The Inoculation Method Could Impact the Outcome of Microbiological Experiments

Area of Science:

• Microbiology
• Bacterial Physiology

Background:

• Liquid batch cultures are standard for bacterial research but often contain aggregates, not just single cells.
• These bacterial aggregates exhibit biofilm-like properties, including increased antibiotic tolerance.

Purpose of the Study:

• To investigate aggregate formation in liquid batch cultures of *Pseudomonas aeruginosa*.
• To determine how inoculation methods influence aggregate development and antibiotic tolerance.

Main Methods:

• Assessed aggregate frequency and size using confocal laser scanning microscopy.
• Compared traditional inoculation methods: frozen cultures, agar-grown cells, and liquid cultures.
• Investigated aggregate recruitment mechanisms and the role of exopolysaccharide Psl.

Main Results:

• Inoculation directly from agar plates yielded the most numerous and largest bacterial aggregates.
• Aggregate presence significantly impacted tobramycin tolerance in cultures.
• A 'snowball effect' mechanism, dependent on Psl, was observed where aggregates recruit single cells.

Conclusions:

• Inoculation method is a critical factor influencing bacterial aggregate formation and antibiotic tolerance in liquid cultures.
• Bacterial aggregation can be a significant, often overlooked, variable in microbiological experiments.
• Consistency in inoculation procedures is essential for reproducible research outcomes.