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

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

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

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

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

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

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

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

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

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

Concentration Chart Control 0 ppb Calcium Nitrate 100 ppb Lead Nitrate Variable (1/1E6th Stock) 0 ml 1 ml 10 μl 0.1 ml 1 mL Staph. E Sterile Dilution Fluid 9.9 ml 8.9 ml ~9.9 ml 9.8 ml Total Volume 10 ml

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

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

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

Biofilm Procedure Quantifying the Biofilm 200 μl of 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

Alpha: 0.05

Dunnett’s Test On Survivorship Groups T-Value T-Critical Conclusions Calcium 100 ppb Vs. Control 0.30 2.89 Not Significant 1 ppb Vs. Control 3.37 Significant 10 ppb Vs. Control 6.60 100 ppb Vs. Control 9.00

Dunnett’s Test on Biofilm Formation Groups T-Value T-Critical Conclusions Calcium 100 ppb Vs. Control 1.10 2.89 Not Significant 1 ppb Vs. Control 1.81 10 ppb Vs. Control 13.46 Significant 100 ppb Vs. Control 25.325

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 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

Limitations and Extensions 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

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.

Survivorship ANOVA

Biofilm ANOVA