Nile blue A for staining Escherichia coli in flow cytometer experiments

Area of Science:

• Microbiology
• Biotechnology
• Cell Biology

Background:

• Nile blue A is a stain traditionally used for detecting polyhydroxyalkanoic acids in microorganisms.
• Its application for general cell identification in bacteria lacking these compounds is less explored.

Purpose of the Study:

• To evaluate Nile blue A as a general stain for Escherichia coli (E. coli) in fluorescence-activated cell sorting (FACS).
• To assess the impact of Nile blue A staining on cell surface peptide display and subsequent labeling.
• To present Nile blue A as a cost-effective alternative for bacterial flow cytometry.

Main Methods:

• Staining of E. coli cells with Nile blue A.
• Analysis using fluorescence-activated cell sorting (FACS).
• Assessment of peptide surface display and secondary fluorescence labeling post-staining.

Main Results:

• Escherichia coli cells, even those not accumulating polyhydroxyalkanoic acids, were successfully stained with Nile blue A.
• Nile blue A staining did not interfere with the surface display of peptides or their labeling with a second fluorophore.
• The staining enabled clear identification of E. coli cells in FACS experiments.

Conclusions:

• Nile blue A is effective for identifying E. coli in FACS, irrespective of polyhydroxyalkanoic acid accumulation.
• It serves as a simple, low-cost alternative to other fluorescent dyes or intracellular reporters like green fluorescent protein for E. coli flow cytometry.
• This method preserves cellular functions like peptide display, making it suitable for various applications.