Fixation procedures for flow cytometric analysis of environmental bacteria

Area of Science:

• Environmental microbiology
• Analytical chemistry

Background:

• Single-cell analysis of environmental bacteria requires effective fixation methods.
• Current methods often struggle with autofluorescence, cell clogging, and surface distortion.
• Fixation must accommodate both aerobic and anaerobic bacterial growth conditions.

Purpose of the Study:

• To develop and evaluate a novel fixation method for environmental bacteria.
• To ensure the method preserves cellular integrity and minimizes analytical artifacts.
• To assess the suitability for diverse bacterial communities and growth conditions.

Main Methods:

• A fixation method combining metal salt solutions (Al, Ba, Bi, Co, Mo, Ni, W) and sodium azide was developed.
• Flow cytometry was used to measure DNA content as a marker for bacterial population/community stability.
• Statistical equivalence testing was employed for reliable flow cytometric data evaluation.
• Tests were conducted on pure cultures and activated sludge samples.

Main Results:

• A mixture of 5 mM barium chloride, 5 mM nickel chloride, and 10% sodium azide proved effective for all tested bacteria.
• The fixative demonstrated good sample stability for at least 9 days.
• The method successfully counteracted issues like autofluorescence and cell distortion.

Conclusions:

• The developed metal salt and sodium azide fixation method is suitable for preserving diverse environmental bacteria.
• This technique enhances the reliability of single-cell analysis, particularly using flow cytometry.
• The method offers improved sample stability, facilitating more flexible experimental designs.