General guidelines for sorting bacteria with fluorescence-activated cell sorting (FACS)

FACS is a powerful tool for high-throughput analysis and manipulation of complex populations. It was designed for use with eukaryotic cells. For this reason, being able to use FACS to sort bacteria is more of a perk than a feature. For example, commonly cultured eukaryotic cells range from 10-100μm in diameter, while bacteria are typically ~1μm. These differences may seem small, but a eukaryotic cell that is 10μm in diameter has 100x greater surface area than most bacteria, and 1000x greater volume. For this reason, there is a blurry line between what is noise and what are actually our cells of interest when working with bacteria. However, complex bacterial populations can be analyzed and sorted with FACS.

This is not supposed to be a broad guide on how to use a FACS machine, since there is plenty of readily available information made by people who use these tools to study big cells. Instead, this will cover some techniques that will help you work with bacteria.

General guide for flow cytometry and bacteria

Buffer for suspensions - Phosphate-buffered saline (PBS) or saline will work fine for this. Some people add a small amount of glucose to their buffer to help their cells survive. For E.coli, this isn't really necessary but may help you if you are having issues with cell viability. Since bacteria are so small, noise may be reduced by filtering your buffer with a 0.22μm filter. This is probably unnecessary, but it can help to reduce noise if your lab has debris in the bottles containing your buffer.

Gating bacteria and minimizing noise - This varies from machine to machine, but side-scatter can be used to resolve differences between cells when working with cells that are small. Forward scatter typically does not offer the same kind of resolution.

• Set threshold on SSC and adjust voltages for SSC and FCS. On an SSC-A vs FCS-A plot (log on both scales), you should see a population when running sample containing bacteria once your voltages and threshold are set right.
• Now, run a blank sample containing only buffer. Adjust voltages/threshold to limit any noise: 100-1000 events per second of noise is acceptable and should not significantly affect your measurements if you keep them out of your population gate. Iterate between this blank sample and the sample containing cells until you have found the right parameters that limits noise while allowing you to get 3,000-10,000 events per second when running a sample with cells.
• Once you have the ideal parameters, finish gating. Gate your population of cells on SSC-A vs FCS-A into a second plot with FCS-H vs FSC-A (both on log again). Gate singlets.

Optimizing sample preparation to avoid swarming

Swarming is a problem that occurs when multiple cells are being interrogated by lasers at the same time. When this occurs, the machine will read these as a single cell. This is mostly an issue for scientists who work with extracellular vesicles or bacteria, since they are small. What does swarming cause, ultimately? For cell sorting, swarms of cells can cause hitchhiking cells to be sorted by accident just because they were adjacent to the cell of interest. You will grow these cells and find out that your sorting was very ineffective. The way to avoid this is to determine the optimal dilution that does not cause swarming. Once cells are dilute enough, they are unlikely to swarm unless you are working with a strain that clumps or does not form single cells. You will need to perform a dilution series and track changes in events per second. Events per second should decrease linearly in relation with dilution within the correct range. If swarming occurs, this relationship will break down—for example, you run a sample 5-fold dilute compared to the last sample, but the events per second decrease only by about half instead of one-fifth. Or worse, the events per second increases.

Performing dilution series

1. If you are growing cells in an autofluorescent media like LB, you will need to wash the cells. Pellet 1mL of stationary culture for at 5k rcf for 5 minutes, decant LB with a pipette tip and resuspend with 1mL of your buffer. If you are growing cells in minimal media like M9 (even with casamino acids), you do not need to wash your cells and can directly add them to your dilution series.
2. Prepare tubes with different volumes of your buffer and make the dilution series of your samples. Begin at a dilution of 10x (adding 100uL of culture to 900uL of buffer), and finish at ~1000x or more. You will have to perform serial dilutions to get a good range. In my experience, a 100x dilution seems to work OK when working with E.coli grown in M9 (0.4% glucose) or ADP1 grown in LB. Include a tube with no bacteria added to know what the background events per second are for your dilution.
3. Prepare a spreadsheet with your dilution series and empty cells for events per second data.

Running samples to determine linear range - Make sure you have set up your parameters to identify cells and limit noise before running your dilution series and recording the events per second rates

1. At the FACS machine, run your buffer-only sample. Record the events per second on your spreadsheet. This is the background events per second.
2. Now, run each of your dilutions, recording the approximate events per second on the spreadsheet. Maintain the same speed and parameters throughout this.
3. Once you have finished your series, create a graph on excel to see the linear range. At high cell densities (10x dilution), it should dip or flatten out.
4. Pick one of the dilutions that remains in the linear range while still having plenty of cells to work with.

Now you know how much to dilute your cells.

General guide for sorting bacteria

• Using bacteria with an antibiotic resistance marker (plasmid or genomic insertion) is advisable, because it is easy to get contamination when sorting cells. Especially if you are performing an evolution experiment that involves sorting, growing, and sorting iteratively.
• Make sure to sort into tubes made of polypropylene. Droplets stick on polystyrene and may not enter the media. These should contain media (saline, LB, or whatever) for your cells to directly fly into. Running through a small tube is probably very stressful (especially twice, see enrichment sorts below), so having some nutrient in the media will help your sorted cells remain viable.
• The nozzle size shouldn't matter so much, as long as your cells are not swarming. A 100μm (common size) nozzle will work fine as long as you do not sort too fast.
• When actively sorting, aim for ~5k events total events per second.
• Some machines allow you to change the sorting stringency. This is advisable. For example, the Sony MA900 has an ultra purity mode that rejects ~20-50% of droplets which do not meet the sorting criteria. This can be adjusted to purity or semi-purity mode, or yield mode if purity is not important.

Enrichment sorts - If your sorts are not working well, you can perform a 2-step sort to achieve sorts with higher 'purity'.

1. Sort a large amount of cells from your population of interest with no rejects at a high rate into an empty tube. Aim for 10-100x the final number of cells you want to sort.
2. Once you have finished sorting the first time, load the SORTED cells (now further diluted in sheath fluid) and begin sorting again, this time with a high stringency. You may have to bump up the speed rate due to the dilution caused by sheath.
   • The 2nd sort should go into a tube with media.

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