Need Help: Bacterial Live/Dead Staining (SYTO9/PI) Populations Identical and Overlapping at 0% Compensation on CytoFLEX
Hi everyone,
I am currently running a bacterial growth curve experiment (Faecalibacterium prausnitzii, a strict anaerobic Gram-positive bacterium) using SYTO 9 and Propidium Iodide (PI) for live/dead quantification on a Beckman Coulter CytoFLEX system.
I've hit a technical wall with template setting and gating, and I would appreciate any insights from the community.
Fig 1-a, b, c. Unstained and single-stained group
Fig 2. Sample acquisition (Red PI 70% EtOH gate is defined by the 2nd time single-stained acquisition)
Fig 3. Compensation adjustment of Fig 2 (FITC - 100% PE)
- Bact treatment: No permeabilization was performed on the bacteria. I only compared two different treatments for the PI dead-cell template: direct staining of intact cells versus 70% ethanol inactivation. The other are fresh bacteria at 0, 24, and 48 hours.
- Channel: FITC for SYTO9; PE for PI
- PMT Gains: FITC: ~180, PE: ~180 (Signal peak natively lands around 10^4 for stained samples).
- Threshold: Manual (FSC-A: 3500, FITC-A: 1000, AND logic) to clear medium debris.
- Dye Concentrations: 1.5 µM SYTO 9 + 10 µM PI (Wash-free protocol, as suggested by Beckman application notes for probiotics). Stained for 15 minutes.
- Compensation Matrix: Currently set to 0% for troubleshooting.
-With compensation completely turned off (0%), I ran three independent control groups, but their fluorescence profiles look identical
-When preparing the PI dead template (using 70% EtOH), the non-viable cluster drastically shifted rightward into the viable gate during a repeated run without any parameter changes. Has anyone experienced EtOH fixation causing such aggressive autofluorescence spectral shifts in Gram-positive anaerobes?
-In my 24-hour dual-stained samples, 99% of events remain in the viable region, but the population splits into two distinct dense sub-clusters (one at FITC 10^4 and an elongated one stretching to 10^5). FSC/SSC shows an elongated diagonal pattern. Is it safe to assume these are single cells vs. bacterial chains/doublets rather than distinct physiological states?
It's a big trouble for me, I can't figure out this machine... And anaerobic bacteria staining is also time-consuming. I would appreciate any advice. Thanks so much