Location: Forage-animal Production ResearchTitle: Thin-layer chromatographic (TLC) separations and bioassays of plant extracts to identify antimicrobial compounds
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2013
Publication Date: 3/27/2014
Citation: Kagan, I., Flythe, M.D. 2014. Thin-layer chromatographic (TLC) separations and bioassays of plant extracts to identify antimicrobial compounds. Journal of Visualized Experiments. 85:e51411. doi:10.3791/51411.
Interpretive Summary: Much research in medicine and agriculture is focused on discovering plant natural products that can kill or slow the growth of microorganisms, such as fungi and bacteria. Bioautography is a simple, relatively inexpensive method to screen for plant products with such antimicrobial properties. The method consists of separating extracts of plant material on a planar surface, leading to discrete spots or bands that can then be seen (possibly with the aid of chemical sprays) under visible light or ultraviolet (UV) light. If the separated compounds are then exposed to bacteria or fungi, the microbes will grow everywhere except on antimicrobial compounds. This paper describes some techniques for separating plant extracts and for determining if the extracts contain compounds antimicrobial to rumen bacteria that ferment amino acids.
Technical Abstract: A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungal spores in nutrient solution or bacteria in liquefied agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography, with an application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face-down onto nutrient agar seeded with the test microorganism. Inhibitory bands are visualized by staining plates with tetrazolium red. The method has been applied to the separation and of red clover (Trifolium pratense cv. Kenland) phenolics and their screening for activity against Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium (HAB). The TLC methods apply to many types of plant extracts, and other bacterial species (aerobic or anaerobic) probably can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.