BMCC Biology Testing Horseweed Plant Extracts Results Paper

The plant used: Horseweed Result Acetone Extract Inhibited E. coli (Y or N) N If yes, clearing zone width (mm) Inhibited Staphylococcus e. (Y or N) 0 N Hexane Extract If yes, clearing Inhibited zone width E. coli (Y (mm) or N) 0 N If yes, clearing zone width (mm) Inhibited Staphylococcus e. (Y or N) 0 N If yes, clearing zone width (mm) 0 PLANT LIFE LAB ANTIMICROBIAL SCREEN Testing Plant Extracts for Antibacterial Activity Plants have a long history of use in traditional medicine, and in some cases the efficacy of these traditional remedies has been confirmed by scientific studies. Digoxin for heart rate regulation and the anti-malarial compound artemisinin are two examples. Two of the most important anti-cancer drug discoveries of the 20th century, vinblastine and taxol, are plant secondary metabolites. Given the large number of plant species in existence, there has been much interest in the last half century in screening additional plants for possible therapeutic properties. We will use a very simple filter disk assay to test crude plant extracts for antibacterial activity. This sort of procedure has been used fairly extensively as a first pass assessment of plant antimicrobial potential. This lab will extend over 3 weeks, not necessarily consecutive. Session 1, Extractions You will work in groups of 3-4 for this lab. 1. Bring in a plant sample to be tested. Dried material is preferred, since the high water content of fresh material may result in concentrations of plant secondary metabolites that are too dilute to be effective. Possibilities are:  Surplus dried, pressed specimens that you don’t need for your plant collection.  Herbs and spices from your kitchen.  We will have a few extras on hand just in case. 2. Crush the material to a fine powder or homogenized pulp. Use mortars and pestles, a razor blade, pipet tips or other tools to chop and crush the plant material as well as possible. Fine particles will admit the solvent more readily. 3. For each type of crushed plant matter, place a small amount (no more than 0.5 mL) in each of 2 microcentrifuge tubes. You will extract one with acetone, which will dissolve many hydrophilic compounds, and the other with hexane, a non-polar solvent, which may extract more highly lipophilic compounds. Label the tubes. Working with gloves and goggles in the fume hood, add the appropriate solvent to each tube up to the 1 ml volume mark. Vortex and/or vigorously shake occasionally for 10 minutes. 4. Centrifuge the tubes at maximum speed to pellet solid matter at the bottom of the tube. This may take several minutes. 5. Pipet off the supernatants and transfer to clean labeled tubes. Place your tubes with their caps open in the tube rack at the back of the fume hood. We will allow the solvent to evaporate to concentrate any compounds that were extracted. 6. Clean up. Discard the tubes with the pellets in the waste collection receptacles. 55 PLANT LIFE LAB ANTIMICROBIAL SCREEN Session 2, Filter Disk Assay 1. Redissolve your dried extracts in a minimum volume of the same solvent as before. Try 50 uL. Vortex to dissolve. 2. Use a pencil to label filter paper disks (or squares) so you can later identify your group and the plant extract applied to each disk. 3. Use a pipeter and new tip to impregnate a labeled disk with the appropriate extract, and place in a sterile petri dish to dry. Avoid removing the lid any more than necessary to place the disk inside. You can leave the lid slightly ajar to promote air exchange and evaporation, but we want to block dust to minimize introducing unwanted microbes to the agar plates (Step 5). 4. Impregnate labeled disks with plain acetone or hexane to use as negative controls. Also impregnate one disk with 50 mg/L kanamycin solution as a positive control. Put in the petri dish to dry. 5. When the disks are dry, dip forceps into 70% ethanol, briefly flame over an alcohol lamp, and use them to place the disks on the surface of agar plates that have been spread with a dilute E. coli suspension. Again, avoid opening the lids any more than necessary to minimize the introduction of unwanted microbes. Be sure to place the disks as far from each other as possible. 6. Turn the plates upside down, label with your name or your group name and any other information. Leave for a minimum of 30 minutes to allow diffusion of the extracts before incubation. 7. We will incubate the plates at 37C overnight, then refrigerate. 56 PLANT LIFE LAB ANTIMICROBIAL SCREEN Session 3, Analysis and Results 1. Examine your plate(s). Carefully measure the width (in mm) of the inhibition zone (the area beyond the edge of the filter paper in which the bacterial do not grow). Compare to your positive and negative controls, and evaluate. 2. We will set up an “assignment” in the Lab folder on Blackboard that will ask you to post your results. We will combine everyone’s results into a single table and re-post to Blackboard. In your notebook, discuss your results and those of the rest of the class. – Did yours or any other tested extract have antibacterial activity? – How valuable could this assay be in discovering antibacterial plant compounds? – What are the limitations, and in what ways could it be improved to make a more sophisticated assay? – Would you expect that this in vitro assay would be a good predictor of antibacterial activity in vivo? Why or why not? 57

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