The agar microdilution method - a new method for antimicrobial susceptibility testing for essential oils and plant...

Area of Science:

• Microbiology
• Natural Product Chemistry
• Pharmacology

Background:

• Increasing antibiotic resistance necessitates exploring new antimicrobial agents, with natural products like plant extracts and essential oils showing promise.
• Standardized and reliable methods for assessing the antimicrobial activity of these complex natural products are lacking.
• Existing methods often struggle with hydrophobic or colored substances, hindering research.

Purpose of the Study:

• To develop and validate a new agar microdilution technique for evaluating the antimicrobial activity of natural plant products.
• To assess the antimicrobial effects of various essential oils and plant extracts using the developed method.
• To address limitations in current methods for testing water-insoluble or colored antimicrobial agents.

Main Methods:

• The study adapted the Clinical and Laboratory Standards Institute (CLSI) agar dilution method for a 96-well microplate format.
• Essential oils and plant extracts were incorporated into molten Mueller-Hinton agar, vortexed, and dispensed into microplate wells.
• The method ensured uniform dispersion of hydrophobic/colored agents and was tested with four reference bacterial strains to determine minimal inhibitory concentrations (MICs).

Main Results:

• The developed agar microdilution method successfully determined minimal inhibitory concentrations (MICs) for hydrophobic essential oils and colored plant extracts.
• The technique provided a uniform and stable dispersion of tested agents, preventing phase separation common in liquid media.
• Preparation of samples was rapid, easy, and economical, combining the convenience of microtitre methods with agar dilution advantages.

Conclusions:

• A new, reliable, cost-effective, and user-friendly agar microdilution method has been established for testing the antimicrobial activity of natural plant products.
• This method effectively overcomes challenges associated with testing water-insoluble, oily, or strongly colored natural antimicrobial agents.
• The validated method facilitates research into plant-derived antimicrobials, crucial for combating rising antibiotic resistance.