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ACTIVE INGREDIENT SCREENING TEST FOR PLANTS

Jane Settle
1993 Woodrow Wilson Biology Institute

INTRODUCTION

There are a million plant species of which only 25 percent have been identified, and only a very small percent have been investigated in any detail. In many areas such as the rainforest the time is running out. In the tropics, where two-thirds of the world's plant species are thought to exist, deforestation is proceeding at a rate estimated at 50 to 100 acres a minute. These and other plants that we may need for our survival, including new sources of treatment for diseases like cancer and AIDS, could be gone forever - before we even know what they are, or what vital roles they might have played. The ethnobotantists or economic botantists collecting plants from the rain forest need a field test to determine possible active ingredients of the collected plants. In this lab students will simulate tests done in the field to determine the active ingredients in plants. This is the first step in a series of biotechnology steps to search for the reactive agents in newly collected plants. Students will be able to test known plants collected locally and also include known herbs for their healing properties. Plants demonstrating clear areas on Petri plates of bacteria would be further screen for the specific ingredients. The presence of clear areas indicates an active ingredient in the plant tested. Active ingredients can include antiseptics, astringents, antibiotics and toxins.

TARGET AGE/ABILITY GROUP:

This could be used by all levels of students from Life Science, to Biology, and including AP Biology (and research projects)

STUDENT/CLASS TIME REQUIRED:

One day for teacher preparation of the inoculum of Escherichia coli. One period for students to prepare their agar plates and plant material discs. The plates will need to be incubated for 48 hours, and then a class period to observe and record results.

MATERIALS:

Teacher preparation the day before the lab: mix up inoculum for the Escherichia coli. Using a notebook paper punch, punch small discs of filter paper and sterilize. Teacher may want to collect various known herbs to be tested. Students can bring them from anywhere including the grocery store.

SAFETY PRECAUTIONS:

Alert students to the fact that although the bacteria is not pathogenic, care should still be taken when used. Sterile techniques should be emphasized.

TEACHER OUTLINE FOR PRESENTATION OF MATERIALS:

The active ingredients in plant material are important in determining whether the plant material has potential active chemicals for further testing. Teachers need to either provide various plant materials or herbal plants. It would be advisable if the students worked in groups of two. The following is a suggested list: yew, golden Alexanders (golden meadow parsnip), parsley, pussy willow leaves and/or bark, wild garlic (wild onion), wild iris, bedstraw, larkspur, blue-eyed grass, penstemon, wild licorice, four o'clock, big bluestem grass, basil, and any local plants and/or house plants. You will need at least 5 leaves from the plant in order to get a concentrated amount of liquid for the sterile discs. Remove the ground up plant material from the mortar by means of a pipette and transfer it to an empty sterile plate. Advanced preparation of materials will require the preparation of Luria-Bertani(LB) broth for the E. coli bacteria (at least 24 hrs. before the lab). Add a loop full of E. coli culture to the broth medium and incubate for 24 hrs. On the morning of the lab prepare the nutrient agar. Take 15 g agar, 1000mL distilled water. Mix and heat until the agar dissolves. Stir constantly to avoid burning. Sterilize in a covered container (cotton plugs), using an autoclave. For an alternative method use a pressure cooker at 15 lb pressure for 20 min.

STUDENT WORKSHEET

ACTIVE INGREDIENT SCREENING TEST FOR PLANT

Objective: To determine if various plant materials contain active ingredients which will inhibit the growth of bacteria.

  1. Preparation Of Petri Dishes:

  2. a. Prepare 3 empty, sterile Petri dishes by marking off the bottom of each Petri dish into quadrants, using a permanent felt-tip pen. Using a code you devise for each plant material, place the letter of that material in each quadrant, near the periphery. Also label the dish with your name, class period and date. See diagram.

    graphic

    Example of Agar Plate.

  3. b. Using a pipette, transfer 1 ml of the inoculum of Escherichia coli to each of 3 sterile Petri dishes. Add 20 ml of the sterile liquid nutrient agar. Gently agitate the plate to diffuse the inoculum and allow to gel and cool.

  4. Preparation Of Plant Materials: (Repeat procedure for each plant)

  5. a. Using a mortar and pestle, grind up the plant material with the addition of a little distilled water.

  6. b. Using a sterile pipette, transfer the liquid from the mortar into an empty sterile Petri dish. Label the Petri dish of each plant material accordingly.

  7. c. Using sterile forceps (that have been flamed in alcohol) place 2-3 sterile disks into each of the liquid plant materials. Allow to soak for one minute.

  8. d. After the agar has gelled, use sterile forceps to carefully place one disk (blot any excess liquid before placing it on the Petri dish) in the correctly labeled quadrant about 2 cm from the outer edge of the Petri dish. Place a control disk saturated with sterile distilled water in the center of each plate.

  9. Incubation: Invert the agar plates and incubate at 35-37'deg.C for 48 hours.

    SAVE THE PLANT MATERIALS IN PETRI DISHES IN THE REFRIGERATOR FOR FUTURE USE.

  10. Observations: After 48 hours, examine the plates with the plant discs and look for zones of inhibition. This is a clear area formed around the disc due to inhibitory action of the substances in the plant material. If present, measure the diameter of this clear area.

For Further Study: Retest the cells found in the zone of inhibition for continued resistance.

STUDENT DATA SHEET

PART A

For measurements of the inhibition zones of the different plant materials, students are to examine the effect of the various plant materials exhibiting active ingredients. Identify the ones that were resistant as slightly sensitive, sensitive and very sensitive. ( no effect =O, slightly sensitive = +, sensitive = ++, very sensitive = +++)

List of Plant MaterialsZone of Inhibition (diameter)Degree of sensitivity
  
  
  
  
  
  
  
  
  


SUMMARY OF DATA

  1. Which of the following plant materials inhibited the bacteria growth?

  2. Which of the following plant materials have no effect on the bacteria growth?

  3. What does the clear zone around the disc indicate in this investigation?

  4. Why is it important to use sterile techniques in this investigation?

  5. What variable factors could affect the zone of inhibition in this investigation?

  6. Why do plants vary so much in their active ingredients? What are the adaptive features of these active ingredients?

SOURCES OF MATERIALS:

All materials and catalogue numbers are from Carolina Biological Supply Company (1-800-334-5551)

Escherichia coli #12-4300

Nutrient agar - Premeasured Packs # 78-9662

Luria Broth Agar #21-6620

Nichrome Wire Inoculating Loop # 21-5826

Disposable Plastic Serological Pipettes #21-4620

Mortar and Pestle #74-2892

Disposable Petri Dishes # 74-1346

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