How can I determine the intact bacterial cell in flow cytometry?

first of all clean your system and filter all solutions in use. Depending on instrument and setup you may struggle to see bacteria on forward scatter but allbut the navios should see them comfortably on side scatter, once you cleaned up everything. Go and buy a clearblue pregnancy test, crack it open and dissolve the blue latex particles deposited on the wick in sterile filtered in 1ml PBS resulting in a faint blue solution. Set trigger on side scatter and display sidescatter versus forward scatter and every other available fluorescence. Insert a sample of sterile filtered water, trigger on side scatter and lower the threshold until you get s rapid increase of events, indicating that you have reached the photon noise level. Now insert a sample with the particles diluted about 1in1000 in filtered PBS. Be prepared to increase the side scatter voltage if possible to position the beads about 2 log from the maximum channel , but you should be able to see a clean cluster of beads. In a well aligned and clean instrument with the suitable forward scatter arrangement the particles should just be visible above noise on the forward scatter. They are about 380nm in diameter but scatter equivalent to bacteria derived from natural environments regarding side scatter. Cells grown in culture might give stronger scatter signals.

Article Standardization in Microbial Cytometry

for some example pictures with the 380nm beads in figure 4 being the second peak from the bottom. You are unlikely to reach that performance on forward scatter but you should on side scatter.

As a model you can stain the particles with fluorescent labelled anti mouse antibody to ensure you can see the level of fluorescent signals you might get from bacteria.

Kieran Mulroney

The previous answers you've received are all excellent advice, and things I might personally recommend to you, but there's a few small points of advice I might add:

You mentioned you've been boiling suspensions of bacteria and not seeing propidium iodide (PI) staining. Depending on the species and strain of the bacteria you are using, the most likely reason for this is that boiling will degrade the cellular envelope to the point that you will be seeing fragmentation of the cells. PI binds nucleic acids, and so, if you fragment and degrade the cells, you will not see significant PI staining in the debris. If you want to heat-kill bacteria, I would personally recommend heating for 60 minutes - 60°C for Gram negative bacteria, 70°C for Gram positive bacteria. You may need to optimise these heat conditions to provide 100% killing, but they are usually sufficient to provide a majority of heat killed cells, that will give you a strong PI signal.

In addition to the information provided, I would emphasise the use of appropriate unstained/stained controls for you experiment. The simplest experiment is to use a freely membrane permeable, nucleic acid fluorophore (such as SYTO® 9, but any dye with the characteristics I've described will do). Prepare one suspension of the bacteria you are interested in that has no stain, and one that is stained with the dye. In the case of SYTO® 9, you will get a strong 488 nm (blue laser) excited signal, emitting around 515 nm (green light). Set aside a block of time (and plenty of test suspension volume) to play around with your cytometer PMT voltages and triggering conditions. Others have made some excellent suggestions where to start. Once you've seen the nucleic acid fluorophore signal, it should help you identify the other parameters (such as the magnitude of your population's forward scatter/side scatter parameters) that will help you develop a generalised experiment template.

In addition to the previous suggestion you've received on filtering your fluids, I'd extend this by suggesting that you not underestimate the usefulness of washing your bacterial cells. We routinely add 2-3 wash steps for suspensions where we suspect heavy non-sample particulate contamination (in the work we do, usually these are clinical sample fluids but many other environmental and culture conditions can be "dirty"). I would recommend centrifuging your test suspension at 9, 800 x g for 5 minutes, carefully removing the supernatant, and then re-suspending in a highly filtered PBS, normal saline or Hank's Balanced Salt Solution. This is usually enough to retain all your bacterial cells, but lose many small particles that may be contributing to background noise in your system.

I hope these suggestions are helpful to you!

Adam B Shapiro

I found this method that might help:

https://www.bdbiosciences.com/documents/Bacterial_Detection_Live_Dead.pdf

Julie Joseph

You could stain your bacteria and then only gate on the +ve events. Just make sure you are not using FITC/AF488 for staining. Alternate strategy would be to use GFP/RFP expressing bacteria. Might not be practical if you intend to do this for diagnostic purposes.

Homa Ahmadi

Many thanks to all researchers for the precious advice and generous response.

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