Comparing the effects of organic and inorganic ...Comparing the effects of organic and inorganic disinfectants on bacteria Regional Math and Science Center, Frostburg State University,

Comparing the effects of organic and inorganic

disinfectants on bacteria

Regional Math and Science Center, Frostburg State University, Frostburg, MD 21532

References

Humans interact with bacteria on a daily basis and have tried to inhibit pathogenic bacteria growth since its discovery. Staphylococcus epidermidis is a gram positive cocci shaped bacteria found on human skin². Escherichia coli is a bacteria found in the stomach or digestive tract of humans; it is a gram negative bacilli shaped bacteria¹. Providencia alcalifaciens is found in the urinary tract in humans as well; this bacteria is a gram negative bacilli shaped bacteria⁵.

In order to control the growth of pathogenic bacteria disinfectants are heavily relied on. Organic and inorganic disinfectants were tested on these bacteria using the disk diffusion method. Antibacterial soap, which is inorganic, contains triclosan which disrupts bacteria's cell membrane, killing some non pathogenic cells in the process⁷. Oxy-Power all-purpose cleaner is another inorganic disinfectant, containing hydrogen peroxide an OTC broad spectrum antiseptic⁶. Turmeric, an organic disinfectant, contains curcumin which kills bacteria on contact by damaging the cell membrane⁷. Tea tree oil is another organic disinfectant containing terpinen-4-ol which is the main antimicrobial ingredient³.

The purpose of this study is to determine which disinfectant would be most efficient in inhibiting pathogenic bacterial growth.

Introduction

• Tea tree oil generated a larger inhibition zone in E.coli than in P. alcalifaciens across all time points. (see Figures 6-8)• Antibacterial soap showcased the largest inhibition zone in E.coli for all examined time intervals. ( see Figure 7)• E.coli was susceptible to antibacterial soap (Table 1).• P. alcalifaciens was susceptible to Oxy Power (Table 1).• S. epidermidis had insufficient growth; therefore we could not evaluate the effects of the disinfectants on it.

Questions & Hypothesis

• Label each petri dish with: Bacterial species, Trial #, Disinfectant (see Figure 1)• Dispense 125 µl of bacterial culture into respective petri dish (see Figure 2)• Dip sterile disks in each disinfectant for 3 seconds• Place disk in designated area of petri dish (see Figure 3)• Incubate at 37°C to encourage bacterial growth. (see Figure 4)

• Record measurements of inhibition zone after 24, 48, and 72 hours. (see Figure 5)

Methods

• Turmeric’s inhibition zone decreased overtime forboth bacteria but decreased more in E.coli. (seeFigure 9 & 10).

• The inhibition zone sizes of antibacterial soap and teatree oil stayed consistent in P. alcalifaciens over 72hours (see Figure 10).

Results (continued)

1. What disinfectants will generate the largest inhibitionzones, organic or inorganic?

• Alternative hypothesis: Organic disinfectants willcause larger inhibition zones than inorganicdisinfectants.

• Null: There will be no significant difference in thesize of inhibition zones produced by organic andinorganic disinfectants.

2. Which bacteria was most resistant to the disinfectants?• Secondary hypothesis: Providencia alcalifaciens

will have a smaller inhibition zone than E.coli andS. epidermidis when introduced to antibacterialsoap.

• Null: There will be no significant difference in theinhibition zones of P. alcalifaciens, E.coli, and S.epidermidis from antibacterial soap.

Results

Figure 3. Placing

disk in petri dish

Figure 2. Micro pipetting

Acknowledgements

Figure 1. Labeled Petri Dish

Figure 4. Incubator Figure 5. Measuring inhibition zones

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Escherichia coli Providenciaalcalifaciens

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Bacteria Species

24 Hours

Boiled DIwaterTea Tree Oil

Turmeric

AntibacterialSoapOxy

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Boiled DIwaterTea TreeOilTurmeric

Antibacterial SoapOxy

Figure 6. Average inhibition zone size of the bacteria species produced by the compounds in 24 hours



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Time of growth (hours)

P. alcalifaciens

Boiled DI water

Tea tree oil

Turmeric

Antibacterial soap

Oxy-power

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E. coli

Boiled DI water

Tea tree oil

Turmeric

Antibacterial soap

Oxy-power

Figure 9. Average inhibition zone size of E. coli generated by the compounds over 72 hours.

Figure 10. Average inhibition zone size of P. alcalifaciens generated by the compounds over 72 hours

Bacterial

Species

Compounds Average Inhibition Zone

(mm)

Bacteria Response Antimicrobial

Agent(s)

Escherichia coli Boiled DI water 10 Resistant Negative Controlled

(None)Tea Tree Oil (organic) 20 Susceptible Terpinen-4-olTurmeric (organic) 8 Resistant CurcuminAntibacterial Soap

(inorganic)

67 Susceptible Triclosan &

Triclocarban Oxy (inorganic) 31 Susceptible Hydrogen Peroxide

Providencia alcalifaciens Boiled DI water 8.5 Resistant Negative Controlled

(None)Tea Tree Oil (organic) 13 Intermediate Terpinen-4-olTurmeric (organic) 8 Resistant CurcuminAntibacterial Soap

(inorganic)

11 Intermediate Triclosan &

Triclocarban Oxy (inorganic) 50 Susceptible Hydrogen Peroxide

Discussion and ConclusionsBased on our limited results, we fail to reject the null

hypothesis and cannot accept the alternative hypothesis of our primary question because organic disinfectants failed to produced a larger inhibition zone than inorganic disinfectants. However, we can accept our secondary hypothesis because the antibacterial soap produced a smaller inhibition zone in the P.alcalifaciens than it did in the E.coli at 24 hours (seeTable 1).

Some limitations included time (restricted to a 5 week program), and materials (limited to FSU Biology Department and RMSC resources).

According to our results, we can conclude that antibacterial soap is efficient at inhibiting E.coli growth. Based on previous research, antibacterial soap could also be dangerous to the skin due to the triclosan. BothE.coli and P. alcalifaciens were susceptible to OxyPower; therefore Oxy Power can be recommended for surface sanitation in kitchen and food-prep sites.

In future studies, we would like to explore the components of antibacterial soap that make it resistant to P.alcalifacians. We would also focus on the growth environment of S. epidermidis to understand why it did not grow in this experiment.

Table 1. This chart displays the average inhibition zone, antimicrobial agents, and bacterial response for E. coli and P. alcalifaciens over the course of 24 hours.

Resistant: <10 mmIntermediate: 11-15 mmSusceptible: ≥16 mm⁴

1. Biology Laboratory Resource Center, Truckee Meadows Community College. Escherichia coli [PDF Document]. Retrieved from website: http://biolabs.tmcc.edu/Micro%20Web/ecoli.pdf2. Biology Laboratory Resource Center, Truckee Meadows Community College. Staphylococcus Genus Characteristics [PDF Document]. Retrieved from website: http://biolabs.tmcc.edu/Micro%20Web/Staph.pdf3. Cox, S.D., Gustafson, J.E., Mann, C.M., Markham, J.L., Liew, Y.C., Hartland, R.P., Bell, H.C., Warminton, J.R. & Wyllie, S.G. (1998) Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli. Letters in Applied

Microbiology 78, 264-269.4. Johnson, T. & Case, C. (1995). "Chemical Methods of Control," adapted from Laboratory Experiments in Microbiology, Brief Edition, 4th ed. Benjamin/Cummings Publishing Co., Redwood City, CA.5. Lumen Learning. (N.d.). Modes of Disease Transmission. Retrieved July 14, 2017, from https://courses.lumenlearning.com/microbiology/chapter/modes-of-disease-transmission/6. Scrubbing Bubbles - Products - Fantastik® Oxy Power Multi-Purpose Cleaner. (2017). Retrieved July 20, 2017, from http://www.scrubbingbubbles.ca/en/products/fantastik-oxy-power-multi-purpose-cleaner.aspx7. Zhou, Dongsheng (2015). Bactericidal Activity of Curcumin I Is Associated with Damaging of Bacterial Membrane Retrieved from website: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374920/

We would like to send a special thank you to our Teachers, T.C.s, and program director:

Sarah KuchinskyAmy Fox

Crestyn WhiteGilbert Cochrum

Rita Hegeman

http://biolabs.tmcc.edu/Micro Web/Staph.pdf

The Effects of Household Disinfectants on Bacteria

William Roberts, Avion Lowery

Regional math and science center

Frostburg.edu

Contact1. Michigan State University. (2011). Pharmacology Module. Retrieved July 14, 2017, from http://amrls.cvm.msu.edu/pharmacology

2. Campbell, N. A. & Reece, J. B. (2008). Biology. Pearson Benjamin Cummings, San Francisco, CA.

3. Johnson, T. & Case, C. (1995). "Chemical Methods of Control," adapted from Laboratory Experiments in Microbiology, Brief Edition, 4th ed. Benjamin/Cummings Publishing Co., Redwood City, CA.

4. Lumen Learning. (n.d.). Modes of Disease Transmission. Retrieved July 14, 2017, from https://courses.lumenlearning.com/microbiology/chapter/modes-of-disease-transmission/

5. Rutala, W. A., Barbee, S. L., Aguiar, N. C., Sobsey, M. D., & Weber, D. J. (2000). Antimicrobial activity of home disinfectants and natural products against potential human pathogens. Infection Control and Hospital Epidemiology, 21, 33-38.

6. Barry, A. L., Coyle, M. B., Thornsberry, C., Gerlach, E. H., & Hawkinson, R. W. (1979). Methods of measuring zones of inhibition with the Bauer-Kirby disk Susceptibility test. Retrieved July 14, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC273288/

7. Providencia alcalifaciens (ID 1997). (2012). Retrieved July 13, 2017, from https://www.ncbi.nlm.nih.gov/genome/?term=txid126385

8. Paul W Sherman; Jennifer Billing, Bioscience; June 1999; 49; 6; Research library, Pg. 453

9. Otto, M. (2009). Staphylococcus epidermidis – the “accidental” pathogen. Retrieved July 13, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/

10. Michigan State University. (2011). Microbiology Module. Retrieved July 14, 2017, from http://amrls.cvm.msu.edu/microbiology.

References

Onion produced an inhibition zone that consistently became smaller over time (Figures 1-3).

Mouthwash produced a small inhibition zone in all three bacteria. Between 24 and 72 hours the

inhibition zone size increased for P. alcalifaciens in mouthwash (Figures 3).

The data was compiled into table 1.

Lysol produced the largest inhibition zone in S. epidermidis, E. coli, and P. alcalifaciens (Figures 4-6).

Baking soda produced the second largest inhibition for S. epidermidis and E. coli (Figures 4-6).

Introduction

We used the Disk Diffusion Method (Michigan State University,

2011).

Using a sharpie, we labeled the petri dish and divided it into 5

sections, one for each disinfectant and one for our negative control

(Figure 7).

Using the micropipette, 125 micro liters of bacteria were dispensed

onto the petri dish (Figure 8).

Using a bacterial spreader, we spread the bacteria around the petri

dishes.

Sterile Forceps were used to moisten the sterile disks in the

disinfectants for 3 seconds (Figure 9 and 10).

Sterile disks were placed on corresponding place of petri dishes.

The forceps were re-sterilized and the plating process was repeated

(Figure 9).

The zone of inhibition was measured after 24, 48, and 72 hours of

incubation (Figure 11).

Methods and Materials

This study was conducted to determine the effectiveness of common

disinfectants’ ability to prevent the growth of pathogenic bacteria. Based on our data,

we failed to reject our null hypothesis and cannot accept our alternate hypothesis.

The data showed that there was no significant difference between organic and in-

organic disinfectants. The gram negativity of the bacteria could be used to explain the

data, since gram negative cells have an extra cell membrane. We also observed that

more basic disinfectants inhibited the growth of bacteria better than their acidic

counterparts. We encountered certain limitations such as material and time

constraints. These limitations prevented us from doing more than two trials which

restricted data analysis.

For future studies, we would identify the ingredient in Lysol that actually kills

the bacteria, and once that is found research ways to convert it into a non-toxic skin

ointment because Lysol is toxic to human skin. Another study could be to identify the

active ingredient in the mouthwash we tested to figure out why it’s inhibition zone

increased after 72 hours. Another study could be to study if the gram of the bacteria

actually affects the resistance of the cell.

Discussion and Conclusion

We encounter bacteria on a daily basis. Pathogenic bacteria can lead

to diseases and death. Consumers rely on different types of compounds

to inhibit bacterial growth. We tested four disinfectants on three

different species of bacteria and used boiled distilled water as a

control.

• Staphylococcus epidermidis (S. epidermidis)

• Escherichia coli (E. coli)

• Providencia alcalifaciens (P. alcalifaciens)

Bacteria can be transmitted in a variety of ways: Vehicle transmission,

direct contact, and indirect contact.

Our objective was to determine which works better at inhibiting

bacterial growth, organic or in-organic disinfectants.

The disinfectants tested:

• Lysol (inorganic)

• Mouthwash (inorganic)

• Baking soda (organic)

• Onion extract (organic)

• Boiled DI water (negative control)

Hypotheses:

Alternate: Inorganic disinfectants will inhibit bacterial growth better

than the organic disinfectants.

Null: There will be no difference between organic and in-organic

disinfectants at inhibiting bacterial growth.

Results

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Compound's effect on S. epidermidis

DI water Lysol Baking soda

Onion Mouthwash

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Compound's effect on E. coli


Onion Mouthwash

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Compound's effect on P. alcalifaciens


Onion Mouthwash

Figure 1: This graph show the effectiveness

of the disinfectants against S. epidermidis

over a 72 hour period of time.

Figure 2: This graph show the effectiveness

of the disinfectants against E. coli over a

72 hour period of time.

Figure 3: This graph show the effectiveness of the

disinfectants against P. alcalifaciens over a 72

hour period of time.

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S.epidermis E. coli P. alcalifaciens

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Onion Mouthwash

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48 hours

DI water Lysol Baking soda Onion Mouthwash

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72 hours


Onion Mouthwash

Figure 6: This graph shows the average

size of the inhibition zone created by

the disinfectants after 72 hours.


size of the inhibition zone created by the

disinfectants after 48 hours.


size of the inhibition zone created by

the disinfectants after 24 hours.


S.epidermis DI water 3 0 0

Lysol 39 38.5 41

Baking

soda 20 17 18

Onion 9 0 0

Mouthwash 6 7 7

E. coli DI water 9 0 0

Lysol 15.5 15.5 16

Baking

soda 9 6 6.5

Onion 4 0 0

Mouthwash 5.5 5.5 6

P. alcalifaciens DI water 0 0 0

Lysol 13 14 12.5

Baking

soda 3 3 3

Onion 9 7 0

Mouthwash 5 6 9

Table 1: A table containing the average sizes for the

substances’ inhibition zones over the course of 72 hours.

This is the interpretation chart used

to determine the resistance of the

bacteria (Johnson & Case, 1995)

Figure 11: Measuring the

zone of inhibition.

Figure 7: A labeled petri dish.

Figure 10: Moistening disk

with Lysol.

Figure 9: Sterilizing forceps

with fire.

Figure 8: Plating bacteria using a

micropipette.

≤10mm Resistant

>10 and <16 Intermediate

≥16mm Susceptible

AcknowledgementsThanks to: Amy Fox, Crestyn White, Dylan Boekmann, Gilbert

Cochrum, Rita Hegeman, Sarah Kuchinsky, and Sammy Lagville-

Graham, Angie Ferguson, and Frostburg State University Biology

Department.

Inhibiting Bacterial Growth:

Comparing the Effects of Organic and Inorganic Compounds

Regional Math/Science Center, Frostburg State University, Frostburg, MD, 21532

• Figures 1-3 show the change in average inhibition zone

over time for each bacterial species.

• Figures 4-6 show the average inhibition zone size for

each bacterial species.

• All three bacteria were susceptible to bleach at 24, 48

and 72 hours (Table 2).

• At 24 hours garlic inhibited the growth of E.coli and

P.alcafaciens and promoted growth of S.epidermidis.

Through this experiment we were able to accept our

hypothesis, because bleach, an in-organic disinfectant,

produced a larger zone of inhibition in all three bacterial

species.

One limitation that arose during our study was the

lack of time; we had less than five weeks to conduct this

experiment. If we had more time we would be able to

practice working with the equipment and be able to master

our plating technique. Due to a limited supply of materials

we could only have two trials. If we had the time and

materials to do 10 or 20 trials, our data would better

represent the effect of the disinfectants on the bacterial

population.

A future study could focus on why garlic inhibited

growth after 24 and promoted growth after 48 hours, unlike

the other disinfectants. Another study could research how

bacteria interacts with the antimicrobial agent in each

disinfectant.

Our study had a few flaws, for example the inhibition

zone for bleach was so immense that it invaded other

inhibition zones and made the zones immeasurable .

Another flaw is that our research question was broad and

so we had to analyze the data in a way that would answer

the question and either accept or reject our hypothesis.

INTRODUCTION

Johnson, T. & Case, C. (1995). "Chemical Methods

of Control," adapted from Laboratory Experiments in

Microbiology, Brief Edition, 4th ed.

Benjamin/Cummings Publishing Co., Redwood City,

CA.

Otto,M (2009) “Stapylococcus epidermidis-the

accidental pathogen” Nat Rev Microbiol.2009

August: 7(8):555-567.dol:10.1038/nrmicr2182.

Providencia alcalifaciens (ID 1997). (n.d.). Retrieved

July 20, 2017, from

https://www.ncbi.nlm.nih.gov/genome/?term=txid12

6385

DISCUSSIONS AND CONCLUSIONS

REFERENCES

Bacteria are single celled organisms that cannot

be seen with the unaided eye. Many bacteria are

present naturally on the human body and do no harm

to us. Some bacteria help digest food and give the

body vitamins. Other bacteria are needed to create

nutritious foods like cheese and yogurt. There are

also infectious bacteria which can cause a human to

become sick. These bacteria are known as

pathogenic bacteria. Staphylococcus epidermidis (S.

epidermidis) is a bacteria known for causing skin

boils, styes, and skin infections. S .epidermidis

colonizes on the skin and mucous membranes of

humans and other mammals (Otto,2009).Escherichia

coli (E. coli) is a bacteria that lives in the digestive

tract and can cause anemia or kidney failure which

can lead to death. Providencia alcalifaciens (P.

alcalifaciens) can cause diarrhea and is found in the

gastro intestinal tract (NCBI, 2012). The purpose of this

study is to compare the effectiveness of organic and

inorganic disinfectants (Table 1) on inhibiting bacterial

growth.

Type Compound Active

ingredients

Antimicrobial Properties

Inorganic Bleach Hypochlorous

acid

Heats up to the same degree as the

proteins in the bacteria and destroys

the bacteria

Inorganic Hand sanitizer Ethyl alcohol Kills bacteria by making the lipids in

the outer cell membrane more

soluble to water which causes the

cell membrane to eventually lose its

structure and fall apart

Organic Aloe Phenol Injure the lipids that contain plasma

membranes of bacteria and cause

the cell to leak its cellular contents.

Organic Garlic Allicin Cause chemical reaction with thiol

groups in bacteria

Control Boiled distilled

water

- -

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E. coli

Boiled DI water Aloe Hand Sanitizer Bleach Garlic

Research Question: Which type of

compounds are better at inhibiting

bacterial growth, organic or inorganic

compounds?

Hypothesis: When the bacterial species

S.epidermidis, E.coli, and P.acalifaciens,

are introduced to organic and inorganic

disinfectants, the inorganic disinfectants

will produce a larger zone of inhibition.

METHODS AND MATERIALS (CONTINUED)

METHODS

Table 2. Classification of bacterial response after 24 hours of growth.

Inhibition zones with a diameter of 10mm or less is classified as resistant, a

diameter of 11mm to 15mm is intermediate, and 16 or more is susceptible

(Johnson and case, 1995).

Table 1: Description of disinfectants used.

RESULTS

Figure 1. The change in average inhibition zone size

over three days for the bacterial strain E.coli.Figure 4. The average inhibition zone size for

each bacterial strain after 24 hours.

Figure 5. The average inhibition zone size for



over three days for the bacterial strain S.epidermidis.


over three days for the bacterial strain P.alcalifaciens.

Figure 6. The average inhibition zone size for


• We used the disk diffusion method to test the

disinfectants on the bacteria.

• A petri dish was labeled with the bacterial strain,

name of researcher, date, disinfectants/control and

trial number (see image 1).

• 125µL of the bacteria strain was plated (see

image 2a and 2b).

• Sterile disks were dipped into each disinfectant

(see image 3).

• The petri-dishes were put in to the incubator at 37°

Celsius for 24 hours (see image 4).

• After 24 hours of incubation the inhibition zones

were measured. The inhibition zones

were measured again at 48 and 72 hours (see

image 5).

Thank you to our teachers Sarah

Kuchinsky and Gilbert Cochrum. We

would also like to thank our tutor

counselors Amy Fox, Sydney Greer and

Crestyn White. Also a special thanks

goes to our program coordinators: Rita

Hegeman, Angie Ferguson and Kayla

Precourt. Thank you to Frostburg State

University for providing the equipment

and facilities needed to conduct this

experiment.

ACKNOWLEDGEMENTS

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Staphylococcus epidermis Echerichia coli Providencia alcalifaciens

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Disinfectants

Growth After 24 Hours


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Disinfectants



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Disinfectants



Bacteria DisinfectantsAverage

Size (mm)

Bacterial

Response

Staphylococcus

epidermis

Boiled DI

water8 Resistant

Aloe 10 Resistant

Hand

Sanitizer9 Resistant

Bleach 70 Susceptible

Garlic N/A N/A

Escherichia coli

Boiled DI

water12 Intermediate

Aloe 10 Resistant

Hand

Sanitizer9 Intermediate


Garlic N/A N/A

Providencia

alcalifaciens

Boiled DI

water8 Resistant

Aloe 9 Resistant

Hand

Sanitizer11 Intermediate


Garlic 8 Resistant

Image 1 Image 2a Image 2b

Image 3 Image 4Image 5

Documents

Comparing the effects of organic and inorganic ...Comparing the effects of organic and inorganic disinfectants on bacteria Regional Math and Science Center, Frostburg State University,