1. The Effects of Vitamins B6 and C on Yeast Growth By: Gianmarco Frezza Pittsburgh Central Catholic HS Grade 9

2. Rationale Humans need vitamins B6 & C from their diets or as supplements Vitamin B6 RDA: 1.3 mg/day Vitamin C RDA males: 90 mg/day, females: 75 mg/day Does vitamin B6 or C affect the microbial flora of the body?

3. Microbial Flora Refers to microorganisms living in or on the body (gastrointestinal tract) Little is known about the association between humans and their flora Effects are mutualistic, parasitic, pathogenic, and commensal Perform functions beneficial to the host, including the manufacture of essential vitamins, and the prevention of colonization by undesirable microbes Human foods and supplements may have unintended effects on the flora populations and their functions

4. Saccharomyces cerevisiae A fungi One of the most intensively studied eukaryotic model organisms Commonly known as yeast Can be used as a model for human microflora

5. Vitamin B6 (Pyridoxine) Important for more than 100 enzymatic reactions involved in metabolism and brain development during pregnancy and infancy Helps the immune system function properly

6. Vitamin C (ascorbic acid) An antioxidant, protects cells from damage from free radicals Helps the immune system function properly Growth and repair of tissues Protects against cardiovascular disease Prevents scurvy

7. Purpose To test if Vitamin B6 or C have an effect on S. cerevisiae growth individually or synergistically

8. Hypothesis Null: the vitamins WILL NOT have an significant effect on yeast growth individually or synergistically. Alternate: the vitamins WILL have a significant effect on yeast growth both individually and synergistically.

9. Materials 36 sidearm flasks Aluminum foil Lab coat Disposable rubber gloves Micropipetters Sterile micropipette tips Sterile dilution fluid (100mMKH2PO4, 100mMK2HPO4, 10mMMgSO4, 1mMNaCl) Macropippetter Sterile macropipette tips 3 Incubators 1% stock solution vitamin B6 1% stock solution vitamin C YEP media (1% yeast extract, 2% peptone, 2% glucose) S. Cerevisiae Klett Spectrophotometer

10. Procedure 1.7.5 mL of YEP media was pipetted into each sterile sidearm flask. 2.Four replicates of the following combinations of variables were prepared as follows. 3.0.5 mL of S. cerevisiae (KU=100) was pipetted into each flask. 4.The flasks were covered with a sterilized aluminum foil cap. 5.The 36 sidearm flasks were placed in incubators (32⁰C), and readings were taken every half hour for six hours in a Klett spectrophotometer.

11. Mixtures by Groups S.Cerevisiae (mL) YEP media (mL) Concentrations (B6, C) % Vitamin C (mL) Vitamin B6 (mL) SDF (mL) Group 1 0.57.5 [0%] B6 [0%] C 002 Group 2 0.57.5 [0%] B6 [0.01%] C 0.101.9 Group 3 0.57.5 [0%] B6 [0.01%] C 101 Group 4 0.57.5 [0.01%] B6 [0%] C 00.11.9 Group 5 0.57.5 [0.01%] B6 [0.01%] C 0.1 1.8 Group 6 0.57.5 [0.01] B6 [0.1] C 10.10.9 Group 7 0.57.5 [0.1%] B6 [0%] C 011 Group 8 0.57.5 [0.1] B6 [0.01] C 0.110.9 Group 9 0.57.5 [0.1%] B6 [0.1] C 110

12. Concentrations of the Vitamins within Groups [0%] (0) B6[0.01%] (L) B6 [0.1%] (H) B6 [0%] (0) C0L0L0H0H0 [0.01%] (L) C0L0LLHLHL [0.1%] (H) C0H0HLHLHH

13. Vitamin Effects on Yeast Growth Time (min.) KU L= [0.01%] H= [0.1%] B, C

14. Groups at 180 minutes Concentrations of vitamin C Klett Units Two factor ANOVA synergy P= 0.1162 Single Factor vitamin B P= 0.958538 Single factor vitamin C P= 0.972004

15. Groups at 360 minutes Concentrations of Vitamin C Klett units Two factor ANOVA synergy P= 0.1785 Single Factor vitamin B6 P= 0.000429 Single factor vitamin C P= 0.16963

16. Conclusion The null hypothesis is accepted for all trials other than vitamin B6 at 360 minutes exposure. Vitamin B6 and C did not have a significant effect on yeast growth individually or synergistically.

17. Limitations and Extensions Limitations Only two concentrations of the variables used Only growth was tested Six hour duration Extensions More concentrations Longer Duration Study survivorship More species

18. Resources http://en.wikipedia.org/wiki/VitaminC www.ods.od.nih.gov/factsheets/VitaminC-HealthProfessional/ http://en.wikipedia.org/wiki/VitaminB6 www.ods.od.nih.gov/factsheets/VitaminB6-HealthProfessional/ http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Mark Krotec, PTEI www.epa.gov/epawaste/nonhoz/municipal/

19. Raw Data at 180 minutes Groups (Replicant 1)Klett units Group 171 Group 273 Group 387 Group 473 Group 559 Group 641 Group 762 Group 879 Group 9101 Groups (Replicant 2)Klett units Group 170 Group 284 Group 380 Group 472 Group 578 Group 635 Group 757 Group 894 Group 9100

20. Raw Data at 360 minutes Groups (Replicant 1) Klett units Group 1128 Group 2107 Group 3127 Group 492 Group 5105 Group 624 Group 7118 Group 8105 Group 9113 Groups (Replicant 2)Klett units Group 1125 Group 2108 Group 3120 Group 494 Group 598 Group 625 Group 7119 Group 8111 Group 9117

21. ANOVA at 180 minutes

22. ANOVA at 360 minutes