An antimicrobial substance is a substance which kills microorganisms or inhibits their growth. In the last few decades, more and more different strains of microorganisms appear. Due to their genetic ability, they became more resistant to antibiotics.
Thus, there is a need for investigation and search for new antimicrobial substances, especially from natural sources such as plants. We need reliable methods to determine if a certain plant extract is an effective microbial agent.
Here, we’ll discuss two methods of determining antimicrobial activity in plant extracts. The first method is the measurement of optical density of a solution after incubation. The second one is the disk diffusion method.
The first method requires a clean culture of some bacteria such as E. coli. It’s necessary to make a calibration curve with solutions of known concentrations of bacteria. This represents the relation between the number of microorganisms and the solution’s absorbance.
Next, the bacteria solution is incubated alone and with plant extract added at 37ºC. Absorbance measurements on spectrophotometer of bacteria alone and bacteria with added plant extract are recorded each hour during the incubation time. Results will show growth of bacteria culture during the time and the percentage of inhibition if present.
In the disk diffusion method, the clean culture of E.coli is seeded on Nutrient Agar plate. Filter paper disks are impregnated with extracts in several dilutions. These are then placed in seeded Nutrient Agar plates and incubated for 24 hours at 37ºC.
One thing to keep in mind is that each experiment should include controls with antibiotic-impregnated discs and solvents of extract. After incubation, growth inhibition zones should be measured in incubated plates and compared to the controls.
Jorgensen, J. H., Ferraro, M. J., Jorgensen, J. H., & Ferraro, M. J. (2009). Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America, 49(11), 1749–55.