ACTIVE INGREDIENT SCREENING TEST FOR PLANTS
1993 Woodrow Wilson Biology Institute
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.
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.
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.
ACTIVE INGREDIENT SCREENING TEST FOR PLANT
Objective: To determine if various plant materials contain active ingredients which will inhibit the growth of bacteria.
- Preparation Of Petri Dishes:
- Preparation Of Plant Materials: (Repeat procedure for each plant)
- 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.
- 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
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 Materials||Zone of Inhibition (diameter)||Degree of sensitivity|
| || |
| || |
| || |
| || |
| || |
| || |
| || |
| || |
| || |
SUMMARY OF DATA
- Which of the following plant materials inhibited the bacteria growth?
- Which of the following plant materials have no effect on the bacteria growth?
- What does the clear zone around the disc indicate in this investigation?
- Why is it important to use sterile techniques in this investigation?
- What variable factors could affect the zone of inhibition in this investigation?
- 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
The Woodrow Wilson National Fellowship Foundation
CN 5281, Princeton NJ 08543-5281