1. Mold Control: Toxicology Experiment Activity Overview Disciplines Covered: Science & Math Prep Time: 60-90 minutes Activity time: 2 class periods, plus incubation time (about 3-5 days)

2. Mold Control: Toxicology Experiment The Dose Makes the Poison LOEL LD100 LD50 100 0 % Mortality LD 50: Lethal Dose 50% LD 100: Lethal Dose 100% LOEL: Lowest Observable Effect Level

3. Mold Control: Toxicology Experiment Serial Dilution Schematic

4. Mold Control: Toxicology Experiment Serial Dilution Preparation Teacher: Yeast dilutions Three batches of 10 -5 yeast solution Students: Cleaners- 100% to 0.0001% Group A – Lysol Kitchen Antibacterial Cleaner Group B – Tilex Mildew Remover Group C- Borax

5. Mold Control: Toxicology Experiment Yeast Serial Dilutions

6. Mold Control: Toxicology Experiment Add yeast solution to film

7. Mold Control: Toxicology Experiment Lay film cover down

8. Mold Control: Toxicology Experiment Press with spreader

9. Mold Control: Toxicology Experiment 1 ml spread on film

10. Mold Control: Toxicology Experiment Incubate in bag

11. Mold Control: Toxicology Experiment Petrifilm after Incubation

12. Mold Control: Toxicology Experiment Part I: Serial dilutions Working in pairs, you have 10 minutes to complete this task: 1.Practice using the disposable pipette with colored water. 2.Create a 10 -6 dilution of yeast a. Mix the 10 -5 yeast dilution well. b. Using a pipette, take 1 ml from 10 yeast dilution. c. Add this to the 9 ml of water in the blank tube. d. Label this new dilution “10 -6 ” e. Mix well.

13. Mold Control: Toxicology Experiment Part II: Petrifilms You have 5 minutes to complete this task. Working in pairs: 1. Mix your 10 -6 dilution well. 2. Draw 1 ml of your solution into a NEW disposable pipette. 3. Take one 3M Yeast and Mold petrifilm. 4. Lift the plastic layer. 5. Squeeze out the 1 ml into one large drop in the middle of the grid 6.Lay the plastic layer back down. 7.Place the spreader over the middle of the drop. 8.Press down with firm pressure for about 5 seconds.

14. Mold Control: Toxicology Experiment Potato Dextrose Agar Plates An alternative to 3M Yeast and Mold Petrifilms are the traditional agar plates. Potato dextrose agar is used because it is a good medium for growing yeast. The alternative student and teacher materials are available on the Hydroville website: www.hydroville.org

15. Mold Control: Toxicology Experiment Yeast solution and plate

16. Mold Control: Toxicology Experiment Draw up about 0.5 ml

17. Mold Control: Toxicology Experiment Add 4 drops to plate

18. Mold Control: Toxicology Experiment Spread with back of spoon

19. Mold Control: Toxicology Experiment Liquid on agar

20. Mold Control: Toxicology Experiment Incubate upside down

21. Mold Control: Toxicology Experiment Agar plate after Incubation Controlvs. 100% Lysol

22. Mold Control: Toxicology Experiment Counting Colonies

23. Mold Control: Toxicology Experiment Part III: Data Analysis and Interpretation You have 10 minutes to complete this task. 1.Work together to count the colonies on the petrifilms for each dilution (Tilex, Lysol, or Borax). 2.Plot your data on the graphs provided. 3.Find the LOEL and LD50 on the dose- response curve. 4.Compare answers with other groups to determine which cleaner is the most effective.

24. Mold Control: Toxicology Experiment Calculations % killed = C-T x 100 C T= Number of colonies in treatment C= Number of colonies in control

25. Mold Control: Toxicology Experiment Results Percent Killed DilutionLysolTilexBorax 0.0001%200 0.001%220 0.01%0560 0.10%34910 1%611000 10%9110019 100%100 50

26. Mold Control: Toxicology Experiment Dose-Response Curve

27. Mold Control: Toxicology Experiment Student Outcomes After doing this activity, students will be able to: Develop a dose-response curve. Identify the Lowest Observable Effect Level (LOEL), LD100 and LD50 on the dose-response curve. Understand the effect disinfectants have on yeast growth. Understand the purpose of using an experimental control.