# Gram differentiation of bacteria by acoustic-enhanced flow cytometry

## TL;DR

This paper shows how SYTO 9 dye and acoustic-enhanced flow cytometry can distinguish Gram-positive and Gram-negative bacteria based on fluorescence signals.

## Contribution

The study introduces a novel single-dye flow cytometry method for Gram differentiation using SYTO 9 and acoustic-enhanced cytometry.

## Key findings

- Gram-positive bacteria showed higher fluorescence intensity in the BL3 channel compared to Gram-negative bacteria.
- SYTO 9 staining combined with flow cytometry successfully segregated bacterial species into distinct quadrants.
- The method works for both single and mixed bacterial cultures.

## Abstract

Introduction. Recently, flow cytometry has gained the attention of clinical microbiologists for its ability to characterize bacterial species. This article shows how acoustic-enhanced flow cytometry, combined with the fluorescent dye SYTO 9, can differentiate between Gram-positive and Gram-negative bacteria.

Gap Statement. SYTO 9 is a cell membrane-permeable dye with a high affinity for nucleic acids in both living and non-living prokaryotic and eukaryotic cells and has been used as a counterstain to discriminate between live and dead cells in combination with other dyes. However, the consistency of its cell permeability in different bacterial species and its potential application to Gram differentiation has not been fully considered.

Aim. We sought to assess the suitability of the fluorescent dye, SYTO 9, to differentiate Gram-positive and Gram-negative bacteria by flow cytometry.

Methodology. A range of common Gram-positive and Gram-negative bacterial species were stained with SYTO 9, then processed using an acoustic-enhanced flow cytometer (Attune NxT, Thermo Fisher). The fluorescence emission data were gated and analysed in quadrant plots.

Results. Single and polymicrobial bacterial suspensions stained with SYTO 9 produced different fluorescence signals in Gram-positive and Gram-negative bacteria in the forward scatter-height/blue 3-height (FSC-H/BL3-H) quadrant. Gram-positive species (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pneumoniae and Streptococcus pyogenes) had higher fluorescence intensities in the BL3 channel than the Gram-negative species (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia thailandensis and Proteus vulgaris) in both single and mixed cultures.

Conclusion. The FSC-H/BL3-H quadrant analysis of flow cytometer emission spectra from SYTO 9-stained bacterial suspensions segregated Gram-positive and Gram-negative bacteria into separate quadrants based on their different fluorescence intensities. This provides a single-dye flow cytometry method for Gram differentiation.