Which Hand Sanitizer Kills Bacteria the Best

Matt Olon

Grade 9

Pittsburgh Central Catholic High School

Background on Purell

• Most commonly used hand sanitizer

• Main use- reduce bacteria on the skin

• Active ingredient: 70% Ethyl Alcohol...Antimicrobial

• Ingredients (inactive): water, isopropyl alcohol, caprylyl glycol, glycerin, isopropyl myristate, tocopheryl acetate, C10-30 alkyl acrylate crosspolymer, aminomethyl propanol, parfum

Background on generic

• Commonly used to replace big brands such as Purell.

• The hand sanitizer used in this experiment was up&up (Target) generic hand sanitizer

• Main use- to decrease bacteria on the skin that could cause disease

• Active ingredient: 70% Ethyl Alcohol...Antiseptic

• Ingredients (inactive): water, glyceryl caprylate/caprate, glycerin, isopropyl myristate, tocopheryl acetate, C10-30 alkyl acrylate crosspolymer, benzophenome-4

Water temperature

• Each test tube was heated in one of the following temperatures

• 40°C

• 48°C- just about the average temperature for washing hands in warm/hot water

• 56°C

Escherichia coli

• Large and diverse group of gram (-) bacteria

• Free living, symbionts, or pathogens

• Live in the intestinal tract of many mammals.

• Most strains are not pathogenic

• Serve as a common prokaryotic cell model.

Purpose

• The purpose of this experiment is to determine whether Purell or Generic hand sanitizer is more effective against E. coli growth at water temperatures of 40, 48, and 56°C.

Hypotheses

• Null hypothesis- Neither hand sanitizer will significantly affect bacterial growth

• Alternative hypothesis 1- Hand sanitizer will significantly affect the amount of E. coli growth

• Alternative hypothesis 2- Exposure to Purell will result in less colonies than Generic at the every temperature

Materials

• LB Media (0.5% yeast extract, 1% tryptone, 1% sodium chloride )

• Purell and generic hand sanitizers

• Micro and Macro-pipettes and sterile tips

• SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)

• Spreader bars

• LB agar plates

• E. coli DH5 alpha

• Bunson Burner

• Ethanol (for cleaning)

• Vortex

• Incubator

• Hot plates with water at 40, 48, and 56°C

• 18 sterile test tubes

Procedure

1. E. coli was grown overnight in sterile LB media.

2. Sample of the overnight culture was added to fresh media in a sterile sidearm flask.

3. The cultures were incubated at 37°C until a density of 50 Klett spectrophotometer units was reached. (Cell density approximately 108 cells/mL).

4. The cultures were diluted in a sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. The selected experimental variables were diluted with sterile water to the chosen concentrations to a total of 9.9 mL.

Purell Chart of Concentrations

Purell 0% 0% 0% 0.01% 0.01% 0.01% 1% 1% 1%

Water temperature 40° 48° 56° 40° 48° 56° 40° 48° 56°

E. Coli 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml

Sterile Water 9.9ml 9.89ml 8.9ml 9.9ml 9.89ml 8.9ml 9.9ml 9.89ml 8.9ml

Purell hand sanitizer 0ml 0.01ml 1ml 0ml 0.01ml 1ml 0ml 0.01ml 1ml

Total 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml

Generic Chart of Concentrations

Generic 0% 0% 0% 0.01% 0.01% 0.01% 1% 1% 1%

Water temperature 40° 48° 56° 40° 48° 56° 40° 48° 56°

E. coli 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml

Sterile Water 9.9ml 9.89ml 8.9ml 9.9ml 9.89ml 8.9ml 9.9ml 9.89ml 8.9ml

Generic hand sanitizer 0ml 0.01ml 1ml 0ml 0.01ml 1ml 0ml 0.01ml 1ml

Total 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10ml

Procedure (continued)

6. 0.1mL of cell culture was then added to the sterile test tubes, resulting in a final volume of 10 mL.

7. The solutions were mixed by a vortex and then sat in water of either 40, 48, or 56°C for 15 minutes.

8. After vortexing again, 0.1 mL of each solution was removed and spread on LB agar plates with 4 replicates for each solution.

9. The resulting colonies were counted and recorded.

Survivorship of E. coli (at 40°C)

682.5

372.5

85

682.5

432.5

190.5

0

100

200

300

400

500

600

700

800

0.00% 0.01% 1.00%

Co

lon

ies

Concentration of Variable

Purell (P value 6.13E-9) Generic (P value 7.11E-8)

P value: 0.0327

P value: 0.015

Survivorship of E. coli (at 48°C)

797.5

447.5

105

797.5

505

137.5

0

100

200

300

400

500

600

700

800

900

0.00% 0.01% 1.00%

Co

lon

ies

Concentration of Variable

Purell (P value 7.68E-8) Generic (P value 1.5E-7)

P value: 0.0124

P value: 0.0369

Survivorship of E. coli (at 56°C)

1017.5

477.5

152.5

1017.5

525

220.5

0

200

400

600

800

1000

1200

0.00% 0.01% 1.00%

Co

lon

ies

Concentration of Variable

Purell (P value 2.73E-7) Generic (P value 6.05E-5)

P value: 0.038

P value: 0.029

Dunnett’s test (40°C)

Concentration T-value Significant? 0.01% 3.46 Significant

1% 4.29 Significant

Concentration T-value Significant? 0.01% 3.12 Significant

1% 3.95 Significant

T crit: 2.5 Alpha: 0.05

Purell

Generic

Dunnett’s test (48°C) T crit: 2.5

Alpha: 0.05

Concentration T-value Significant? 0.01% 3.58 Significant

1% 4.33 Significant

Concentration T-value Significant?

0.01% 3.09 Significant

1% 3.91 Significant

Purell

Generic

Dunnett’s test (56°C) T crit: 2.5

Alpha: 0.05

Concentration T-value Significant? 0.01% 3.67 Significant

1% 4.52 Significant

Concentration T-value Significant? 0.01% 3.4 Significant

1% 4.12 Significant

Purell

Generic

Conclusions

• Reject null hypothesis and accept alt. hypothesis 1, because all concentrations of both hand sanitizers proved to have a significant effect on E. coli survivorship after an ANOVA and Dunnett’s test.

• Alternative Hypothesis 2: Purell hand sanitizer did result in less colonies than Generic at all concentrations

Limitations

• Only two kinds of hand sanitizers

• Only 3 different, and close temperatures

• Only one microbe

• Between aliquots, the test tubes may have been contaminated or changed

• There is controversy on whether Purell’s function is affected or changed by water

• Only survivorship was tested

Extensions

• More replicates

• Wider temperature ranges

• Various sanitizers

• Infusion of agar plates

• Growth curve analysis

References

• http://www.purell.com/

• http://www.colorado.edu/eeb/courses/1230jbasey/abstracts%202007/14.htm

• http://www.cdc.gov/ecoli/general/index.html

• http://www.universityofcalifornia.edu/everyday/agriculture/ecoli.html