1. Effects Of Air Fresheners on Yeast Cell Survivorship
By, Peter Koltas, Pittsburgh Central Catholic, 10th grader, 2nd Year in pjas

2. Problem
Many people around the world use air fresheners often, and nobody knows it’s affects on the skin microflora.

3. Microbial Flora
Little is known about the association between humans and their flora
Effects are mutualistic, parasitic, pathogenic, and commensal
Perform functions beneficial to the host
Human foods, supplements, and medicinal products, may have unintended effects on the flora populations and their functions.

4. Yeast Saccharomyces cerevisiae. Easy to manipulate in laboratories.
Most commonly studied cell.
Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells, like human cells.
Used in this study to represent eukaryotic skin flora.

5. Air Fresheners
Di-ethly Phthalate (DEP), Di-n-butyl Phthalate (DBP), Di-isobutyl Phthalate (DIBP), Di-methyl Phthalate (DMP), and Di-isohexyl Phthalate (DIHP) were found to be in some air fresheners and are reproductive toxins.
Do not clean the air, often make it worse quality, only makes the room smell better.

6. Air Fresheners Being Tested
Febreeze- Mediterranean Lavender
Airwick- Lavender and Chamomile
Glade- Lavender and Peach Blossom
Nice- Lavender Vanilla

7. Air Freshener Ingredients
Fragrance, Solubilizers, Emulsifier, Stabilizer, Corrosion Inhibitor, Propellants, Water, Non-Flammable Natural Propellant, Quality Control Ingedients.

8. Purpose
The purpose of this experiment is to test the effects of four commercial air fresheners on microbial survivorship, specifically Saccharomyces cerevisiae.

9. Hypotheses
Null hypothesis: None of the air fresheners will have an effect on yeast cell survivorship.
Alternate hypothesis: All air fresheners will have significant effects on yeast survivorship in all tested air fresheners in all tested concentrations.

10. Materials Saccharomyces cerevisiae Sterile pipette tips Micropipettes
64 YEPD Agar plates (1%yeast extract, 2% glucose,1.5% agar)
Micro Rack
Micro Tubes
YEPD Media (1% yeast extract, 2% peptone, 2%glucose)
Agar Plates
Vortex
Incubator
sterile dilution fluid (10mMKH2PO4, 10mM K2HPO4, 1mM MgSO4, 0.1 mM CaCl2,100mM NaCl)
Spreader Bars
Ethanol
Bunsen Burner
Sterile water

11. Procedure
1. Saccharomyces cerevisiae were grown overnight in sterile YEPD media.
2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask.
3. The culture was placed in an incubator (30C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^7 cells/mL.
4. The cell culture was diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.
5. Test tubes were made with concentrations of 0%, 0.1%, 1% and 10% air freshener.(Four replicates, one for each type of air freshener.)

12. Test Tube Concentrations0%
0.1% 1%
10%
Sterile Water
9.9 ml
9.89 ml
9.8 ml
8.9 ml
Yeast
0.1 ml
Air Freshener
0 ml
0.01 ml
1 ml

13. Procedure cont.
6. The tubes were incubated at room temperature for 15 minutes.
7. Tubes were vortexed and 0.1mL aliquots were plated onto YEPD agar.
8. Plates were incubated at 30 degrees Celsius for two days and colonies were counted. Each colony was assumed to have arisen from a single cell.

14. graph
P Value for all data =
Control Group

15. ANOVA
Statistical test that allows for the comparison of means of different groups, to determine significant variation
Single factor ANOVA
Utilizes p-values as measure of significance
p>0.05: not significant
p<0.05: significant

16. Dunnett’s Test
A test used to find out which variable groups produced significant variation compared to a control.
If T-value is greater than the T critical, variations are considered significant.

17. Dunnett’s Test Results
T Value
Result
0% vs. .1% Glade
5.07
Significant
0% vs. .1% Breeze
5.99
0% vs. .1% Nice
7.13
0% vs. .1% Wick
4.15
0% vs. 1% Glade
8.40
0% vs. 1% Breeze
7.83
0% vs. 1% Nice
8.86
0% vs. 1% Wick
6.22
0% vs. 10% Glade
10.12
0% vs. 10% Breeze
9.78
0% vs. 10% Nice
9.89
0% vs. 10% Wick
6.46
T Critical is 3.48

18. Interpretation of Results
Null hypothesis can be rejected
Alternate hypothesis can be accepted
All air fresheners at all concentrations produced significant negative effects on yeast cell survivorship.
All air fresheners had similar effects at given concentrations

19. Limitations and Inconsistencies
Slight positioning differences in the incubation process
Slightly desynchronized plating
Only one time of exposure (liquid pulse)
Limited concentration exposures
Only survivorship tested, not growth or other health parameters
Study does not account for other factors that might affect the skin microbial flora.

20. Extensions
Addition of more microbial models such as Staphylococcus and E-coli.
Other brands of air fresheners and different scents could be used for further analysis.
Different concentrations of the variable could be tested.
Varied exposure times
Growth curve analysis

21. Bibliography http://www.toxipedia.org/display/toxipedia/Air+Fresheners