Disinfectant Concentration Effects on Bacteria

Joey ZiccarelliGrade 9

Central Catholic High School

Many disinfectant manufacturers claim that their product effectively kills microbes.

These disinfectants are commonly used in society to either sterilize a wound or to reduce the concentration of pathogens on surfaces.

How effective are these products?

Problem

To determine the effects of common antiseptic solutions on the survivorship of a gram positive and gram negative bacteria: specifically Staphylococcus Epidermidis and Escherichia coli

To assess the antibacterial properties of common household disinfectants

Purpose

Iodine was discovered by the French chemist Barnard Courtois in 1811. Courtois was extracting sodium and potassium compounds from seaweed ash.

Iodine is used as an indicator test for starch and turns a deep blue when it comes in contact with it.

Potassium iodide (KI) is used to make photographic film. When mixed with iodine in alcohol, as an antiseptic for

external wounds.

Antiseptic Iodine

Vinegar is a liquid substance consisting mainly of acetic acid and water, the acetic acid being produced through the fermentation of ethanol by acetic acid bacteria.

Produced either by fast or slow fermentation processes. Types of vinegar: Malt, Wine, Sherry, Apple Cider, Fruit,

Balsamic, Rice, Wine vinegar, Distilled vinegar Any type of vinegar may be distilled to produce a colorless

solution of about 5% to 8% acetic acid in water. This is variously known as distilled spirit, or white vinegar,

and is used for medicinal, laboratory, and cleaning purposes, as well as in cooking, baking, meat preservation, and pickling.

Vinegar

Louis Jacque Thenard discovered hydrogen peroxide in 1818. Hydrogen peroxide consists of oxygen and hydrogen atoms. In the environment, hydrogen peroxide can be found in very low concentrations.

Gaseous hydrogen peroxide is produced by photo chemical reactions in the atmosphere surrounding the earth. It can also be found in water in small quantities.

The peroxide ion consists of a single bond between two oxygen atoms: (O-O)2-. It is a strong oxidizer. Hydrogen peroxide has the chemical formula H2O2 and the following structural formula: H-O-O-H

3% H2O2 - One of the most common disinfectants for minor cuts

Hydrogen Peroxide

Null Hypothesis: Hydrogen Peroxide, Antiseptic Iodine, Vinegar will have no effect on E. coli and Staph e survivorship.

Alternative Hypothesis: Hydrogen Peroxide, Antiseptic Iodine, Vinegar will significantly reduce survivorship of E. coli and Staph e.

Hypothesis

Glass Test Tubes with SDF (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)

Macropipettes Micropipettes + tips Disinfectants (Iodine

Antiseptic, Vinegar, Hydrogen Peroxide)

Vortex Ethanol, Spreaders

LB Agar Plates LB media (0.5% yeast

extract, 1% tryptone, 1% sodium chloride)

Incubator Escherichia coli (DH5

Alpha) Staphylococcus

epidermidis Test tube rack Side Arm Flask

Materials

Most commonly studied historical microbe Gram negative Found in animal intestines (including humans) Necessary to keep body healthy Sometimes found in food as a result of cross

contamination (many times during the slaughtering process)

Symptoms: Diarrhea, Abdominal cramping, Nausea and vomiting

E. coli (Escherichia coli)

E. coli: Gram Negative Bacteria Cell wall is thin

extra layer of lipopolysaccharide which adds extra level of protection.

If the toxin enters the circulatory system it causes a toxic reaction.

This outer membrane protects the bacteria from several antibiotics.

Professor Duman Lab at CMU

Often found as an external symbiont on human skin

Gram positive Round (Coccid) Forms grape-like clusters Symptoms: Boils, Impetigo, Cellulitis Staphylococcal scalded skin syndrome

Staph (Staphylococcus Epidermidis)

Staph: Gram Positive Bacteria

Most pathogenic bacteria in humans are gram-positive organisms.

Simple cell wall.

Antibiotics such as penicillin work against the formation of the cell wall.

Carolina Company

A: Liquid Exposure Escherichia coli and Staphylococcus epidermidis were grown overnight

in sterile LB media. A sample of the overnight culture was added to fresh media in a sterile

sidearm flask. The culture was incubated until a density of 50 Klett spectrophotometer

units was reached. This represents a cell density of approximately 108-109 cells/ml.

The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/ml.

The disinfectants were diluted with sterile dilution fluid to concentrations of 0%, .1%, 1%, and 10% to total 9.9 ml.

0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 103 cells/ml.]

The pH of each disinfectant was recorded during the experiment. The pH of antiseptic iodine was 6. The pH of Hydrogen Peroxide was 6.

The pH of distilled vinegar was 3.

Procedure

Procedure

Stock Solution 0% 0.1% 1% 10%

SDF 9.9 mL 9.89 mL 9.8 mL 8.9 mL

Disinfectant 0 mL 0.01 mL 0.1 mL 1 mL

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Total 10 mL 10 mL 10 mL 10 mL

The tubes were vortexed and allowed to incubate at room temperature for a 20 minute time period.

After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on LB agar plates.

B: Infusion Test 200 microliters of disinfectant (or SDF) were spread onto LB agar

plates. The plates were inverted and incubated for two hours at 37 C for infusion.

As in the liquid experiment, bacteria was spread onto the infused plates.

The plates were left to sit overnight. The resulting colonies were counted. Each colony is assumed to

have arisen from one cell. It is assumed that the 200 microliters diffused into the agar plate’s

20 milliliters evenly at a 1/100 variable concentration in the agar.

Procedure (cont.)

0.00% 0.10% 1.00% 10.00%0

10

20

30

40

50

60

70

80

Disinfectant Effects on E. coli Survivorship

E-coli Antiseptic

E-coli Vinegar

E-coli Hydrogen Peroxide

Concentration of Disinfectant

Resu

lting

Num

ber o

f Col

onie

s

2.91E-08

3.31E-20

0.392186

3.75E-192.59E-211.31E-21

0.00% 0.10% 1.00% 10.00%0

10

20

30

40

50

60

70

80Disinfectants Effect on Staph Survivorship

Staph Antiseptic

Staph Vinegar

Staph Hydrogen Peroxide

Concentration of Disinfectant

Resu

lting

Num

ber o

f Col

onie

s

1.89E-397.34E-391.9E-27

1.48E-26

1.64E-26

0.020846

Dunnett’s Test

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

Vinegar Concentration

T-Value Interpretation

E. coli Staph E. coli Staph

0.1% 1.35 58.26 Not Significant Significant

1% 8.32 59.25 Significant Significant

10% 17.32 59.5 Significant Significant

T-critical = 2.76Alpha value= .05

Dunnett’s Test

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

Antiseptic Concentration

T-Value Interpretation

E. coli Staph E. coli Staph

0.1% 8.9 60.25 Significant Significant

1% 18.62 60.76 Significant Significant

10% 18.75 61.01 Significant Significant

T-critical = 2.76Alpha value= .05

Dunnett’s Test

𝑡𝑑=𝑀𝑖−𝑀𝑐

√ 2𝑀𝑆𝐸𝑛h

Hydrogen Peroxide Concentration

T-Value Interpretation

E. coli Staph E. coli Staph

0.1% 9.04 .91 Significant Not Sig.

1% 19.52 5.15 Significant Significant

10% 19.55 31.5 Significant Significant

T-critical = 2.76Alpha value= .05

Overall, the three disinfectant variables used in this experiment had a significant effect on the survivorship of Escherichia coli and Staphyloccocus Epidermidis.

Therefore, the null hypothesis was rejected.

Conclusion

The disinfectants were somewhat insoluble and needed to be inverted repeatedly

Only one time of exposure was utilized Difficult to synchronize plating

Test various concentrations of disinfectants Test the disinfectants effects on numerous cell models

(yeast, algae) Test the effects of different disinfectants

Limitations and Future Experimentation

Limitations

Future Experimentation

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1057892/

http://cmr.asm.org/content/12/1/147.short http://www.academicjournals.org/ajpp/pdf/

%20pdf2009/May/El-Mahmood%20%20and%20%20Doughari.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88971/

http://aem.asm.org/content/10/5/458

References

Anova• Abbreviation for analysis of variance• Statistical test to see variance between and

within groups

•If the P- value is larger than the alpha value (.05), then the result is significant

Sample ANOVA used in experiment

Single Factor Analysis of Variance (Antiseptic Staph)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 721 72.1 26.32222

Column 2 10 1 0.1 0.1

Column 3 10 4 0.4 0.266667

Column 4 10 10 1 1.111111

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 38453.4 3 12817.8 1844.288 1.89E-39 2.866266

Within Groups 250.2 36 6.95

Total 38703.6 39

Single Factor Analysis of Variance (Vinegar Staph)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 721 72.1 26.32222

Column 2 10 16 1.6 2.044444

Column 3 10 10 1 0.666667

Column 4 10 7 0.7 0.455556

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 37811.7 3 12603.9 1709.647 7.34E-39 2.866266

Within Groups 265.4 36 7.372222

Total 38077.1 39

Single Factor Analysis of Variance (Hydrogen Peroxide Staph)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 721 72.1 26.32222

Column 2 10 741 74.1 46.98889

Column 3 10 608 60.8 32.4

Column 4 10 31 3.1 9.877778

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 33599.68 3 11199.89 387.5768 1.9E-27 2.866266

Within Groups 1040.3 36 28.89722

Total 34639.98 39

Single Factor Analysis of Variance (Vinegar E-coli)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 698 69.8 213.0667

Column 2 10 644 64.4 71.82222

Column 3 10 365 36.5 34.72222

Column 4 10 5 0.5 0.277778

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 30245.4 3 10081.8 126.0663 3.75E-19 2.866266

Within Groups 2879 36 79.97222

Total 33124.4 39

Single Factor Analysis of Variance (Antiseptic E. coli)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 698 69.8 213.0667

Column 2 10 431 43.1 57.87778

Column 3 10 9 0.9 0.766667

Column 4 10 4 0.4 0.266667

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 34702.1 3 11567.37 170.1222 2.59E-21 2.866266

Within Groups 2447.8 36 67.99444

Total 37149.9 39

Single Factor Analysis of Variance (Hydrogen Peroxide E. coli)

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 10 698 69.8 213.0667

Column 2 10 376 37.6 40.48889

Column 3 10 3 0.3 0.233333

Column 4 10 2 0.2 0.177778

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 33753.28 3 11251.09 177.2058 1.31E-21 2.866266

Within Groups 2285.7 36 63.49167

Total 36038.98 39

Dunnett’s Test