Lead Nitrate Suppression of Staph. E Biofilm Formation

1. Lead Nitrate Suppression of Staph. E Biofilm Formation John Lynch Pittsburgh Central Catholic High School February 3, 2018

2. Lead (II) Nitrate • Pb(NO3)2 • Industrial waste often found in water • EPA action level 15ppb • Most water – 1-5 ppb • Flint – 27 ppb • East St. Louis - 10,000 ppm

3. Biofilms • Coherent and generally adherent cells • Extracellular Polymeric Substance (EPS) • Often aquatic niches • Phenotypic shift in gene regulation • More resistant – 1000x • Lateral gene transfer • Quorum sensing

4. Biofilm Inhibition • Adherence – conditioning films, polysaccharides • Anti-biofilm agents – chemical inhibitors to adherence • Other major methods: • Surface modification (anti-microbial coatings) • Hydrophobicity

5. Staphylococcus Epidermidis • Gram positive • Non-pathogenic • Common surface symbiont • Forms biofilms

6. Importance • Lead (II) Nitrate is a notorious poison • Poisons water • Biofilms common in humans • Also the most common cause of disease • Biofilms are crucial to aquatic ecosystems • A main biomass food source for invertebrates

7. Purpose • To test for the effects of Lead (II) Nitrate on biofilm formation • To test for the effects of Lead (II) Nitrate on Staph. E survivorship • To compare the effects of biofilm formation and cellular survivorship

8. Hypothesis Null Lead (II) Nitrate will not significantly affect Staph E. survivorship or biofilm formation Alternative Lead (II) Nitrate will significantly reduce Staph. E survivorship and biofilm formation

9. Materials • Microtiter plate absorbance reader • Acetic acid • Crystal violet • 96 well tissue culture treated microtiter dish • Ethanol • Sterile spreader bars • Sidearm flask • Incubator (37 Co) • Vortex • Lead (II) Nitrate • Calcium Nitrate • Sterile pipette tips • Sterile dilution fluid (100mM KH2PO4, 10mM MgSO4, 1 mM NaCl) • LB media (.5% yeast extract, 1% tryptone, 1% sodium chloride) • LB agar plates • Staph. E culture • Micropipettes

10. Survivorship Procedure • Staph. E was grown overnight in sterile LB Media • The culture was added to fresh media in a sterile sidearm flask • The cultures were placed in an incubator (37oC) until a density of 50 Klett spectrophotometer units was reached. This is a cell density of ~108 cells/mL • The cultures were diluted in sterile dilution fluid to a concentration of ~105 cells/mL • The variable was mixed with the appropriate amounts of SDF to create concentrations of 0ppb, 1ppb, 10 ppb, and 100 ppb

11. Concentration Chart

12. Survivorship Procedure • The tubes were vortexed and allowed to sit at room temperature for 15 minutes • 100 μl aliquots were removed from the tubes and spread on LB-agar plates; 10 replicates were used • The plates were incubated at 37oC for 48 hours • Colonies were counted visually

13. Biofilm Procedure Growing the Biofilm • Tubes were prepared with the same dilution chart • 200 μl from the tubes were added per well in a 96 well microtiter plate; 10 replicates were created • The microtiter plates were incubated for 48 hours at 37oC

14. Biofilm Procedure Staining the Biofilm • After incubation, the cells were gently removed by flipping the plate and drip drying overnight • The plate was submerged in water, then drip-dried overnight • 200 μl of a 0.1% solution of crystal violet in water was added to each well of the microtiter plate • The microtiter plate was incubated at room temperature for 10 minutes • The plate was rinsed by dipping in water • The plate was upturned and drip-dried overnight

15. Biofilm Procedure Quantifying the Biofilm • 200 μlof 30% acetic acid in water was added to each well • The microtiter plate was incubated at room temperature for 10 minutes • The absorbance was quantified in a microtiter plate reader at 550 nm • 30% acetic acid in water was the blank

16. Alpha: 0.05

17. Dunnett’s Test On Survivorship

19. Dunnett’s Test on Biofilm Formation

21. Conclusions • Did Lead (II) Nitrate have a significant effect on survivorship? • Yes, p-value 2.22E-12 • Negative Effect • Did Lead (II) Nitrate have a significant effect on biofilm formation? • Yes, p-value 1.83E-29 • Negative Effect • Low concentration • Biofilms were inhibited at a higher rate than survivorship • Lead (II) Nitrate appears to have an isolated effect on biofilm formation • Further research necessary

22. Limitations and Extensions • Limitations • Micropipette accuracy (10 μl) • Replicates • Lack of higher concentrations • One exposure time • Technique • Extensions • Higher concentrations • Different nitrates to test • More replicates • More bacteria to test • Further experimentation on isolated biofilm effect

23. Bibliography • Dr. Carrie Doonan of CMU for usage of lab materials • 'Toole, George A. "Microtiter Dish Biofilm Formation Assay." Journal of Visualized Experiments : JoVE. MyJove Corporation, 2011. Web. 31 Dec. 2016. • “Biofilms in the Medical Field.” Biofilms in the Medical Field - Microbewiki, 12 Oct. 2001, microbewiki.kenyon.edu/index.php/Biofilms_in_the_Medical_Field. • “Clean Water for Flint Michigan.” LaunchGood, www.launchgood.com/project/clean_water_for_flint_michigan#!/. • “How Do Biofilms Impact Our World?” Beneficial Biofilms, www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter001/section005/green/page001.html • “Kinetics of Sodium Hydroxide-Crystal Violet Reaction Photos.” Kinetics of Sodium Hydroxide-Crystal Violet Reaction · Metallacycle, 23 May 2007, www.metallacycle.com/chemistry/laboratory/gallery/1310/kinetics/. • “Public Health Image Library (PHIL).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 17 Dec. 2017, phil.cdc.gov/default.aspx. • “Staphylococcus Epidermidis.” Staphylococcus Epidermidis - ZipcodeZoo, zipcodezoo.com/index.php/Staphylococcus_epidermidis.

24. Survivorship ANOVA

25. Biofilm ANOVA