Bacteria Labelling Protocols

 

Hoechst 33342

 

1. Harvest bacteria
2. Add 10 mg/ml Hoechst 33342 to 1ml of bacteria
3. Incubate at 37C for 10 minutes
4. Analyse 50,000 events by flow cytometry
5. Change FSC and SSC to Log scale and adjust Threshold to 200 on BD LSRII
6. Excite at UV or 405nm wavelengths collect emission at 461 nm

Bis-oxonal DiBAC4(5)

1. Harvest bacteria
2. Add 10 mg/ml of Bis-oxonal dye to 1 ml of 0.55 OD of bacteria
3. Incubate for 10 minutes at Room Temperature on a rotating mixer
4. Wash twice in PBS
5. Analyse 50,000 events by flow cytometry
6. Change FSC and SSC to Log scale and adjust Threshold to 200 on BD LSRII
7. Excite with 488nm laser collect emission at 575nm