Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry

Materials and Reagents

1. Pipette tips (2-200 µl and 100-1,000 µl tips)
   
2. 1.5 ml Eppendorf tubes
   
3. 12 x 75-mm Falcon^® 5 ml uncapped Round Bottom Polystyrene Test tubes (Corning, catalog number: 352052 )
   
4. Bacterial samples for analysis
   Note: The bacteria can be harvested from multiple sources (e.g., liquid cultures, colonies on semisolid agar plates, natural environments). However, it must be taken into account that adaptations in the steps for cell preparation should be considered, depending on the specific source of the cells.
   
5. Absolute ethanol (e.g., Sigma-Aldrich, catalog number: E7023 )
   
6. Sterile deionized water (e.g., Sigma-Aldrich, catalog number: W3500 )
   
7. Sodium chloride (NaCl) (e.g., Sigma-Aldrich, catalog number: S9888 )
   
8. Potassium chloride (KCl) (e.g., Sigma-Aldrich, catalog number: P3911 )
   
9. Sodium phosphate dibasic (Na₂HPO₄) (e.g., Sigma-Aldrich, catalog number: S9763 )
   
10. Potassium phosphate monobasic (KH₂PO₄) (e.g., Sigma-Aldrich, catalog number: P0662 )
    
11. Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D8418 )
    
12. Nile red stain (e.g., Sigma-Aldrich, catalog number: N3013 )
    
13. 1 N HCl and 1 N NaOH solutions for pH adjustments
    
14. Cytometer sheath fluid (e.g., BD FACSFlow^TM solution, BD, catalog number: 342003 )
    
15. Full-strength bleach (for waste disposal)
    
16. Phosphate-buffered saline (PBS; see Recipes for 10x and 1x solution preparation)
    
17. Permeabilization solution (see Recipes)
    
18. Nile red stock solution (see Recipes)
    

Equipment

1. Pipets (e.g., Gilson Pipetman Classic) (Gilson, models: P20 , P200 , P1000 )
   
2. Centrifuge for 1.5 ml Eppendorf tubes (e.g., Eppendorf, model: 5424 , catalog number: 5424000010)
   
3. pH meter
   
4. Spectrophotometer suitable for measurement of optical density at 600 nm (OD₆₀₀)
   
5. FACSCalibur flow cytometer (BD, model: FACSCALIBUR ) equipped with an excitation laser of 488-nm-wavelength and an emission filter with bandwidth of 585 ± 45 nm
   Note: The suitability of other flow cytometers than FACSCalibur (BD, USA) for this assay is subjected to the availability of a proper excitation laser source and of the filter for the emitted fluorescence, as indicated above.
   

Software

1. Flow cytometer acquisition software
   
2. FlowJo [FlowJo, USA]
   

Procedure