I will try!

First: make sure to use 0.2µm filtered growth media if you are running live cells, growth media contain lots of debris. When measuring live cells from culture, it is also  good to dilute the cell culture 1/50 in 0.2µm filtered PBS directly before measurement, or centrifuge and wash like Richard suggested. (I usually perform my experiments with dye in the growth media, but this can be tricky with toxic dyes)

Second: the type of flow cytometer is important. Most flow cytometers I have encountered are made for eukaryote cells, 1000 times larger than bacteria. Therefore, careful adjustment is necessary, for example the default FSC or SSC cutoff can be pre-set above the bacterial peak. There exist flow cytometers that are better for small particles, like the Partec Cube and Apogee A50 Micro, if possible use these instead of your BD FACSAria, LSR / similar usual suspects.

Third: Always use SSC cutoff with bacteria, and set the cutoff low. The bacterial peak is usually part of the noise peak, which is why labelling is crucial. I start my analysis with an SSC/FL dotplot, where the bacterial peak is easy to spot when comparing with negative control.

Fourth: If separation is still hard, use ethanol deactivated bacteria and stain with PI (in the live/dead kit) - this dye is really strong, and you will find the bacterial peak easily after juggling the PI gain. Dead cells are similar size to living, so by comparing with non-deactivated you will know where the peak is.

Good luck with your experiments!

Hey, 

If you wanna use flow cytometry to detect the live/dead bacteria, you may try fluorescent dye. Attached is the related paper"Reliable Detection of Dead Microbial Cells by Using Fluorescent Hydrazides". 

Good luck

Dear Jiansheng,

Thank you very much...

Karuppusamy,

I second Jiansheng's suggestion.  You can also use the LIVE/DEAD BacLight Bacterial kit from Life Technologies.  Their paperwork on this can be found below.

We typically pellet the cells and wash twice with PBS (resuspending and pelleting each time) before adding the components and analyzing using Flow Cytometry.  

https://tools.lifetechnologies.com/content/sfs/manuals/mp07007.pdf

I have extensive experience in viability staining and FCM of bacteria. There exist many possible options to discriminate live/dead cells, such as PI, FDA, TO-PRO-3, the whole SYTOX line, CTC, DiOC2(3) etc. It is important to realize that none of these dyes produce consistent results to the others, and none correlate well with plating for CFU. This is due to the dyes marking indirect signals of cell death like membrane integrity, metabolic activity or membrane polarity. Each individual dye will mark populations differently depending on which drug has been used for inactivation as well. Even with these limitations, FCM can be very powerful, see the attached protocol by Shapiro for some ideas. I have personally had the most success with simple PI staining, especially when looking at physical inactivation like heat or ethanol.

With regards to S. aureus detection: for unspecific bacterial stain, SYTO BC is a good alternative, as well as FM-4-64. DNA stains like DAPI have to be used after inactivation, defeating the purpose of live/dead discrimination. Nile Red A works fairly well for a very low cost alternative, see the other attached article for a protocol.

For specific detection I don't have hands on experience, but I would direct you towards labelled antibodies against S. aureus surface proteins, like protein A.

Good luck!

Dear,

Christer. Thanks for your sharing of research experience.

Christer, we are currently trying to run S. aureus through our flow cytometer, we stained the cells with Live/Dead (syto) but we are having a difficult time with what parameters to use for the cytometer as we have a lot of background, and cannot distinguish our cells from the background. Could you offer any insight? Thanks!

I will try!

First: make sure to use 0.2µm filtered growth media if you are running live cells, growth media contain lots of debris. When measuring live cells from culture, it is also  good to dilute the cell culture 1/50 in 0.2µm filtered PBS directly before measurement, or centrifuge and wash like Richard suggested. (I usually perform my experiments with dye in the growth media, but this can be tricky with toxic dyes)

Second: the type of flow cytometer is important. Most flow cytometers I have encountered are made for eukaryote cells, 1000 times larger than bacteria. Therefore, careful adjustment is necessary, for example the default FSC or SSC cutoff can be pre-set above the bacterial peak. There exist flow cytometers that are better for small particles, like the Partec Cube and Apogee A50 Micro, if possible use these instead of your BD FACSAria, LSR / similar usual suspects.

Third: Always use SSC cutoff with bacteria, and set the cutoff low. The bacterial peak is usually part of the noise peak, which is why labelling is crucial. I start my analysis with an SSC/FL dotplot, where the bacterial peak is easy to spot when comparing with negative control.

Fourth: If separation is still hard, use ethanol deactivated bacteria and stain with PI (in the live/dead kit) - this dye is really strong, and you will find the bacterial peak easily after juggling the PI gain. Dead cells are similar size to living, so by comparing with non-deactivated you will know where the peak is.

Good luck with your experiments!

depending how you kill your cells they may well change scatter. Heat killed cells tend to go lower forward higher side scatter.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20696/epdf

gives you some idea were to look if you got small beads as reference.

and

http://bitc2-preview.sr.unh.edu/TRC/REFERENCES/FC/NebevonCaron2000.pdf

to get some concept of viability. If you kill your cells by radiation they may retain integrity or esterase activity.

Great thank you Christer and Gerhard!