Lead Compound Effects on Microbial Survivorship. Joseph Ga; Central Catholic High School 9 th grade

Transcription

1 Lead Compound Effects on Microbial Survivorship Joseph Ga; Central Catholic High School 9 th grade

2 Microbial Flora is known about the associacon between humans and their flora. Effects are mutualiscc, parasicc, pathogenic and commensal. Provide nutriconal and digescve benefits, secrete vitamins, scmulate ancbody produccon, and protect against pathogenic microbes. Environmental agents might have unintended effects on the flora populacons and their funccons.

3 Problem Many people use paints and other products containing lead [Lead(II)nitrate]. This could potencally effect microbes in the body if exposed for periods of Cme. Lead compounds found in paints, nylon, polyesters, and in coacngs of photothermographic paper.

4 Lead-Nitrate (Pb(NO 3 ) 2 Lead(II)nitrate Inorganic Compound Commonly occurs as a colorless crystal or white powder Soluble in water used in lead paints and other products

5 Escherichia coli (E. coli) Gram- NegaCve Bacteria OVen found in intescnes of warm blooded animals Most forms are harmless but some can become pathogens Normal Flora or indigenous bacteria Widely used in many experiments

6 Purpose To determine if the survivorship of E. coli cells will be significantly affected by Lead- Nitrate.

7 Null/AlternaCve Hypotheses Null Hypothesis- The survivorship of E. coli will not be significantly affected by Lead- Nitrate. Alterna4ve Hypothesis- Lead Nitrate will significantly reduce E. coli survivorship.

8 Materials Escherichia coli (DH5 alpha) LB Agar Plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Glass Test Tubes with sterile dilucon fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCL) Macropipe@es Micropipe@es + Cps Lead- nitrate Vortex Ethanol Spreader bar (for sterilizacon) Sharpie Burner Incubator Test tube rack

9 Liquid Pulse Procedure 1. E. coli 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 cultures were placed in an incubator (37 C) uncl a density of 50 Kle@ spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/ml 4. The culture was diluted in sterile dilucon fluid to a concentracon of approximately 10 3 cells/ml 5. The tubes were prepared as follows:

10 Liquid Pulse ConcentraCons (ml) 0% % 0.001% 0.01% 0.1% Sterile Fluid E. Coli Lead Nitrate Total

11 Liquid Procedure (concnued) 6. The E. Coli was exposed to the Lead- Nitrate concentracons for 20 minutes µl was removed from the tubes and spread on LB plates (8 replicacons per concentracon) 8. The plates were incubated at 37 C for 24 hours 9. The resulcng colonies were then quancfied. Each colony is assumed to have arisen from one cell.

12 Infusion Procedure microliters of (Lead- Nitrate + SDF combinacon) were infused into the LB agar media by spreading, invercng, and incubacng at 37 C for 60 minutes. Approximate concentracons of 0.1% and 0.001% µL of cells from the Control tube (0% Lead) were spread on the infused plates. 3. The plates were incubated at 37 C for 24 hours 4. The resulcng colonies were then quancfied. Each colony is assumed to have arisen from one cell.

13 Survivorship (Liquid Pulse) P- Value= 7.89E- 16 Number of Colonies % % 0.001% 0.01% 0.1% Concentra4on of Lead- Nitrate

14 s Test (Liquid Pulse) T- crit value=3.26 Alpha value=.05 T > T- crit: Significant VariaCon T < T- crit: No Significant VariaCon Concentra4on T value Result % 9.04 Significant 0.001% Significant 0.01% Significant 0.1% Significant

15 Survivorship (Infusion) Number of Colonies % 0.1% 0% Concentra4ons P- Value=

16 s Test (Infusion) T- crit value=2.86 Alpha value=.05 T > T- crit: Significant VariaCon T < T- crit: No Significant VariaCon Concentra4on T value Result 0.001% 2.52 Not Significant 0.1% 6.25 Significant

17 Liquid Pulse LD Number of Colonies Average of Surviving Colonies LD50 was not met 0 0% % 0.001% 0.01% 0.10% Concentra4ons

18 Infusion LD Number of Colonies % 0.10% Concentra4ons Number of Surviving Colonies LD50 was not met

19 Conclusion The null hypothesis was rejected for: %, 0.001%, 0.01%, 0.1% (liquid pulse) The null hypothesis was rejected for: - 0.1% (infused plates) The null hypothesis was accepted for: % (infused plates)

20 LimitaCons Slight variacon in synchronizacon of placng Only one species of bacteria was tested Other cellular effects? Growth rate? High concentracons not tested Extensions Using different species of bacteria (gram (+) posicve) Other variables (other forms of lead) Vary exposure Cme Growth curve experiment planned Increase concentracon range LD50 planned

21 References

22 Data *8 replicates were performed, only 6 were counted

23 Liquid Pulse ANOVA

24 Infusion ANOVA