A Step-by-Step Guide to Antibacterial Testing of Plants

Studying medicinal plants for their antibacterial properties is what my lab excels at! Today, I share the basic steps to our research process.

Mar 11, 2025

Steps to collecting an herbarium voucher specimen. Select a representative plant sample and flatten it on dry newspaper (a), overlay with the other side of the newspaper and add cardboard (b), add a wooden frame and straps (c), then place the press in open air and check daily for drying (d). Image source.

Medicinal plants have long been used to address infections and inflammation, making them promising sources of new antimicrobial remedies. In 2021, my lab published a protocol describing how to scientifically test these plants’ anti-infective properties. It begins with collecting specimens in the field and preserving them as herbarium vouchers. Next, it explains how to dry, grind, and extract the relevant parts of each plant. Finally, the protocol describes how to test these extracts against common bacteria that cause human disease. By following this approach, researchers can confirm whether traditional plant medicines truly deliver antimicrobial benefits.

Chassagne F, Quave CL. Collection, Extraction, and In Vitro Antibacterial Evaluation of Plants Used in Traditional Medicine. Methods Mol Biol. 2021;2296:19-41. doi: 10.1007/978-1-0716-1358-0_2. PMID: 33977440.

While the protocol is available as a scientific book chapter online, it is unfortunately behind a paywall. So, here I offer a summary of some of the key steps in the process.

Plant Collection

First, obtain all necessary permits from local and national authorities. In the field, locate a healthy and abundant plant population that can handle harvesting. Create a herbarium voucher by choosing a representative sample, photographing it from multiple angles, and documenting essential information, such as location, environment, local name, and notable plant features. Collect required parts—leaves, flowers, fruits, and stems—while keeping each specimen intact as much as possible. Label the material, and press it between newspaper sheets with cardboard and blotters to absorb moisture. Monitor the specimens daily, replacing the blotter paper until each plant part is completely dried, then freeze to eliminate insects. Deposit the preserved specimen at a recognized herbarium.

For biological analysis, gather each part when it is freshest and most suitable: leaves after morning dew evaporates, flowers as they open, seeds once fully ripened, and roots or rhizomes while leaving sufficient material to regenerate. Harvest bark in small strips from non-critical areas of the trunk. Label everything with a collection number, transport it in appropriate containers, and begin drying by the end of the day. This process preserves plant integrity while maintaining detailed records for future scientific research.

(a) Take pictures of the plant specimen to be collected and assign a collection number. (b) If the entire plant is needed, dig around it with a shovel. (c) Collect multiple specimens of the same species and place them in a heavy-duty bag labeled with the collection number. (d) Remove any debris from the plant materials and cut them into small pieces. (e) Spread the cut materials in a drying cabinet rack and allow them to dry for 24–72 hours. (f) Once dried, place the plant material into a labeled paper bag, staple it, and store it at room temperature (20–25 °C). Image source.

Preparing the Bulk Plant Material for Extraction

Remove any debris and rinse if needed. Cut the plant material into smaller pieces and spread it on a well-ventilated surface or place it in a low heat dryer or dehydration apparatus for 24–72 hours until it snaps when bent. Label each batch with an accession number, plant name, parts used, and collection date. Once dried, store the pieces in labeled paper bags, away from sunlight and high humidity. For grinding, use a suitable tool (e.g., Wiley® Mill, coffee grinder, or blender), ensuring the plant is processed to a consistent size. Collect the resulting powder in labeled paper bags. Finally, clean and dry all equipment thoroughly between uses to prevent contamination and rust. Store plant material in airtight containers for long-term preservation.

Preparing a Plant Extract

Measure the ground plant material and place it in a flask. Add solvent (such as ethanol or 80% ethanol in water) at a 1:10 ratio (grams to milliliters), then stir or shake. Cover and label the container. If possible, use continuous stirring for 72 hours at room temperature and away from sunlight. Filter the mixture twice (coarse then fine filter), and keep the filtered liquid. Repeat extraction by adding fresh solvent to the leftover plant material. Combine both filtrates, then remove the solvent with a rotary evaporator at under 40 °C. Record the yield by weighing the dried extract, place it in a labeled vial, and store at −20 °C.

Antibacterial Testing

Liquid culture of bacteria turn cloudy as the bacteria grow. We can measure the cloudiness (optical density) as an indicator for bacterial cell growth in the presence or absence of the plant extract to determine the minimum inhibitory concentration (MIC).

First, grow bacteria on nutrient agar plates and pick a single colony to start a fresh liquid culture, allowing it to grow overnight. Then, place varying amounts of the extract (I like to test a range of 4-256 µg/mL to generate a dose-response curve) into a 96-well plate, add a measured amount of bacteria, and incubate the plate. Monitor any changes in growth using optical density readings. The smallest extract dose that clearly stops bacterial growth is the Minimum Inhibitory Concentration (MIC). To find the Minimum Bactericidal Concentration (MBC)—or the concentration at which the bacteria is killed by the extract components, transfer liquid from growth-free wells onto fresh agar plates. If no colonies develop the next day, the extract is bactericidal at that concentration. By following this approach, it becomes possible to see exactly how much of the plant extract is needed to limit growth or kill bacteria, providing a clearer picture of its antimicrobial potential.

The Takeaway

Identifying plant extracts with antibacterial activity is just the first step in the discovery process. Subsequent testing focuses on isolating and identifying the active compounds, evaluating them against a broad range of resistant bacterial strains, and assessing their cytotoxicity in human cells (an early check for safety). Because each plant tissue contains hundreds to thousands of unique metabolites, there is a vast chemical space to explore!

Want to learn more? Visit the Quave Research Group’s main website for links to our research papers on this topic.

Yours in health, Dr. Quave