Jake Rocchi CCHS, 9 th grade 1 st year in PJAS Bleach Effects on Microbial Life
Clorox Bleach Ingredients Sodium hypochlorite Sodium chlorite Sodium carbonate Sodium hydroxide Sodium polycarbonate Effective at killing Staphylococcus aureus (MRSA), Influenza A2 and Rhinovirus Type 17 Laundry detergent Household cleaner
Escherichia coli (E. coli) Gram-Negative Bacteria Located in the intestines of warm-blooded animals Most forms are harmless, but some can become pathogens Normal Flora or indigenous bacteria Widely used in many experiments
Gut Flora Consists of microorganisms that live in the digestive tracts of humans and many animals. Have many useful functions including preventing growth of harmful pathogenic bacteria. Studied more than any other type of flora in the body. Includes Bacteroides, Escherichia, and Clostridium.
Recent Studies A study was performed on the effects of water treatment compounds on the growth rate and mortality of E. coli. The results suggested that the compounds significantly decreased the growth rate of E. coli and increased the mortality of the E. coli. This experiment could show a similarity to the Clorox experiment because the water treatment compounds and the Clorox compounds both have hazardous properties.
Purpose The purpose of the experiment was to determine if the survivorship of E. coli cells will be significantly affected by the chemicals in the Clorox bleach. This experiment was broken up into three different types of experiments. Liquid Pulse Infusion Confluent Lawns
Null/Alternative Hypothesis Null Hypothesis- The growth rate and the mortality of the E. coli colonies will not be significantly affected by the bleach. Alternative Hypothesis- The growth rate and the mortality of the E. coli colonies will be significantly reduced by the bleach.
Materials Escherichia coli (DH5 Alpha) Glass Test Tubes with Sterile Dilution Fluid (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl) Macropipettes Micropipettes + tips Clorox bleach Vortex Ethanol, Spreaders LB Agar Plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Incubator Test tube rack Klett spectrophotometer Sidearm flask
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) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/ml. 4) The culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/ml. 5) The tubes were prepared as follows:
Liquid Pulse Concentrations (ml) 0% 0.01% 0.1% 1% Sterile Fluid 9.9 9.899 9.89 9.8 E. coli.1.1.1.1 Bleach 0.001.01.1 Total 10 10 10 10
Liquid Procedure (continued) 6) The E. coli was exposed to the Clorox concentrations for 20 minutes. 7) 100 µl was removed from the tubes and spread on LB plates (5 replicates per concentration). 8) The plates were incubated at 37 C for 24 hours. 9) The resulting colonies were quantified. Each colony is assumed to have arisen from one cell.
Infusion Procedure 1) 200 microliters of Clorox bleach were infused into the LB agar media by spreading, inverting, and incubating at 37C for 60 minutes. 2) 100 µl of cell culture was then added to an SDF solution of 9.9mL, yielding a final volume of 10 ml and a cell density of approximately 10 3 cells/ml. 3) After vortexing to evenly suspend the cells, 100 µl of cell suspension was spread on the infused LB plates. 4) The plates were incubated at 37 C for 24 hours. 5) The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Confluent Lawns Procedure 1) 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. 2) 0.2 ml of cell suspension was spread onto LB plates, and incubated for 60 minutes to create confluent lawns. 3) Sterile discs were dipped into various concentations of bleach and carefully placed on the lawn surfaces. 4) Plates were incubated at 37C for 24 hours, and the resulting zones of inhibition measured.
ANOVA Abbreviation for analysis of variance Statistical test to see variance between and within groups If the P- value is smaller than the alpha value (.05), then the result is significant Sample ANOVA used in experiment
Number of Colonies E. Coli Survivorship (Liquid Pulse) 500 450 400 350 300 250 200 150 100 50 0 P value= 2.03E-07 (Significant) 0% 0.01% 0.10% 1% Concentration of Clorox
Dunnett s Test (Liquid Pulse) T-crit value= 3.29 Alpha value=.05 T > T-crit: Significant Variation T < T-crit: No Significant Variation Concentration T value Result 0.01% 1.70 Not Significant 0.1% 7.54 Significant 1% 9.88 Significant
Number of Colonies E. coli Survivorship (Infusion) 250 200 P value= 0.00288 (Significant) 150 100 50 0 0% 0.01% 0.10% 1% Concentration of Clorox
Dunnett s Test (Infusion) T-crit value= 2.69 Alpha value=.05 T > T-crit: Significant Variation T < T-crit: No Significant Variation Concentration T value Result 0.01% 1.56 Not Significant 0.1% 6.87 Significant 1% 8.32 Significant
Measurement of Zone Zones of Inhibition (mm) 18 16 14 12 10 8 6 4 2 0 P value= 3.98E-14 (Significant) 0% 0.01% 0.10% 1% Concentration of Clorox
Dunnett s Test (Confluent Lawns) T crit value= 2.88 Alpha value=.05 T > T-crit: Significant Variation T < T-crit: No Significant Variation Concentration T value Result 0.01% 6.56 Significant 0.1% 9.38 Significant 1% 14.03 Significant
Number of Colonies Liquid Pulse LD50 500 450 400 350 300 250 200 150 100 50 0 Average Growth of E. coli LD50= about 0.50% 0% 0.01% 0.10% 1% Concentration of Clorox
Infusion LD50 250 Average Number of Colonies Grown 200 150 100 Average Number of Colonies Grown 50 LD50 was not met 0 0% 0.01% 0.10% 1%
Conclusions The results of all the experiments (except for the Dunnett s Tests for Liquid Pulse and Infusion at 0.01%) suggest that the Clorox bleach significantly decreased the survivorship of E. coli cells. Therefore, the results support a rejection of the null hypothesis.
Extensions and Limitations Extensions More concentrations Testing another type of bleach to compare with the Clorox More plates can be used for each concentration Longer incubation time Trypan exclusion assay Test the ph effects of the Liquid Pulse Limitations Only four concentrations Only one variable was tested Limited amount of plates for each experiment Limited incubation time Synergistic effects?
References E. coli Experiment. AquaLiv.com 22 Dec 2012. <http://www.aqualiv.com/experiments/87-ecoli-experiment> Phillips, Theresa. Top 5 Reasons E. coli is used for Gene Cloning. About.com. 21 Dec 2012. http://biotech.about.com/od/technicaltheory/tp/ecoli.htm Research Article: Escherichia coil. BookRags.com 22 Dec 2012. http://www.bookrags.com/research/escherichia-coli-gen-02/ Todar, Kenneth. The Normal Bacterial Flora of Humans. Textbook of Bacteriology.net 2009. University of Wisconsin. 21 Dec 2012. <http://www.textbookofbacteriology.net/themircobialworld/normalflora.html> What s in Clorox Regular Bleach? Clorox.com. 22 Dec 2012. http://www.clorox.com/products/clorox-regular-bleach/ingredients-and-safety/
Liquid Pulse ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 5 2167 433.4 1237.3 Column 2 5 1953 390.6 2580.8 Column 3 5 1217 243.4 1424.3 Column 4 4 738 184.5 941.6667 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 191997.1 3 63999.03 40.34468 2.03E-07 3.287382 Within Groups 23794.6 15 1586.307 Total 215791.7 18
Infusion ANOVA SUMMARY Groups Count Sum Average Variance Column 1 2 458 229 338 Column 2 2 417 208.5 24.5 Column 3 2 278 139 162 Column 4 2 240 120 162 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 16712.38 3 5570.792 32.45909 0.00288 6.591382 Within Groups 686.5 4 171.625 Total 17398.88 7
Confluent Lawns ANOVA Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 0 0 0 0.01% 8 13 1.625 1.982143 0.10% 8 75 9.375 6.267857 1% 8 123 15.375 11.125 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 1224.094 3 408.0313 84.23871 3.98E-14 2.946685 Within Groups 135.625 28 4.84375 Total 1359.719 31