Determination of antibacterial activity 

For determination of antibacterial activity of natural products various methodological approaches are used. Among the most reliable methods, we use:

  • Radial diffusion test
  • Microdilution test based on the minimal inhibitory concentration (MIC) of the tested honey sample

The radial diffusion test is a qualitative test that uses the diffusion process of tested antimicrobial / substance mixture through an agar matrix in which bacterial cells are incorporated, resulting in zones of inhibition. This method is particularly suitable for testing lower molecular weight compounds such as antibiotics or isolated antibacterial agents.

For complex natural products, such as honey, with multiple mechanisms of antibacterial action, the diffusion test is not an appropriate choice as it does not take into account the overall antibacterial potential of the natural product (honey). 

Inhibition zones on agar matrix - radial diffusion test
Inhibition zones on agar matrix - radial diffusion test

The microdilution test is a quantitative test that provides more accurate and more comprehensive results than diffusion tests. This test serves to determine the MIC value, the minimal inhibitory concentration that is able to stop bacterial growth. Testing is performed in 96-well plates, diluting the test substance / product with liquid culture medium at various concentrations. Bacterial growth is manifested by turbidity, and when growth is stopped, the well is clear (turbidity does not form). This methodology is suitable for various antimicrobial substances, including antibiotics, but also complex natural products with several effective antibacterial substances, such as honey.

Diluted sample and determination of the minimum inhibitory concentration (MIC) in 96-well plate
Diluted sample and determination of the minimum inhibitory concentration (MIC) in 96-well plate

Determination of hydrogen peroxide concentration

The antibacterial effect of honey does not depend only on the activity of one active ingredient, as in the case of antibiotics, but is based on the action of various substances and mechanisms that result in the multifactorial antibacterial action of honey. When honey is diluted, hydrogen peroxide becomes the main antibacterial factor. The production of hydrogen peroxide and its accumulation in diluted honey is extremely important with regard to its biological properties. Hydrogen peroxide not only plays a role in the antibacterial effect of honey, but also participates in the regeneration process in wound healing. After neutralization of hydrogen peroxide in diluted honey by the action of catalase enzymes, the total antibacterial activity also decreases significantly.

A commercially available test kit (GOX assay kit from Megazyme, Ireland), which has been optimized for honey testing, is used to determine hydrogen peroxide concentration. The honey to be tested is first diluted with buffer, creating a 40% (w / w) honey solution, and incubated for 24 hours in the presence of peroxidase. 

Determination of physicochemical parameters

In the Honey laboratory, we also determine the basic physicochemical quantities of honey, which include the pH value and electrical conductivity (conductivity). Both parameters are determined using calibrated digital instruments.