2016 Final SIRS Paper

ABSTRACT

The Illinois Junior Academy of Science This form/paper may not be taken without IJAS authorization.

CATEGORY Microbiology STATE REGION # 6

SCHOOL Niles North High School IJAS SCHOOL # 6038 CITY/ZIP Skokie, IL 60077 SCHOOL PHONE # 847.626.2258

SPONSOR RJ Thielsen

MARK ONE: EXPERIMENTAL INVESTIGATION DESIGN INVESTIGATION NAME OF SCIENTIST* Jennifer Goodfriend GRADE 12

NAME OF SCIENTIST GRADE

NAME OF SCIENTIST GRADE

NAME OF SCIENTIST GRADE * If this project is awarded a monetary prize, the check will be written in this scientist's name, and it will be his/her responsibility to distribute the prize money equally among all participating scientists. PROJECT TITLE The Effect of E. coli Concentration on Graphene Oxide Antibacterial Properties Purpose: The purpose of this experiment is to determine if there is a concentration of Escherichia coli where the antibacterial properties of graphene oxide will no longer work and to determine this concentration.

Procedure: LB agar plates were made. E. coli cells were washed with isotonic saline solution in the centrifuge. Bacterial dilutions were created. A portion of each dilution was suspended with graphene oxide (experimental group) and an equal amount was suspended with saline solution (control group). After a shaking incubation, each tube was plated on a separate plate. Plates were incubated overnight and colonies were counted to compare the experimental plates to the control plates.

Conclusion: Based on the results of the trials, the hypothesis was supported because there is a concentration of E. coli that is too large to be affected by the antibacterial properties of graphene oxide. The purpose of the experiment, however, could not be reached due to the already low concentrations of E. coli. A turning point concentration could not be determined. Due to the strange outcomes, the results are inconclusive. These results are inconclusive due to the supposed definite antibacterial properties of graphene oxide that were unable to be completely demonstrated through this type of experimentation and specific procedure.

1) Limit Abstract to 3 paragraphs (about 200 words or less). a) Purpose what you set out to investigate; b) Procedure how you did

it; c) Conclusion based on your results. Label each paragraph. 2) Must be typed, singlespaced on the front of this form. Do not write on the back of this form. 3) Three copies of your complete paper are required at the State Science Project Exposition. Four copies of your complete paper are required for the State Paper Session Competition. This form must be used. This form must be displayed on the front of the exhibitor’s display board. It may be reduced to half a sheet of paper; 8.5inches (vertical) X 5.5 inches (horizontal).

SAFETY SHEET The Illinois Junior Academy of Science

Directions: The student is asked to read these introductions carefully and fill out the bottom of this sheet. The science teacher and/or advisor must sign in the indicated space. By signing this sheet, the sponsor assumes all responsibilities related to this project. Safety and the Student: Experimentation or design may involve an element of risk or injury to the student, test subjects and to others. Recognition of such hazards and provision for adequate control measures are joint responsibilities of the student and the sponsor. Some of the more common risks encountered in research are those of electrical shock, infection from pathogenic organisms, uncontrolled reactions of incompatible chemicals, eye injury from materials or procedures, and fire in apparatus or work area. Countering these hazards and others with suitable safety practices is an integral part of good scientific research. In thechart below,list the principal hazards associated with your project, if any, and what specific precautions you have used as safeguards. Be sure to read the entire section in the Policy and Procedure Manual of the Illinois Junior Academy of Science entitled "Safety Guidelines for Experimentation" before completing this form. Possible hazards Precautions taken to deal with each hazard

E. coli

Graphene oxide

Gloves and goggles were worn. Materials were

sprayed with bleach and disposed of or put through the autoclave and disposed of.

Gloves and goggles were worn. Contaminated materials were autoclaved.

Specific safety practices related to materials requiring endorsement sheets should be detailed on the specific endorsement sheet and not included on this safety sheet. Please check off any other possible endorsements needed. Include these documents in your paper and on your board. _____Humans as Test Subjects –for any projects involving humans including survey administration; __X___Microorganismsfor any projects involving bacteria, viruses, yeasts, fungi or protazoa; _____NonHuman Vertebrates for any projects involving fish, amphibians, reptiles, birds or mammals; _____Tissue Culturefor any projects involving growing eukaryotic tissues or cell cultures; _____Letter from institution where research was done or IJAS SRC, if an exception to the IJAS rules has been granted… SIGNED

Student Exhibitor(s) SIGNED

Sponsor *

*As a sponsor, I assume all responsibilities related to this project.

This Sheet Must Be Typed and This form must be displayed on the front of the exhibitor’s display board. It may be reduced to half a sheet of paper 8.5 inches (vertical) X 5.5 inches (horizontal).

Microorganism Endorsement The Illinois Junior Academy of Science

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No region should send a project to the State Exposition that does not meet these regulations. Students and sponsors doing a microorganism project must complete this form. The signature of the student or students and the sponsor indicates that the project was done within these rules and regulations. Failure to comply with these rules will mean the disqualification of the project at the state level. This form must follow the Safety Sheet in the project paper and on the project board. 1. This area of science may involve many dangers and hazards while experimenting. It is the sole responsibility of all teacher(s)/sponsor(s) to teach students proper safety methods and sterile techniques. 2. The Illinois Junior Academy of Science prohibits the use of primary or secondary cultures taken from humans or other vertebrate animals in any project because of the danger from unknown viruses or other diseasecausing agents that may be present. Pure cultures of microorganisms known to inhabit vertebrate animals may be obtained from reputable suppliers and used in proper settings. 3. Microorganism experiments must be conducted in a laboratory such as science classroom or research facility. 4. Projects involving viruses and recombinant DNA should be done with the help of a professional and should comply with the National Institutes of Health (NIH) Guidelines unless the project is limited to a kit obtained from a legitimate supply house. 5. All cultures should be destroyed by methods such as autoclaving or with a suitable NaOCl (bleach) solution before disposal. Complete all boxes of the following chart. Genus and species of organism(s) being used.

E. coli

Name of the reputable source of the organism(s) being used.

Carolina Biological

Method of disposal of the organism(s) being used.

Spray bleach on all infected objects and put plates/test tubes in the autoclave and dispose in a hazardous bag.

List the location where the lab work was conducted.

Niles North High School

Describe the use of microorganisms in this project.

The E. coli was used to test the antibacterial properties of Graphene Oxide.

Other precautions taken to ensure microorganisms are used safely in this investigation.

Gloves, goggles

The signatures of the student or students and sponsor below indicate that the project conforms to the above rules of the Illinois Junior Academy of Science.

(Sponsor)* (Student)

(Date) (Student)

*As a sponsor, I assume all responsibilities related to this project.

This Sheet Must Be Typed

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Check box if exception/approval letter from an institution where research was done, or the IJAS SRC is required and attached.

Jennifer Goodfriend

NILES NORTH HIGH SCHOOL

THE EFFECT OF E. COLI

CONCENTRATION ON GRAPHENE

OXIDE ANTIBACTERIAL PROPERTIES

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Table of Contents

Acknowledgements…………………………………………………………………….. 2

Purpose…………………………………………………………………………………. 3

Hypothesis………………………………………………………………………............ 3

Review of Literature……………………………………………………………............ 4-7

Materials……………………………………………………………………………….. 8-9

Procedure………………………………………………………………………………. 10-13

Variables……………………………………………………………………………….. 14

Results………………………………………………………………………………….. 15-19

Data Analysis…………………………………………………………………………... 20-21

Conclusion………………………………………………………………………........... 22-23

Experimental Error……………………………………………………………………... 24

References………………………………………………………………………............ 25-26

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Acknowledgements

I would like to thank Ms. Hadallia Bergeron for acting as my mentor through

Northwestern University. She provided continuous guidance and support through the

developmental and experimental phases of my research. I appreciate the time that she dedicated

to my project and my countless questions. In addition, I would like to thank Mr. Richard

Thielsen for providing extra lab hours and guidance with my project. I would also like to thank

my family for supporting my research and providing me with constructive criticism on my work.

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Purpose

The purpose of this experiment is to determine if there is a concentration of Escherichia

coli where the antibacterial properties of graphene oxide will no longer work and to determine

this concentration.

Hypothesis

If the concentration of E. coli increases, then a standard solution of graphene oxide of

concentration 100 µg/mL will only continue to kill E. coli until a plateau is hit which will

saturate the antibacterial effect of the graphene oxide solution.

Rationale: While the amount of E. coli that is killed is directly related to the graphene oxide

solution concentration, there will be a regime in which the graphene oxide does not work as an

antibacterial agent since the graphene oxide will be saturated by the concentration of E. coli.

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Review of Literature

Antibacterial substances are being heavily studied in the field of medicine. For example,

silver is currently being used on catheters to act as an antibiotic. However, it has been

determined that silver decomposes the drugs that are used in the catheters for

chemotherapy. Graphene oxide (GO) has been found to have antibacterial properties and is

being tested as an alternative for silver on catheters (“Graphene coating on catheters may

improve chemo effectiveness,” 2015). “Graphene is a single atomic plane of graphite” and

graphene oxide is an oxidized-graphene sheet which can be exfoliated from graphite oxide.

Graphene materials have unique electronic and thermal properties and have the potential to be

used in nanosensors and nanomedicine (Liu et al, 2011). E. coli is a common bacteria that can

cause diseases (Escherichia coli, 2015). The antibacterial properties of GO were recently tested

with E. coli and the E. coli activity decreased after incubation (Hu et al., 2010). Dilutions were

performed to obtain a specific concentration of graphene oxide solution from a stock

concentration ("Dilutions: Explanations and Examples of Common Methods," n.d.). Once the

antibacterial properties of graphene oxide are fully examined, GO has the potential to sterilize

medical equipment without decomposing the drugs.

Pure graphene is expensive which is why researchers are finding ways to make

inexpensive yet effective graphene derivatives ("Graphene Oxide: An Introduction," n.d.). High

quality equipment is needed to produce viable sheets of graphene through chemical vapor

deposition (CVD), so a graphene oxide solution can be used as an alternative. (“Materials

Science Products,” n.d.). Graphene oxide is a single atomic-layered material which is made by

the oxidation of graphite ("Graphene Oxide: An Introduction," n.d.). Graphene oxide has been

found to exhibit antibacterial properties which is attributed to “membrane stress induced by

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sharp edges of graphene nanosheets, which may result in physical damages on cell membranes,

leading to the loss of bacterial membrane integrity and the leakage of RNA” (Liu et al, 2011).

“Graphene oxide is cheaper and easier to manufacture than graphene, and so may enter

mass production and use sooner” ("Graphene Oxide: An Introduction," n.d.). It is very easy to

make graphene oxide because it is dispersible in water and other solvents. This material is very

similar to carbon nanotubes as carbon nanotubes are made of rolled graphene. Because of the

ability to use graphene oxide “for the production of transparent conductive films,” ("Graphene

Oxide: An Introduction," n.d.) graphene oxide is especially used for solar cells and chemical

sensors. Unfortunately, there are not many health or environmental studies on graphene in

comparison to carbon nanotubes (Liu et al, 2011).

“Escherichia coli (abbreviated as E. coli) are bacteria found in the environment, foods,

and intestines of people and animals” (“Escherichia coli,” 2015). A great deal of E. coli are

harmless, however, some are pathogenic and they cause diseases in their hosts (“Escherichia

coli,” 2015). E. coli are gram-negative; they have thick and strong external walls (Beveridge,

n.d.). In order to culture E. coli, sterile LB nutrient broth and sterile tubes must be used. The E.

coli is incubated at 37ºC in a shaking incubator ("Microbial Growth Protocols," n.d.).

The antibacterial activity of graphene oxide (GO) nanosheets was tested with E. coli and

after a two hour incubation, the “cell metabolic activity for E. coli deceased” (Hu et al.,

2010). This suggests the “strong inhibition ability of GO nanosheets to E. coli” (Hu et al.,

2010). In addition, these GO nanosheets fully suppressed the growth of E. coli. Transmission

Electron Microscopy revealed that the cellular integrity was lost and the cell membrane was

completely destroyed. This caused the cytoplasm to flow out of the cells. It is suggested that

“GO induced cellular damage of E. coli might arise from the effects of either oxidative stress or

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physical disruption that have been observed in cellular effects of related carbon nanomaterial

(CNTs and fullerene)” (Hu et al., 2010).

It is necessary to prepare solutions in order to obtain a diluted solution from a stock

solution ("Dilutions: Explanations and Examples of Common Methods," n.d.). Solutions are

often prepared “by diluting a more concentrated solution.” The number of moles between the

concentrated and diluted solutions remain the same but the amount of solvent is increased in the

diluted solution. The moles in a solution is calculated by multiplying the molarity of the solution

times the volume of the solution. Dilutions can be performed by the equation M1V1=M2V2,

where M is molarity and V is volume ("Dilution Calculations," n.d.).

Serial dilutions are a series of simple dilutions. The solute for each dilution comes from

the solution from the previous dilution step. For example, a ten-fold dilution takes 10 µL of the

previous solution and 90 µL of saline solution or distilled water ("Resource Materials: Making

Simple Solutions and Dilutions," n.d.). Ten-fold dilutions decrease the concentration of a

suspension by a factor of ten. A ten-fold dilution is one-tenth the original concentration

("Appendix 5. Ten-fold Serial Dilutions," n.d.).

Through previous experimentation, it was determined that graphene oxide and graphene-

based products show antibacterial activity. Graphene oxide has the highest antibacterial

activities, and this activity is time and concentration dependent. “Most bacterial inactivation

happens in the first hour of incubation, and cell death rate increases continuously with the

increase of material concentration” (Liu et al, 2011). “Large GO sheets more easily cover cells,

and cells cannot proliferate once fully covered, resulting in the cell viability loss observed in the

followed colony counting test. In contrast, small GO sheets adhere to the bacterial surfaces,

which cannot effectively isolate cells from environment” (Liu et al, 2012).

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In summary, a graphene oxide solution is inexpensive and is an easier to manufacture

alternative to graphene (“Materials Science Products,” n.d.). It has been seen that graphene

oxide exhibits antibacterial properties in terms of bacteria, but unfortunately health and

environmental standards have not been fully reviewed for this substance (Liu et al, 2011). In

order to test bacteria, a common bacteria, E. coli, is used. It is a single-celled organism; a large

amount of E. coli are pathogenic (Escherichia coli, 2012). In order to create a diluted solution of

graphene oxide or E. coli from a highly concentrated solution, one must create dilutions, and the

equation M1V1=M2V2 is used ("Dilution Calculations," n.d.). Serial dilutions are a set of

dilutions where the solute comes from the previous dilution. In the medical world, graphene

oxide is currently being tested on catheters for antibacterial purposes. Unfortunately,

environmental and health issues have not been studied adequately for graphene oxide, so

applications cannot be finalized until these standards are established (Liu et al, 2011). Through

experimentation, a standard solution of graphene oxide will be added to varying concentrations

of E. coli to determine the amount of E. coli that a (previously experimented) mid-range

concentration of graphene oxide is successfully able to destroy. Once there is an amount of E.

coli that is able to be destroyed, researchers can determine if it is safe to use graphene oxide as

an antibiotic in a medical setting.

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Materials and Instruments

(Exact amounts per trial.)

Materials:

E. coli strain

11.5 g LB agar powder

4.0 g nutrient broth powder

1000 mL distilled water

500 mL Erlenmeyer flask (2)

Stirring rod (2)

Heat protectant gloves

Petri dishes (6)

Parafilm

Sterile test tubes (4)

100 µL and 200 µL micropipettes

100 µL and 200 µL micropipette tips

Small centrifugation tubes (1)

Isotonic saline solution (1,280 µL)

Large centrifugation tubes (14)

Test tube rack

500 mg/L solution of graphene oxide (200 µL)

Sterile spreaders (6)

Permanent marker

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Instruments:

Microwave

Refrigerator

Incubator

Shaking incubator

Centrifuge

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Procedure

1. Prepare LB agar plates:

a. Measure 11.5 g agar nutrients and mix with 500 mL of distilled water.

b. Bring to a boil in the microwave periodically agitating the flask to mix the nutrient

agar with the distilled water.

c. Let sit to cool for 15 minutes.

d. Pour agar in petri dishes (use the clam shell method). Only add enough to cover the

bottom of the dish.

e. Let cool and leave in refrigerator overnight.

2. Culture E. coli in nutrient broth

a. Measure 4.0 g of the nutrient broth powder and mix with 500 mL of distilled water.

b. Bring to a boil in the microwave periodically agitating the flask to mix the nutrient

broth with the distilled water.

c. Let sit to cool and cover with parafilm to keep the nutrient broth sterile.

d. Pour nutrient broth into a sterile test tube.

e. Add E. coli to the test tube containing nutrient broth and agitate.

f. Place in shaking incubator at 37 ºC and 250 rpm; incubate overnight.

g. Use a micropipette to take 200 µL of the nutrient broth with E. coli. Transfer to a

centrifugation tube.

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3. Wash procedure:

a. Place tube in the centrifuge for 5 minutes at 9000 rpm to pellet E. coli cells from the

nutrient broth.

b. Discard the supernatant and resuspend cells with 100 µL isotonic saline solution.

c. Place in the centrifuge for 5 minutes at 9000 rpm to pellet cells from the saline

solution.

d. Discard the supernatant.

e. Complete steps b through d two additional times to wash the cells.

4. Create E. coli dilutions:

a. After discarding the final supernatant, to create a 10x dilution, resuspend E. coli cells

with 90 µL isotonic saline solution in a large centrifugation tube.

b. 100x dilution: suspend 10 µL of the 10x dilution with 90 µL isotonic saline solution

in a large centrifugation tube.

c. 1,000x dilution: suspend 10 µL of the 100x dilution with 90 µL isotonic saline

solution in a large centrifugation tube.

d. 10,000x dilution: suspend 10 µL of the 1,000x dilution with 90 µL isotonic saline


e. 100,000x dilution: suspend 10 µL of the 10,000x dilution with 90 µL isotonic saline


f. 200,000x dilution: suspend 50 µL of the 100,000x dilution with 50 µL isotonic saline


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g. 400,000x dilution: suspend 50 µL of the 200,000x dilution with 50 µL isotonic saline


h. 600,000x dilution: suspend 50 µL of the 400,000x dilution with 50 µL isotonic saline


5. Prepare the standard concentration of graphene oxide (100 µg/mL):

a. Use a micropipette to suspend 200 µL of the 500 mg/L solution of graphene oxide

with 800 µL of isotonic saline solution.

6. Preparing control and experimental tubes:

a. Use a micropipette to transfer 25 µL of the 200,000x E. coli dilution to an

experimental tube and 25 µL of the 200,000x E. coli dilution to the control tube.

b. Complete step a with the 400,000x dilution and the 600,000x dilution.

c. Use a micropipette to suspend each control dilution with 100 µL isotonic saline

solution.

d. Use a micropipette to suspend each experimental dilution with 100 µL graphene

oxide solution.

e. Place control and experimental tubes in a shaking incubator and incubate at 37ºC and

250 rpm for 4 hours.

7. Plate each tube:

a. Use a micropipette to transfer all the contents of the 200,000x control dilution to a

labeled plate.

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b. Use a sterile spreader to spread the solution around the entire plate.

c. Complete steps a through b with each control and experimental dilution, plating each

tube on a separate plate.

d. Incubate the plates at 37 ºC for 24 hours.

e. Use the colony counting method to count the number of E. coli colonies on each plate

after incubation (only large E. coli colonies were counted for accuracy).

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Variables

Independent variable: Concentration of E. coli

Dependent variable: Number of colonies of E. coli

Control: E. coli plates containing isotonic saline solution only

Constants: Graphene oxide solution concentration, amount of E. coli plated, LB agar, nutrient

broth, time of incubation, temperature of incubation

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Results

Example plates used to determine the number of colonies on experimental and control plates:

Figure 1: Example 200,000x dilution

control plate

137 colonies

Figure 2: Example 200,000x dilution

experimental plate

16 colonies

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Graph 1: Results and comparison of the 200,000x control and experimental colony numbers.


0

20

40

60

80

100

120

140

1 2 3 4

Nu

mb

er o

f co

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Trials

1 2 3 4

200,000x Control 137 65 80 61

200,000x Experimental 16 62 96 23

200,000x E. coli Dilutions

200,000x Control 200,000x Experimental

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120

140

1 2 3 4

Nu

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Trials

1 2 3 4

400,000x Control 120 95 134 66

400,000x Experimental 99 65 116 41



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Graph 4: Average number of colonies for the 200,000x control and experimental trials.

-10

10

30

50

70

90

110

130

150

1 2 3 4

Nu

mb

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f co

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Trials

1 2 3 4

600,000x Control 95 62 59 32

600,000x Expperimental 99 49 48 36


600,000x Control 600,000x Expperimental

0

50

100

150

1

Nu

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f co

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1

200,000x Control 85.75

200,000x Experimental 49.25

200,000x E. coli Dilutions Average


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0

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100

120

1

Nu

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400,000x Control 103.75

400,000x Experimental 80.25



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150

1

Nu

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600,000x Control 62

600,000x Experimental 58



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Graph 7: Comparison of the average colony numbers for the 200,000x, 400,000x, and

600,000x dilutions.

0

20

40

60

80

100

120

200,000xControl

200,000xExperimental

400,000xControl


600,000xControl


Nu

mb

er o

f C

olo

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200,000xControl


400,000xControl


600,000xControl


Series1 85.75 49.25 103.75 80.25 62 58

Average E. coli Dilutions

200,000x Control 200,000x Experimental 400,000x Control

400,000x Experimental 600,000x Control 600,000x Experimental

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Data Analysis

In order to determine the amount of E. coli that the graphene oxide successfully killed,

control and experimental plates were observed 24 hours after they were plated. According to the

data, there was no substantial decrease on the experimental plates containing graphene oxide. In

addition, the decrease in E. coli concentration did not lead to a decreased number of

colonies. Four trials were performed for each concentration of E. coli. Each trial consisted of a

control plate containing isotonic saline solution and an experimental plate containing a solution

of graphene oxide.

The colony numbers for the 200,000x control concentration varied greatly. The number

of colonies on the 200,000x control plates were 137, 65, 80, and 61. The colony numbers on the

experimental plates also varied; the number of colonies on the 200,000x experimental plates

were 16, 62, 96, and 23. In the third trial, the experimental plate contained a greater number of

colonies (96 colonies) than the control plate (80 colonies). The average number of colonies for

the 200,000x concentration with isotonic saline solution was 85.75 while the average number of

colonies for the 200,000x concentration with graphene oxide was 49.25.

Again, the colony numbers for the 400,000x control and experimental plates were not

consistent. Each 400,000x dilution contained a greater number of colonies than the 200,000x

dilutions even though there was a smaller concentration of E. coli on the 400,000x plates. The

number of colonies per trial on the 400,000x control plates were 120, 95, 134, and 66 and the

number of colonies on the 400,000x experimental plates were 99, 65, 116, and 41. The average

number of colonies for the 400,000x concentration with isotonic saline solution was 103.75

while the average number of colonies for the 400,000x concentration with graphene oxide was

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80.25. The average number of colonies on the control plate are greater than the average number

of colonies on the experimental plates, but this difference is not very significant.

Following the pattern, the numbers of E. coli colonies were not consistent on the

600,000x control and experimental plates either. The number of colonies per trial on the

600,000x control plates were 95, 62, 59, and 32 and the number of colonies per trial on the

600,000x experimental plates were 99, 49, 48 and 36. The experimental plates in trial 1 and trial

4 resulted in a greater number of E. coli colonies than their control plates. The average number

of colonies on the 600,000x control plates was 62 while the average number of colonies on the

600,000x experimental plates was 58. The average number of colonies again decreased on the

experimental plates, but not by a statistically significant amount.

Overall, the number of colonies was not consistent with the dilution factors because the

400,000x dilutions produced more E. coli colonies than the 200,000x dilutions. While most of

the experimental trials of graphene oxide incubation resulted in less E. coli colonies, they did not

demonstrate the significant systematic difference from the control plates necessary to prove

antibacterial properties.

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Conclusion

The purpose of this experiment was to determine the concentration of E. coli that a

standard solution of 100 µg/mL of graphene oxide is able to kill. Four trials were performed and

each trial consisted of three concentrations of E. coli made from serial dilutions of a stock

solution: 200,000x, 400,000x, and 600,000x. Each concentration was plated twice: once with a

standard solution of graphene oxide and once with isotonic saline solution. The number of E.

coli colonies were counted 24 hours later and the experimental plates were compared with the

control plates. Based on the results of the trials, the hypothesis was supported because there is a

concentration of E. coli that is too large to be affected by the antibacterial properties of graphene

oxide. However, a turning point concentration of E. coli could not be determined since all

concentrations seemed to be within the graphene oxide saturation regime. The purpose of the

experiment, thus, could not be reached due to the already low concentrations of E. coli and the

observation of the turning point requires further serial dilutions.

Furthermore, in each trial, the 400,000x plates resulted in a greater number of colonies

than the 200,000x plates. Due to this strange outcome, the results are inconclusive. Graphene

oxide has been previously proven to have antibacterial properties with E. coli, but in this

procedure, each experimental concentration of E. coli did not yield substantially less colonies

than the control plates. Though the average number of colonies for the most experimental plates

with graphene oxide is less, it is not significant enough to indicate strong antibacterial

properties. The E. coli concentrations that were used are already low in comparison to the initial

concentrate, but they could still be too large to have a substantial effect by the graphene

oxide. This proves that there is a concentration of E. coli where graphene oxide does not have

successful antibacterial properties, though it may be lower than expected.

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Multiple procedures were tested with graphene oxide, however the antibacterial

properties could not be consistently demonstrated. In conclusion, these results are inconclusive

due to the supposed definite antibacterial properties of graphene oxide that were unable to be

completely demonstrated through this specific procedure.

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Experimental Error

Due to the inconclusive results, experimental error was highly controlled to try and

minimize uncertainty. This procedure required precise measurements and dilutions. Each

amount was measured using a micropipette and each ten-fold dilution was completed with a

micropipette to reduce experimental error. The micropipette tips used were sterile as were each

test tube, centrifugation tube, LB agar plate, and spreader to prevent the contamination of other

bacteria. Each time a new dilution was worked with, a separate spreader and micropipette tip

was used to prevent the contamination of different E. coli concentrations.

To obtain qualitative results, the E. coli colonies were counted after a 24 hour incubation

at 37ºC. Due to high numbers of miniscule colonies, only large colonies were counted. In

addition, due to the tiny colonies, a standard size was not measured but was estimated. This

could lead to an incorrect number of colonies if smaller colonies were counted on some plates

but not others. To eliminate this uncertainty, an average of the number of colonies on each

control and experimental dilution was taken.

There were three pairs of plates that resulted in a greater number of E. coli colonies on

the experimental plates than the control plates. The antibacterial properties of graphene oxide

did not work at all in these cases and no reasonable explanation can be found besides the fact that

this specific procedure is not conducive to exemplifying antibacterial properties in graphene

oxide.

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References

Appendix 5. ten-fold serial dilutions. (n.d.). Retrieved February 25, 2016, from

http://www.fao.org/docrep/005/ac802e/ac802e0r.htm

Beveridge, T. J. (n.d.). Structures of gram-negative cell walls and their derived membrane

vesicles. Retrieved November 30, 2015, from http://www.ncbi.nlm.nih.gov

/pmc/articles/PMC93954/

Dilution calculations. (n.d.). Retrieved October 28, 2015, from http://dl.clackamas.edu/ch105-

04/dilution.htm

Dilutions: explanations and examples of common methods. (n.d.). Retrieved October 28, 2015,

from http://www.quansysbio.com/dilutions

E. coli (Escherichia coli). (2015, January 07). Retrieved October 26, 2015, from

http://www.cdc.gov/ecoli/

Graphene coating on catheters may improve chemo effectiveness. (2015, July 10). Retrieved

October 26, 2015, from http://www.medicalnewstoday.com/articles/296601.php

Graphene Oxide: An introduction. (n.d.). Retrieved October 28, 2015, from

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2016 Final SIRS Paper

Documents

Transcript of 2016 Final SIRS Paper