Isolation of Naturally
Occuring Pesticides

Developers:

Andrea Martin
Abington Friends School
Jenkintown, PA

Dr. Paul Reibach
Dr. Diana Bender
Rohm and Haas Company
Spring House, PA

Grade
Levels:

Grades 7 - 12

Discipline:

Biology, Environmental Science, Ecology

Goals:

To show that insecticides occur in nature.

Materials:

Mortar and pestle (for plants)
Sand
Acetone
Corn Oil (Mazola�)
4-oz. jars
Drosophila melanogaster
Instant Drosophila food
Cotton
Squirt bottle containing water
1 package of cheesecloth
125-ml Erlenmeyer flasks
7-cm funnel
Gloves
Electric coffee or spice grinder (for spices)

Spices: peppercorns 5 g., fennel seeds 5 g, cumin seeds 5 g.

Plants: Chrysanthemum flowers 10 g, Marigold leaves 5 g,
Marigold flowers 5 g.

Procedure:

1. Make up stock solution of corn oil/acetone: add 0.5 ml corn oil to 100 ml acetone
2. Weigh out 5 grams of each sample - fennel, cumin, pepper, marigold leaves, flowers and a 10-gram sample of chrysanthemum flowers.
3. Processing of Samples
   
   a. Plants
   Put plant sample in mortar, sprinkle some sand over the sample and grind until thoroughly crushed, about 2 minutes.
   
   Add 10 ml of acetone and grind thoroughly, being careful not to splash liquid. If the mixture is too thick, add 5 more ml of acetone. Strain the acetone/extract mixture by pouring though cheesecloth in a funnel on top of a 125-ml Erlenmeyer flask. After most of the liquid has drained into the flask, extract the remaining liquid by forming a bag around the plant material and squeezing (make sure that you wear gloves). The liquid in the flask is your extract.
   
   
   b. Spices
   Grind spice sample in grinder until pulverized. Transfer ground sample to a jar using a spatula. Add 10 ml of acetone and stir mixture well (about thirty seconds). Strain the acetone/extract mixture by pouring through cheesecloth in a funnel, on top of a 125-ml Erlenmeyer flask. After most of the liquid has drained into the flask, extract the remaining liquid by forming a bag around the plant material and squeezing (make sure that you wear gloves). The liquid in the flask is your extract.
   
   
4. Mix 1 ml of the extract with 0.5 ml of corn/acetone stock solution. Pour into a 4-oz jar.
5. Quickly twirl jar in a hood to evaporate the acetone. The oil/extract mixture should coat the sides of the jar.
6. Add dry instant food so that it just covers the bottom of the jar. Carefully add water until the food has been hydrated.
7. Add 20 Drosophila melanogaster adults to the jar and stopper with cotton. They should be awake (see Teacher Note). Record results after 5, 10, 15, 60 minutes and 24 hours.
8. Control: 0.5 ml corn oil and 1 ml acetone. Coat sides of jar and add food.

Extensions:

1. Try different plants and different plant parts.
2. Try different spices.
3. Capture different species of wild Drosophila and test them.
4. Use different types of insects.
5. Try chromatography of extracts.
6. Instead of corn oil use Kimwipes�. A Kimwipe� is saturated with 1 ml of extract. The Kimwipe� is then spread out in the hood and dried for 10 minutes. The Kimwipe� is then placed in the bottom of the jar and saturated with 1.5% sugar (sucrose) solution. Be careful not to get any water on the sides of the jar. Add 20 adult Drosophila melanogaster to the jar and put in cotton stopper. Record results as above.

Safety Considerations:

Teacher should advise students to avoid breathing acetone vapors or getting acetone on their skin.

Teacher Note:

Anesthetize flies ahead of time and place 20 in a fly vial for each extract. Transfer of live flies is best achieved by placing a funnel on the jar with the extract. The fly vial is then tapped to cause flies to drop to the bottom, and the cotton is removed. The vial is quickly inverted onto the funnel. Gently tap the jar to cause the flies to drop into it. (see figure).

Drosophilia Insecticide
Data Sheet

Test Substance_____________Scientist Names:______________________

Initial numbner of flies:____________

	Time in Hours	Dead Flies	% Dead Flies
Study start time
_______________	___________	___________	___________
Observation times
_______________	___________	___________	___________
_______________	___________	___________	___________
_______________	___________	___________	___________
_______________	___________	___________	___________
_______________	___________	___________	___________
_______________	___________	___________	___________
_______________	___________	___________	___________
Visual Symptoms or Comments _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________

Drosophilia Insecticide
Data Sheet

Test Substance_CUMIN_Scientist Names:_Martin, Bender, Reibach_

Initial numbner of flies:___25______

	Time in Hours	Dead Flies	% Dead Flies
Study start time
_1:30 pm _8-8-91_	____0______	____0______	____0______
Observation times
_______________	___________	___________	___________
_2:30 pm _8-8-91_	____1______	____3______	____12_____
_3:30 pm _8-8-91_	____2______	____8______	____32_____
_8:30 am _8-8-91_	___19______	___15______	____60_____
10:30 am _8-8-91_	___21______	___25______	___100_____
_1:30 am _8-8-91_	___24______	___25______	___100_____
Visual Symptoms or Comments _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________