Griffitts:Bacterial Staining

Solutions

• LB Broth
• 0.85% NaCl
• 20 mM propidium iodide (in kit in freezer)
• 3.34 mM SYTO 9 (in kit in freezer)

Procedure

• Grow the bacterial cells to an OD₆₀₀ of 0.5
  • If the OD₆₀₀ is > 0.5, dilute appropriately
• Pellet at 13,200 rpm for 1 minute
• Resuspend in 1 mL 0.85% NaCl
• Incubate 10 minutes
• Pellet at 13,200 rpm for 1 minute
• Resuspend in 500 μL 0.85% NaCl
• Add 0.75 μL of 20 mM propidium iodide
• Add 0.75 μL of 3.34 mM SYTO 9
• Incubate in the dark at room temperature for 15 minutes
• Proceed to fluorescent microscopy
  • PI (red) stains dead cells
  • SYTO 9 (green) stains live cells