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

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

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.

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.

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.

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

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

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

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.

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

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.)

Test Tube Concentrations 0% 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

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.

graph P Value for all data = 0.0013 Control Group

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

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.

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

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

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.

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

Bibliography http://www.toxipedia.org/display/toxipedia/Air+Fresheners http://www.ct.gov/dph/lib/dph/environmental_health/eoha/pdf/air_freshener_fs.pdf http://www.graphpad.com/guides/prism/6/statistics/index.htm?stat_the_methods_of_tukey_and_dunne.htm http://davidmlane.com/hyperstat/B112114.html