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 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

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 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

16. Alpha: 0.05

17. 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

19. 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

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
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
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, Web. 31 Dec
“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,
“How Do Biofilms Impact Our World?” Beneficial Biofilms, /green/page001.html
“Kinetics of Sodium Hydroxide-Crystal Violet Reaction Photos.” Kinetics of Sodium Hydroxide- Crystal Violet Reaction · Metallacycle, 23 May 2007,
“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