Elizabeth Michalochick Pennsylvania Junior Academy of Science (Region 2)

Hazleton Area Academy of Sciences: 12th Grade Sponsor: Boris, R/ Koch, J

Background Practice: Serial Dilution

A dilution is a process that reduces the concentration of a substance in a solution.

A serial dilution’s goal is to achieve a geometric dilution of the original solution.

It is used to accurately create highly diluted solutions.

In order for a dilution to be serial, it must be a dilution where the concentration decreases by the same quantity in each successive step.

Background Term: Colloidal Silver

A liquid mixture of silver nanoparticles and silver ions suspended in distilled water.

Silver itself has been shown to be toxic to bacteria and other microbes.

Silver ions can interrupt important chemical bonds and cellular functions that bacteria need to survive.

Silver nanoparticles can be taken up, or absorbed, by

bacteria, essentially causing this interruption.

It is easier to incorporate silver nanoparticles into products for its antimicrobial benefits.

Background Term: Ampicillin

ß-lactam antibiotic that is part of the aminopenicillin family.

Used to treat certain infections caused by

bacteria. Taken either orally or intravenously.

Commonly used to treat infections like

pneumonia, bronchitis, and ear, lung, skin, and urinary tract infections.

Background Concept: Antimicrobial Resistance

Resistance of a microorganism to an antimicrobial drug that was originally effective for treatment of the infection caused by the microorganism.

Resistant microorganisms are able to withstand attack by antimicrobial drugs.

The evolution of resistant strains is a natural phenomenon that occurs when microorganisms replicate themselves erroneously or when resistant traits are exchanged between them.

Background Concept: Kirby-Bauer Antibiotic Testing

In a Kirby-Bauer test, the size of the zone of inhibition indicates the sensitivity of bacteria to a drug.

A bigger area of bacteria-free media surrounding an antibiotic disk means the bacteria are more sensitive to the drug the disk contains. The zone of inhibition is an area of media where

bacteria are unable to grow, due to presence of a drug that inhibits their growth.

KB tests are performed under standard conditions, so the minimum inhibitory concentration for a given antibiotic can be calculated by comparing the observed zone of inhibition’s size to known values.

Do silver nanoparticles increase the strength of ampicillin against ampicillin resistant bacteria?

Problem:

Purpose: Determine if a collaboration of silver nanoparticles and ampicillin are effective at eliminating ampicillin resistant E. coli, and at what concentration the silver is most effective.

Hypothesis: If colloidal silver at a concentration of 500,000 µg/L is added to ampicillin, then it will increase the strength of the ampicillin and create an inhibition zone, greater than 3 mm.

Independent Variable: Concentration of colloidal silver added to a 10 µg ampicillin disk .

Dependent Variable: The measured width of inhibition zone.

Control Group: Group with ampicillin resistant E. coli and ampicillin disks only.

Disposable gloves Paper towels 70% isopropyl alcohol 250 mL beaker (3) Permanent marker 25mL Graduated cylinder 90 mL Distilled water 25 mL Colloidal silver, 500

PPM Micropipette and tips Large sterile Petri dishes

(22) Labeling or masking tape Hot Plate 1000 mL beaker 250 mL water

Materials: 500 mL Nutrient agar,

prepared media Heat protective gloves Filter paper 1-hole punch Bunsen burner 150 mm sterile cotton

swabs MM294 p/AMP slant

culture of E. coli Tweezers (Sterilized, 3

pairs) 10 µg Ampicillin magazine Metric ruler 10% bleach solution

Procedure: Preparation: Serial Dilution

1. Wear gloves for the entirety of the project and wipe the surface to be worked on with 70% isopropyl alcohol.

2. Label each 250 mL beaker #1 to 3 and with the concentration of silver solution that it will contain. (50,000 µg/L, 5,000 µg/L and 50 µg/L)

3. Put 24.75 mL of distilled water in beakers #2 and #3.

4. Perform a 1:100 serial dilution.

5. Pour 25 mL of colloidal silver into Beaker 1.

6. Take 250 µL from Beaker 1, put into Beaker 2, and swirl.

7. Take 250 µL from Beaker 2, put into Beaker 3, and swirl.

Procedure: Preparation: Agar Plates

8. Label the bottom of the Petri dishes with their Group name (1-7) and Trial number (1-3).

9. Fill the 1000 mL beaker with 250 mL of water.

10. Loosen the cap on the Nutrient agar bottle and place it the hot water bath.

11. Every two minutes, put on heat protectant gloves and swirl the bottle.

12. When the nutrient agar has liquefied, take the bottle out of the beaker, take the beaker off of the hot plate, and turn the hotplate off.

13. Open the bottle and pour it into the Petri dishes.

Procedure: Streaking the Plates

15. Punch 30 circles from a piece of filter paper with a 1-hole punch.

16. Take off gloves and replace them with a new pair.

17. Streaking the Plates: a. Take a sterile 150 mm cotton swab. b. Open the tube of MM294 p/AMP E. coli slant

culture. c. Sterilize the top of the tube. d. Dip the swab into the bacteria tube. e. Rub the tip of the cotton swab against the

agar slant and spread the E. coli on the surface of the agar plate as shown.

15. Repeat step 17 until all 21 plates are streaked.

Procedure: Placing the Disks

20. Using sterile tweezers, place two ampicillin disks, 4 cm away from each other in all Petri dishes in Group 1 (control).

21. For Group 2, use tweezers to individually dip two ampicillin disks in Beaker 1. Place the ampicillin/silver disks 4 cm away from each other in all Petri dishes in Group 2.

22. For Group 3, use tweezers to individually dip two filter paper disks in Beaker 1. Place two silver disks 4 cm away from each other in all Petri dishes in Group 3.

23. For Group 4 using a fresh pair of sterilized tweezers, repeat step 12 but dip the ampicillin disks in Beaker 2 instead of Beaker 1.

24. For Group 5, repeat step 13 but dip the filter paper disks in Beaker 2 instead of Beaker 1.

25. For Group 6 using a fresh pair of sterilized tweezers, repeat step 12 but dip the ampicillin disks in Beaker 3 instead of Beaker 1.

26. For Group 7, repeat step 13 but dip the filter paper disks Beaker 3 instead of Beaker 1.

Procedure: Finish

26. Make sure all plates are closed and secure lid with a few pieces of tape.

27. After 5 minutes flip the plates over so that they are upside down.

28. Place the dishes in an incubator that is set to 37° C.

29. After moving the plates clean up the project area by wiping it with 70% isopropyl alcohol.

30. Check on the plates every day after starting the experiment and look for any bacterial colonies.

31. After bacterial colonies are seen, let the bacteria grow one more day before analyzing.

32. Measure the radius of the inhibition zone with a mm ruler and record in a data table.

33. After observations have been made, sterilize the plates by soaking them in a 10% bleach solution for at least 2 hours and then throw them away.

Procedure: Pictures

Group 5 Group 7

Group 2

Group 3

Group 4 Group 6

Data Analysis: Table

Measurement of Inhibition Zones (in mm) Group 1

(Control) Group 2

(Ag+Amp) Group 3

(Ag) Group 4

(Ag+Amp) Group 5

(Ag) Group 6

(Ag+Amp) Group 7

(Ag)

Ag Concn. ---------- 50,000 𝜇𝑔

𝐿 50,000 𝜇𝑔

𝐿 5,000 𝜇𝑔

𝐿 5,000 𝜇𝑔

𝐿 50 𝜇𝑔

𝐿 50 𝜇𝑔

𝐿

Trial 1 0.00 mm 5.50 mm 1.50 mm 3.50 mm 0.00 mm 0.50 mm 0.00 mm

Trial 2 0.00 mm 6.00 mm 1.00 mm 3.00 mm 0.00 mm 1.00 mm 0.00 mm

Trial 3 0.00 mm 6.00 mm 1.00 mm 2.50 mm 0.00 mm 0.50 mm 0.00 mm

Standard Deviation 0.00 mm 0.34 mm 0.19 mm 0.34 mm 0.00 mm 0.29 mm 0.00 mm

Data Analysis: Graph

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1

2

3

4

5

6

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Zone

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Group and Trial Number

Zone of Inhibition in Groups 1-7

Conclusion: Overall, the undiluted colloidal silver did prove to create a larger zone of inhibition and enhance the antibiotic properties of the ampicillin, against its resistive bacteria.

Hypothesis: If colloidal silver at a concentration of 500,000 µg/L is added to ampicillin, then it will increase the strength of the ampicillin and create an inhibition zone, greater than 3 mm.

The experiment supported the hypothesis.

Possible Errors: Contamination of the Petri dishes. Over/under saturation of the disks.

Practical Applications: Undiluted colloidal silver does increase an

antibiotic’s strength. Silver nanoparticles may be able to be used in

conjunction with a more natural antibiotic like penicillin to treat resistant infections instead of harsh antibiotics.

Further Researching: Research different strains of antibiotic resistant

bacteria in collaboration with different antibiotics.

Repeat the research with more diverse concentrations of colloidal silver.

Resources: Benn, T., & Westerhoff, P. (2008, March 24). Nanoparticle Silver Released into

Water from Commercially Available Sock Fabrics. Retrieved January 12, 2015, from http://www.sludgenews.org/resources/documents/benn_nanosilver.pdf

Brown, A., Smith, K., Samuels, T., Lu, J., Obare, S., & Scott, M. (2012). Nanoparticles Functionalized with Ampicillin Destroy Multiple-Antibiotic-Resistant Isolates of Pseudomonas aeruginosa and Enterobacter aerogenes and Methicillin-Resistant Staphylococcus aureus. Applied and Environmental Microbiology, 78(8), 2768-2774. Retrieved January 12, 2015, from http://aem.asm.org/content/78/8/2768.full

Dilutions: Explanations and Examples of Common Methods. (2013, January 1). Retrieved January 12, 2015, from http://www.quansysbio.com/dilutions

Kirby-Bauer Disk Susceptibility Test. (2014, December 9). Retrieved January 12, 2015, from https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/antimicrobial-drugs-13/measuring-drug-susceptibility-157/kirby-bauer-disk-susceptibility-test-791-6152/

Shrivastava, S., Bera, T., Roy, A., Singh, G., Ramachandrarao, P., & Dash, D. (2007). Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology, 18, 225103-225103. Retrieved February 12, 2015, from http://core.ac.uk/download/pdf/1559971.pdf

The toxicity of antimicrobial silver in products can be reduced. (2010, February 24). Retrieved January 12, 2015, from http://www.eurekalert.org/pub_releases/2010-02/uoh-tto022410.php