1. The Effects Of Temperature and Antibiotic on E.coli and Staph epidermidis James Shaver Grade 9 Central Catholic High School

2. Ampicillin Antibiotic belonging to the amino penicillin group of penicillin Used to treat urinary tract infections, Salmonella, some Influenza strains, and Meningitis. Commonly used in the lab and the medical world. Has Gram-positive coverage but limited Gram- negative coverage. The Amino group in the antibiotic helps it penetrate the membrane of Gram-negative bacteria.

3. - Beta-lactam antibiotic which is part of the aminopenicillin family.antibiotic aminopenicillin - Roughly equivalent to its successor, amoxicillin in terms of spectrum and level of activity. amoxicillin The most common side effects include: -rash -fever -diarrhea -nausea The Effects of Ampicillin

4. Bacterial Heat Stress - Bacterial heat-shock response allows for bacteria to change (stress) in the environment. - Bacteria use various response mechanisms for coping with the stress and for adapting to the new conditions.

5. -Most pathogenic bacteria in humans are Gram (+) organisms. -Simple cell wall. -Some antibiotics work against the formation of the cell wall. -Cell was contains an extra layer of lipopolysaccharides for extra protection. -Outer membrane protects bacteria from several antibiotics. Gram (-) Vs. Gram (+) Bacteria

6. - Large and diverse group of gram (-) bacteria -Free living, symbiotes, or pathogens -Live in the intestinal tract of many mammals. -Most strains are not pathogenic -Serve as a common prokaryotic cell model. Escherichia coli

7. Staphylococcus epidermidis -Common surface symbiont in many mammals (Human). -Gram (+) bacteria -Most strains considered non- pathogenic. -Pathogenic strains can be life- threatening.

8. Purpose ● To determine if Ampicillin and temperature have a synergistic effect on Staphylococcus epidermidis and Escherichia coli survivorship.

9. Hypotheses Null: 1. Ampicillin and Heat individually will not reduce bacterial survivorship. 2. Ampicillin and Heat will not have synergistic effects at reducing bacterial survivorship. Alternative: 1. The effects of Ampicillin and Heat individually will significantly reduce bacterial survivorship. 2. Heat and Ampicillin in synergy will significantly reduce bacterial survivorship.

10. Materials - Escherichia coli DH5-alpha(E.Coli) (Obtained from Doonan Lab, CMU) - Staphylococcus epidermidis (Staph) (Obtained from Carolina Science Supply) - Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) - Thermometer - Latex Gloves - Micro pipettes - Micro tubes - Ampicillin - 72 LB agar plates - 72 infused LB agar plates - Spreader Bar - Incubator - Ethanol - Matches - Bunsen Burner - Vortex - Sharpie - hot plate - Luria Broth(LB) Agar Plates ( 0.5% yeast extract,1% tryptone, 1% NaCl)

11. Procedure for Liquid Pulse 1.Bacteria (E.coli/Staph epidermidis) were grown overnight in sterile LB Media. 2.Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3.The cultures were 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 cultures were diluted in sterile dilution fluid to a concentration of approximately 10 ⁵ cells/mL. 5. Ampicillin Stock of AMP 200 (this stock represents 10x or 10 times the recommended working concentration) was sterile filtered. 6.The Ampicillin Stock was mixed with the appropriate amounts of SDF to create concentrations of 0x, 0.01x,0.1x and 1x.

12. Chart of Concentration Control0.01x0.1xx E. coli0.1 ml Ampicillin0 ml0.01ml0.1ml1ml Sterile Water9.9 ml9.899.88.9 Total Volume10ml

13. Procedures Continued 7. After addition of 100 microliters of bacterial suspension, the tubes were incubated at the selected temperatures; 40,50,60 degrees F. 8. 100 microliter aliquots from each tube were pipetted on LB agar plates. 9. The LB agar plates were placed in the incubator overnight and the number of colonies were then counted.

14. Procedures for Infused Plates 1.The following volumes of ampicillin were spread onto Lb agar plates to create the following concentrations; 60 microliters(X), 6 microliters of sub stock ( 0.01x). 2. The plates were placed in the incubator for one hour to be infused. 3.Each E. coli population subjected to the various temperatures was then pipetted onto the infused plates. 4.The plates were then incubated for 24 hours at 37 C.

15. Anova -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)

16. Dunnett’s Test - A procedure for comparing the mean of each experimental group individually against the mean of the control group. - A Dunnett’s Test allows you to see if there was a significant difference from the control. - If the T Value is greater than the T Critical value than the difference is significant.

17. Anova P- Value Staph- 0.01289E coli- 4E-07 Dunnett’s TestT- Crit (3.25) T- Value 50 C 1.2265 Not Sig 2.259 Not Sig T- Value 60 C 2.313 Not Sig 14.407 Sig The Effects of Temperature on Bacterial Survivorship

18. The Effects of Ampicillin on Bacterial Survivorship 40 C Anova P- Value Staph- 0.203322E coli- 8.52E-08 Dunnett’s TestT-Crit (3.25) T Value of 0.01x 0.50861 Not Sig 3.88418 Sig T- Value of 1x 2.14252 Not Sig 17.2157 Sig 210 178.8

19. Interaction P- Value 0.000414 Synergistic Effects on Staph Survivorship(Infusion) Concentration of AmpicillinNumber of Bacteria Colonies 0x 0.1x 1x

20. P-Value Interaction 3.75E-11 Synergistic Effects on E coli Survivorship(Infusion) Number of Bacteria Colonies Concentration of Ampicillin 0x 0.1x 1x

21. Effects of Temperature on E coli Survivorship Anova P-Value 5.29E-05 Dunnett’s TestT-Crit-2.86 50 C 1.14947(Not Sig) 60 C 3.31763(Sig)

22. Dunnett’s TestT- Crit(3.10) 0.01x 3.680174 Sig 0.1x 11.771 Sig 1x 11.8156 Sig Anova P-Value 4E-06 Effects of Ampicillin on E coli Survivorship

23. Ampicillin Effects on E coli (Liquid Pulse) Number of Bacteria Colonies Concentration of Ampicillin P-Value Interaction 5.59E-09 0x 0.01x 0.1x 1x

24. 0.1x1x E coli40 C17%77% 50 C32%85% Staph Epidermidis 40 C36%71% 50 C18%63% Percent Decrease In Survivorship Compared to Control

25. Conclusion Experiment 1(Liquid Pulse) - 1st null hypothesis is rejected because temperature and ampicillin individually had effects on bacterial survivorship. - 2nd null hypothesis is rejected because temperature and ampicillin had synergistic effects on bacterial survivorship. Experiment 2(LB Agar Infusion) - 1st null hypothesis is rejected because temperature and ampicillin individually had effects on bacterial survivorship. - 2nd null hypothesis is rejected because the temperature and ampicillin had synergistic effects on bacterial survivorship.

26. Limitations And Future Studies - Plating could have been unsynchronized. - Only three concentrations of the variable were tested. - Only survivorship examined. - Use more Antibiotics - Use a greater range of temperatures including those that are closer to eachother -Perform a Trypan blue exclusion essay to test for dead cells - Explore growth curve effects

27. References - http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidi s/sepidermidis.html http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidi s/sepidermidis.html -http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con- 20032105http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con- 20032105 -http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0000105/http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0000105/

28. Data Infused Plates Infused PlatesE coli ControlLow ConcentrationHigh Concentration 40 degrees224,228,236,204168,176,204,18954,44,39,68 50 degrees144,156,168,224104,110,123,13124,16,31,29 60 degrees0,0,0,0

29. Data Infused Plates Infused PlatesStaph epidermidis ControlLow ConcentrationHigh Concentration 40 degrees,256,360,224,236,256,22096,88,92,99 50 degrees252,234,208,204156,176,193,20781,96,78,76 60 degrees0,0,0,0

30. Data Liquid Pulse Regular PlatesE coli 0mg Stock0.01x0.1x1x 40 degrees208,352,284,333, 305,252 421,532,327,554,366, 496 276,175,180,189,2 01,222 184,184,304,209,258, 196 50 degrees176,208,304,222, 258,202 240,288,311,225,233, 316 240,220,176,220,2 28,248 211,144,156,202,178, 198 60 degrees0,0,0,0,0,0

31. Data Liquid Pulse Regular PlatesE coli 0mg Stock0.01x0.1x1x 40 degrees,352,284,333, 305,252 421,327,366,,175,180,189,201, 222 184,184,209,196 50 degrees176,208,304,222, 258,202 240,288,311,225,233, 316 240,220,176,220,2 28,248,144,156,202,178,198 60 degrees0,0,0,0,0,0