum concentrator (e.g. Savant SPEEDVAC SVC 200H: 200 x g)
   
8. Tabletop centrifuge
   
9. Vortex mixer
   

Procedure