A new fluorescence staining assay for visualizing living microorganisms in soil

Area of Science:

• Microbiology
• Soil Science
• Microbial Ecology

Background:

• Assessing microbial viability in soil is crucial for understanding ecosystem functions.
• Traditional methods often struggle with distinguishing live from dead cells in complex soil matrices.
• Autofluorescence from soil minerals and detritus can interfere with microscopic observations.

Purpose of the Study:

• To evaluate 5-(and 6-)Sulfofluorescein diacetate (SFDA) as a vital stain for microorganisms in soil.
• To determine the specificity of SFDA for live microbial cells.
• To demonstrate the utility of SFDA for direct visualization and enumeration of soil bacteria.

Main Methods:

• SFDA (1 mM in ethyl alcohol) was applied to intact soil samples.
• Epifluorescence microscopy was used to examine stained microbial cells.
• Heat-killed Escherichia coli and microbial colonies from natural soils were used for specificity and efficacy testing.

Main Results:

• SFDA successfully stained live bacteria, Saccharomyces cerevisiae, and fungi in soil without interference from soil autofluorescence.
• Heat-killed Escherichia coli did not fluoresce, confirming SFDA stains only viable cells.
• 151 out of 154 colonies from natural soils were stained, and most cultured strains showed positive staining.

Conclusions:

• SFDA is a reliable and specific vital stain for visualizing and quantifying live microorganisms in soil.
• The method is effective in overcoming interference from soil autofluorescence.
• SFDA enables direct microscopic assessment of microbial populations in environmental samples.