Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria

Materials and Reagents

1. Lactococcus lactis IL1403
   
2. Enterococcus faecalis JH2-2
   
3. Lactobacillus casei (ATCC, catalog number: 334 )
   
4. 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) (acid form) (Life Technologies, Molecular Probes^®)
   
5. Luria-Bertani broth (Sigma-Aldrich, catalog number: L3147 )
   
6. MRS broth (Sigma-Aldrich, catalog number: 69966 )
   
7. Citrate (Sigma-Aldrich, catalog number: C7254 )
   
8. Malic acid (Supelco, catalog number: 46940U )
   
9. Pyruvate (Sigma-Aldrich, catalog number: P2256 )
   
10. D-(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
    
11. D-(+)-galactose (Sigma-Aldrich, catalog number: G0750 )
    
12. Electrode filling solution (Orion, catalog number: 900011 )
    
13. Na₂HPO₄ (Merck KGaA, catalog number: 1006559 )
    
14. NaH₂PO₄ (Merck KGaA, catalog number: 106349 )
    
15. HCl (Merck KGaA, catalog number: 100317 )
    
16. NaOH (Merck KGaA, catalog number: 106462 )
    
17. Triton X100 detergent (Merck KGaA, catalog number: 648466 )
    
18. Valinomycin (Sigma-Aldrich, catalog number: V0627 )
    
19. Nigericin (Sigma-Aldrich, catalog number: N7143 )
    
20. BCECF stock solution (see Recipes)
    
21. 50 mM potassium phosphate (KPi) buffer (see Recipes)
    

Equipment

1. Stove Lab Tech (LIB-080M)
   
2. Centrifuge for Eppendorf tubes (Eppendorf, model: 5418 )
   
3. Centrifuge Sorvall ST 16R for centrifugation of Falcon tube under refrigerated conditions (Thermo Fisher Scientific)
   
4. Fluorescence spectrophotometer (PerkinElmer, model: LS55 )
   
5. Thermostatic bath (Lauda Alpha, model: RA8 )
   
6. pH/MV meters (Orion, model: 420A )
   
7. 4 M KCl saturated with AgCl for combination Ag/AgCl pH electrode (Orion, catalog number: 900011)
   
8. 5 ml Quartz cuvette (optical path 1 cm)
   
9. pH microelectrode (Horiba, model: 9669-10D )
   

Procedure