The Effects of Shampoo on Microbial Flora Andrew Walker Grade 9 Central Catholic High School
Shampoo Hair care product used to clean hair of unwanted build up Combined soap, water, and herbs to make hair shine and create a pleasant fragrance Liquid shampoo was invented in 1927, by German Hans Schwarzkopf Salt is used to adjust viscosity
Variable (V05 Shampoo) Contains: Vitamin A Vitamin H Vitamin C Vitamin B5 Vitamin B3
Gram (+) vs. Gram (-) Bacteria Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall Some antibiotics work against the formation of the cell wall. Cell was contains an extra layer of lipopolysaccharides for extra protection. Outer membrane protects bacteria from several antibiotics.
Escherichia coli Rod-shaped, 2 micrometers diameter Gram negative bacteria Reproduction rate: 20 min Survival, growth, and replication require only a single carbon source and ammonium salts. Common symbiont found in the lower intestines of warm blooded animals. Few pathogenic - Almost 73,000 cases of infection and 61 deaths per year in the United States. Most common prokaryote model
Staphylococcus epidermidis Gram positive bacteria Common surface symbiont in many mammals (including humans) Most forms considered non-pathogenic. Potentially pathogenic upon systemic entry Forms biofilms
Rationale The human micro flora performs many functions vital to human health. Almost every person the world uses shampoo when they bathe. Does shampoo effect the micro flora on the human scalp?
Hypothesis Null Hypothesis- The shampoo will have no effect on the survivorship of E. coli and Staph Alternate Hypothesis- The shampoo will have an effect on the survivorship of E. coli and Staph
Materials Variable (V05 Shampoo) Micro pipettes + Sterile tips Spreader bars LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli DH5- alpha(e.coli) (Obtained from Doonan Lab, CMU) Staphylococcus epidermidis (Staph) (Obtained from Ward s Scientific) Burner Sidearm Flask Vortex Incubator SDF (Sterile Dilution Fluid) Sterile Test Tubes Ethanol Klett spectrophotometer
Procedure Bacteria (E.coli and Staph) were grown overnight in sterile LB Media. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 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 10⁸ cells/ml. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/ml. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%,0.1%, 1%, and 10%.
Procedure (cont.) Concentration Chart 0% 0.1% 1% 10% 0.1 ml microbe 0.1 microbe 0.1 microbe 0.1 microbe 0 ml variable 0.01 ml variable 0.1 ml variable 1 ml variable 9.9 ml SDF 9.89 ml SDF 9.8 ml SDF 8.9 ml SDF
Procedure (cont.) 100 µl of cell culture was then added to shampoo solutions, yielding a final volume of 10 ml and a cell density of approximately 103 cells/ml. The solutions were vortexed and allowed to sit at room temperature for 15 minutes. After vortexing to evenly suspend the cells, 100 µl aliquots were removed from the tubes and spread on LB-agar plates. The plates were incubated at 37 C for 24 hours. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
ANOVA and Dunnett s Test ANOVA Statistical Test that allows the compares the means multiple groups. Dunnett s Test A follow-up test to an ANOVA, Comparison of an experimental and a control group to determine significant variation.
Number of Colonies 900 800 E. Coli Averages P-value: 1.67859E-06 700 600 500 400 300 200 100 0 0% 0.10% 1% 10% Number of Colonies 820 464.8 498.8 221.6 Concentration of Shampoo
Dunnett s Statistical Analysis (E. coli) Shampoo Concentration T Value Significance 0.1% 5.32 Significant 1% 4.81 Significant 10% 8.96 Significant T-crit= 3.29
Number of Colonies 900 Survivorship Curve (E. coli) 800 700 600 500 400 Number of Colonies 300 200 100 0 0% 0.10% 1% 10% Concentration of Shampoo
Number of Colonies 450 400 S. epidermidis Averages P-value: 2.51049E-12 350 300 250 200 150 100 50 0 0% 0.10% 1% 10% number of colonies 381.6667 72.5 2.3333 1.5 Concentration of Shampoo
Dunnett s Statistical Analysis (Staph) Shampoo Concentration T Value Significance 0.1% 11.82 Significant 1% 14.49 Significant 10% 14.53 Significant T-crit= 3.29
Number of Colonies 450 Survivorship Curve (Staph) 400 350 300 250 200 number of colonies 150 100 50 0 0% 0.10% 1% 10% Concentration of Shampoo
LD50 LD50- Lethal Dose 50%, what concentration kills 50% of the colonies Type of Bacteria LD50 Escherichia coli 3% concentration Staphylococcus epidermidis 0.065% concentration
Conclusions The null hypothesis was rejected, and the alternate hypothesis was accepted for both E. coli and Staph. The shampoo (V05 Shampoo) did significantly reduce on the survivorship of E. coli and Staph.
Limitations Future Studies Plating slightly unsynchronized Only one kind of test, liquid pulse Only 4 concentrations of variable tested Only 1 exposure time Use more types of energy drinks. Use more concentrations and exposure times Agar infusion Synergistic effects
Thank you for your time and attention. Are there any questions?
ANOVA (E. coli) Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0% 5 4100 820 26836 0.10% 5 2324 464.8 11457.2 1% 5 2494 498.8 4265.2 10% 5 1108 221.6 2121.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 905701.4 3 301900.5 27.0276737 1.67859E-06 3.238871517 Within Groups 178720.8 16 11170.05 Total 1084422.2 19
ANOVA (staph) Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0% 6 2290 381.6667 7321.066667 0.10% 6 435 72.5 337.5 1% 6 14 2.333333 1.066666667 10% 6 9 1.5 0.7 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 590954.3333 3 196984.8 102.8596377 2.51049E-12 3.098391212 Within Groups 38301.66667 20 1915.083 Total 629256 23