The Effects of Pesticide on Microbial Life

Jose Esquivel11th Grade

Central Catholic High School

Pesticide Use! Its active ingredient is diethyltoluamide, or DEET! DEET is the most active ingredient in insect

repellents across the market ! toxic for species of freshwater zooplankton

Pesticide! Primary factors affecting the effectiveness of

pesticides include:1. Type of pesticide2. Degradation3. Low temperatures

Pesticide! 25% DEET, 75% other ingredients (not revealed)! Stored in room temperature! Provides 8 Hours of protection

Yeast! Single celled fungi! Most commonly studied organism- similar

biochemistry and genes as other Eukaryotes! Saccharomyces cerevisiae (Yeast)

Purpose! To examine the effects of OFF DEEP WOODS on

Yeast cell survivorship

Hypothesis! Null Hypothesis: The pesticide will not have a

significant effect on Yeast cell survivorship

! Alternative Hypothesis: The pesticide will significantly reduce Yeast cell survivorship

Materials! YEPD agar plates (YEPD media + 1.5% agar)! YEPD media (1% yeast extract, 2% peptone, 2% glucose)! Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4,

1mM NaCI)! Sterile pipette tips! Micropippettes! Vortex! Incubator! Sidearm flask! Spreading turntable! Spreader bar! Ethyl alcohol! Sterile capped test tubes with Sterile distilled water! Saccharomyces cerevisiae (Yeast)! Pesticide

Procedure1. Pesticide was obtained from local Home Depot 2. The pesticide was sprayed into a 50 mL to achieve a fluid

form of the pesticide3. The pesticide was sterile filtered4. Yeast cells were grown overnight in sterile YEPD media5. The cultures were placed in an incubator until a density of

50 Klett spectrophotometer was reached. This is a density of approximately 10^7 cells/mL

6. The cultures were then diluted in Sterile Dilution Fluid to a concentration of 10^5 cells/mL

Procedure (Continued)7. The different concentrations were made:

0% 0.1% 1% 10%

Yeast 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Dilution Fluid 8.9 mL 8.9 mL 8.9 mL 8.9 mL

Pesticide Solution 0 mL 0.01 mL 0.1 mL 1 mL

Sterile Water 1 mL .99 mL 0.9 mL 0 mL

Final Volume 10mL 10 mL 10 mL 10 mL

Procedure (Continued)8. The tubes were allowed to incubate at room

temperature for 15 minutes9. After vortexing to evenly suspend cells, 0.1 mL

aliquots were removed from the tubes and spread on the YEPD agar plates

10. The Yeast cells incubated for 48 hours at 30C11. The resulting colonies were counted, each colony is

assumed to have arisen from one cell

Pesticide Effect on Yeast Cells

Col

onie

s

0% 0.1% 1% 10%

848

521.14

334.29

131.43

0

225

450

675

900

P Value- 0.00

Dunnett’s Test Table! T crit.=3.01 Alpha=0.05! Yeast T Value Interpretation! 0.1%. 11.693 Significant ! 1%. 18.378 Significant! 10%. 25.635 Significant

Yeast Analysis! Anova revealed significant variation between the

means ! The Dunnett's test revealed significant variation

Limitations and Extensions! Limitations:

! Synergizing the spreading of plates more precisely! Limited exposure times! Only tested liquid pulse! Only survivorship was measured- not growth

! Extensions! Increase trial size to 20 or 30 for more accurate results! exposure time to the water ! Conduct test on different micro organisms

References! http://www.gmasw.com/chemlist.pdf ! http://www.ncbi.nlm.nih.gov/! http://www.biotecharticles.com/. ! http://www.off.com/en-US/Products/Pages/off-

deep-woods.aspx#off-deep-woods-insect-repellent-v

Yeast ANOVA