I have to determine the dead cells in culture and I have propidium iodide in powder form and I have diluted 2mg with 2mL sterile filtered double Distilled water and to stain bacteria I use 10micro liters of PI to stain 1mL of the bacterial suspension.
Do I need to fix the cells after antibacterial treatment?
Is my PI stain solution is Ok or Do I need to make it more concentrated?
You should use fresh live bacterial suspensions with 0.01%-0.1% Propidium Iodide in culture medium, and analyse them with flow-cytometer immediately. You will see dead bacteria in red channel.
If you really need to fix the bacteria, you should wash the culture twice by PBS before re-suspending and fixing it with 2% formaldehyde.
Fixed bacteria in formaldehyde can be kept in the fridge for weeks, but Propidium Iodide staining will fade, so you should add PI just before (5-10 minutes) acquisition on Flow-cytometry or Analysis on Microscopy.
Hope this is helpful
You should use fresh live bacterial suspensions with 0.01%-0.1% Propidium Iodide in culture medium, and analyse them with flow-cytometer immediately. You will see dead bacteria in red channel.
If you really need to fix the bacteria, you should wash the culture twice by PBS before re-suspending and fixing it with 2% formaldehyde.
Fixed bacteria in formaldehyde can be kept in the fridge for weeks, but Propidium Iodide staining will fade, so you should add PI just before (5-10 minutes) acquisition on Flow-cytometry or Analysis on Microscopy.
Hope this is helpful
Thank you Jitendra Kumar Shandilya can you tell me what will be best to dissolve the PI in?
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