By: Luke Beck Pittsburgh Central Catholic HS PJAS 2012 Grade 11 Synergistic Drug Effects on Microbial Flora

 Accompanied with drug side effects, combination of over the counter drugs can have dire side effects  Do common daily over the counter drugs cause adverse effects on human cells? Problem

 A nonsteroidal anti- inflammatory drug  Used to treat such symptoms as headache, muscular aches, the common cold and arthritis pain.  Third most consumed drug in the entire world.  Can be taken on a daily basis to prevent symptoms. Ibuprofen

 Active Ingredient: Diphenhydramine Hydrochloride  Used as an Antihistamine  Over 25 over the counter drugs share the same active ingredient as Benadryl  Taken on a daily basis to prevent allergy symptoms Benadryl

 Has been the most studied type of bacteria in biological research.  Gram negative bacteria  Symbiont in intestinal tracts of many mammals, including humans.  Mostly non-pathogenic. Escherichia Coli

 Staph. species consisting harmless skin-dwelling microorganisms  Species of Staph. are commonly used in microbiological experiments  Gram Positive Bacteria Staphylococcus epidermidis

 To determine if differing concentrations of Ibuprofen, the antihistamine Benadryl and/or synergy of the two have an adverse effect on E. Coli and Staph. Survivorship. Purpose

Hypothesis  Null: The presence of Ibuprofen, Benadryl, and a combination of both will not cause a change in bacterial population outside of chance  Alternative: The presence of Ibuprofen, Benadryl, and a combination of both will cause a change in bacterial population outside of chance

Materials  Ibuprofen (200mg liquid gels)  Benadryl (25mg liquid gels)  Escherichia coli bacteria  Staphylococcus epidermidis bacteria  Ethanol  20 mL tubes  Sterile  Micro burner  Ethanol  Test tubes  Test Tube Rack  Microtubes  Vortex  Incubator  Gloves  Goggles  Klett Spectrophotometer  SDF  LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl)  Sidearm flask  Spreader bar  SDF Test Tubes

1.Bacteria (E. coli and Staph) was grown overnight in sterile LB 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 (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4.The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. Procedure

Concentrations (Ibuprofen, Benadryl) Control(0 x, 0.01x) (0 x, 0.1 x)(0.1 x, 0 x)(0.1 x, 0.01 x) (0.1 x, 0.1 x) (1 x, 0 x)(1 x, 0.01 x) (1 x, 0.1 x) Microbe E. Coli or Staph. 0.1 mL SDF9.9 mL9.89 mL9.8 mL 9.79 mL9.7 mL8.9 mL8.89 mL8.8 mL Ibuprofen0 mL 0.1 mL 1.0 mL Benadryl0 mL0.01 mL0.1 mL0 mL0.01 mL0.1 mL0 mL0.01 mL0.1 mL Total10 mL

 100 µL of cell culture was then added to each solution, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL.  The solutions were vortexed and allowed to sit at room temperature for 15 minutes.  After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates.  The plates were incubated at 37°C for 48 hours.  The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. Procedure

 Abbreviation for analysis of variance  Statistical test comparing variation within and between experimental groups  If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence)  Two factor: verifies interaction between two variables. ANOVA

Staph Survivorship P-Value:.0508 Concentration (Ibuprofen, Benadryl) Number of Colonies

E. Coli Survivorship Concentration (Ibuprofen, Benadryl) Number of Colonies

 The presence of Ibuprofen, Benadryl, and a combination of the two drugs did not significantly change the number of Staphylococcus epidermidis or E. Coli.  The null hypothesis can be accepted.  The alternative hypothesis can be rejected Conclusion

Extensions Limitations  Try prescription medication combination.  Test synergy effects of more harmful drugs.  Use human cells rather than bacteria.  These bacteria were only models for the human cell.  Plates were not plated simultaneously.

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