Hi Marjan,

You need to run controls that are single stained for pi and single stained for syto9 and then use these values to perform compensation to eliminate Chanel bleed through. There is considerable spectral overlap between the 2 markers.

Hi Marjan,

I absolutely agree with Lora Benoit. Indeed, you can do live vs. dead cell staining for bacteria by that kit. But you have to run a control sample first, then you can proceed with the compensation.

Also, you can try HOECHST3342, if your machine permits.

Pi channel and syto9 generally not overlap. But still, you can try what Lora told you. or you can use HOECHST3342.

Best,

Utsav

Lora Benoit Hi Lora, thanks for your advice. Could you please explain more about applying compensation. I will run live and dead cells using PI and SYTO individually, (4 samples in total), then do I need to play with voltage to be able to separate the signals of each stain on a histogram plot for example?

Utsav Sen thanks for your response. HOECHST3342 is an alternative stain for syto9, right? is it common to be used for bacteria as well? I see mostly used for eukaryotic cells

Hi Marjan,

Most flow cytometers have settings that allow you to compensate. Compensation is separate from voltage, basically, the electronics allow you to substract syto9 bleed through in the PI channel and PI from the syto9 channel so that you can see the separate populations a quick explanation can be found here: https://www.bio-rad-antibodies.com/flow-cytometry-fluorescence-compensation.html

A 40 min tutorial can be found here https://www.youtube.com/watch?v=uV6rkOs8H50

Lora Benoit thank you so much for the helpful info.

Lora Benoit did you try to see the picture with a fluorescent microscope instead of a flow cytometry? Do you have an idea which one is better for the visualization of live and dead bacterial cells?

Never looked under the microscope to discriminate live/dead. If I did not have a set up for flow, I would look at colony counts post treatment as an alternative.

Lora Benoit Can I use only DAPI stain for visualization? I know it will dye the dead cells but what about the live cells too?

I had Live and dead stains (SYTO9 and PI) and I always use a fluorescent microscope for visualization but I ran with PI; Can I instead mix DAPI with SYTO9 and what will be the ratio?

Thanks