Question

I am trying to count populations of cells in a co-culture. One organism is Pichia pastoris and the other organism is a gram-negative bacterium. Pichia stains gram-positive using crystal violet and the bacterium is negative. These two populations are reasonably distinct in front and side scatter but not so much that I can gate the entire population. I can pick a region in FSC-SCC for each organism that I know will only include that organism, which is 'good enough' for my purposes, but I'd love to know if there is a quick, easy, cheap way to distinguish these two populations more decisively. To clarify my requirements:

Cheap: Ideally something widely available in the price range of <$1/sample e.g. Hoechst/Van-Bodipy/WGA-647
No processing steps: I just want to add the dye/stain to my sample automatically using the flow cytometer before running the samples
Things I have considered but have ruled out:
Antibodies: They are too expensive for my case. I would need a custom conjugated antibody or to add processing steps between primary and secondary antibody that is too complicated.
Tracker/tracer dyes (add to one cell population before mixing the co-culture together): Also expensive, and I will probably want to track my population over more generations than most of these dyes work for.
Intracellular markers that require permeabilisation: I don't want to do any processing
Things I'm considering and would welcome input on:
Conjugated lectins, specific to glycoproteins on Pichia pastoris
DNA dyes, using the fact that Pichia genome is approx 2x larger than the bacterial genome
Differing autofluorescence: There's nothing obviously different here from my data so far, but I will go back and tweak the voltages etc. if this is a very promising avenue
Any combination of stains/techniques that are not specific alone, but then the populations can be separated using e.g. PCA
Is there anything out there that will work for me? It seems to me that the cell types are so different there must be an easier solution here than expensive antibodies etc. An answer of No would be just as valid as yes! I can continue just using front and side scatter if the only solutions are going to be too complicated.

Answer 1

You could use negative staining using India ink or nigrosin to capitalize on the distinct capsular properties of Pichia pastoris to differentiate it from gram-negative bacteria in a co-culture. Viability stains which distinguish live and dead cells and autofluorescence adjustments are other avenues to explore.

Considering your requirements for a quick, easy, and cheap method to distinguish between Pichia pastoris and gram-negative bacteria in a co-culture using flow cytometry, I would suggest exploring the use of differential staining techniques that can be applied directly without additional processing. Specifically, you could utilize the principles of negative staining to capitalize on the unique capsule properties of certain yeasts and bacteria. For example, staining with India ink or nigrosin can create a halo effect around encapsulated cells like Pichia pastoris, distinct from gram-negative bacteria which would not produce a halo.

Differential staining such as the viability stains that differentiate live and dead cells based on their membrane integrity might also offer a potential solution. Live cells could be stained green, while dead cells would appear red. However, this approach may not exclusively distinguish between your two cell types unless there are differences in membrane integrity that can be exploited. Lastly, experimenting with autofluorescence by adjusting flow cytometer voltages might reveal differences not immediately apparent. The difference in the genomic size could potentially be leveraged using DNA-binding dyes. Keep in mind that the sample concentration and processes should keep costs below your threshold.