A toluidine blue-membrane filter method for the quantitative staining of bacteria

Area of Science:

• Microbiology
• Analytical Chemistry
• Biotechnology

Background:

• Accurate bacterial quantification is crucial for various scientific and industrial applications.
• Existing methods for bacterial enumeration can be time-consuming or require specialized equipment.
• Developing efficient and reliable methods for bacterial cell concentration determination is an ongoing research area.

Purpose of the Study:

• To develop and validate a novel method for measuring toluidine blue dye uptake by bacteria immobilized on membrane filters.
• To assess the applicability of the developed method for quantifying specific bacterial species, namely Streptococcus cremoris and Escherichia coli.
• To investigate the adsorption kinetics of toluidine blue by bacterial cells using established isotherm models.

Main Methods:

• Bacteria were isolated from solutions onto cellulose acetate membrane filters.
• Filters were stained with toluidine blue at 50°C in a citrate-phosphate buffer (pH 4.0).
• Stained filters underwent destaining, clearing using a 1,4-dioxan and cyclohexanone mixture, and spectrophotometric absorbance measurement at 590 nm.

Main Results:

• The developed method successfully measured toluidine blue uptake by bacteria on membrane filters.
• Bacterial dye uptake for Streptococcus cremoris and Escherichia coli followed the Freundlich adsorption isotherm.
• The method demonstrated the ability to determine the cell concentrations of these bacterial species.

Conclusions:

• A robust and sensitive method for quantifying bacterial cell concentrations via dye uptake has been established.
• The Freundlich adsorption isotherm effectively models toluidine blue uptake by Streptococcus cremoris and Escherichia coli.
• This technique offers a viable approach for bacterial enumeration in relevant applications.