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Bacteria

Bacteria can be analysed flow cytometrically when in suspension without fluorescent staining, numerous fluorescent dyes can be used to indicate the presence of bacteria, viability, DNA content and membrane potential.

Labelling bacteria
The presence of bacteria for image analysis or interactions with mammalian cells can be achieved by labelling with FITC, Bis-oxonol dye, DiBAC4(5) or Hoechst 33342.

Viability
Bacterial viability can be assessed by commercial kits but DAPI and PI can be used to determine bacterial viability. This can be used in conjunction with Hoechst 33342. The Live/Dead BacLight kit from Invitrogen uses two nucleic acid dyes, PI to stain dead bacteria and SYTO9, a green nucleic acid stain which will only stain bacteria with an intact membrane. Gram negative and positive bacteria will take up these two dyes to differing degree's and so each species/strain of bacteria has to be titrated to show live (SYTO9+ve), dead (PI+ve) populations of bacteria, as well as a double stained population of unknown viability, see figure. 

DNA content
Bacterial DNA content can be assessed by the use of Hoechst 33342 or PI on fixed bacteria.

Membrane potential
Measurement of bacterial membrane potential by Bis-oxonol is useful in combination with determination of bacterial viability giving a differential analysis.

Fluorescent Protein (FP)
Bacteria can be transfected with FPs including Cyan or CFP,Green or GFP and Yellow or YFP that can be detected by flow cytometry.

Counting
Bacteria can also be counted using the Invitrogen BacLight kit which is also supplied with calibrated 6 mm microspheres at 100 million/ml. Thus 1 ml of the bead solution is also added to 1 ml of bacteria to enable the counting of bacteria per volume, see figure.


  • Labelling protocol 

    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
  • Viability protocol 

    Propidium Iodide (PI) 

    1. Harvest bacteria
    2. Add 5 mg/ml PI 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 488nm wavelengths collect emission at 610 nm

    DAPI

    1. Harvest bacteria
    2. Add 200ng DAPI to 1ml of bacteria
    3. Incubate for 10 minutes at Room Temperature
    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 with UV or 405nm laser collect emission at 461nm
  • DNA content protocol 

    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 wavelength collect emission at 461 nm on linear
  • Membrane potential protocol 

    Bis-oxonal DiBAC4(5) with DAPI

    1. Harvest bacteria
    2. Add 10mg/ml of Bis-oxonal dye to 1ml 0.55 OD of bacteria
    3. Incubate for 10 minutes at Room Temperature
    4. Wash twice in PBS
    5. Add 200ng/ml DAPI
    6. Analyse 50,000 events by flow cytometry
    7. Change FSC and SSC to Log scale and adjust Threshold to 200 on BD LSRII
    8. Excite with 488nm laser collect emission at 575nm
  • Live Dead BacLight Kit (L34856) 

Publication.

VA Gant, G Warnes, I Phillips, GF Savidge. The application of flow cytometry to the study of bacterial responses to antibiotics. J Med Microbiol 39,1475-154, 1993.

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