Which Acne Medication Works Best

Which Acne Medication Works Best? | Brett Larabie

3/7/2015

Which Acne Medication Works Best? Proactive, Simple, or Cucumber Water

Brett Larabie SAMANTHA HORNELL, CANADORE COLLEGE, NORTH BAY, ONTARIO


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Which Acne Medication Works Best?

1 INTRODUCTION

This experiment will test 3 liquid forms of acne medication on Staphyloccocus aureus, and Staphyloccus

epidermis using the Paper Disk Method. There are several types of acne, which are all caused by

different bacteria. However, this experiment will be based around two strains of Staphylococcus, and

how effective Simple, Natural Cucumber Toner, and Proactive are at eliminating the bacteria, and

therefore the acne caused by these strains.

2 METHOD

The experiment will be conducted via the Kirby-Bauer Paper Disc Method (available on page 231 of the

lab manual). Staphylococcus aureus and Staphylococcus epidermis will be applied to 4 labelled Tryptic

Soy Agar plates via a cotton swab (2 plates for each bacteria). There will be 1 paper disc applied to each

half of each plate. Each disc will have been soaked in its associated medication, except for the control

disc which will be soaked in DI water. The plates will be left for 48 hours and then observed, and

measured for their zone of inhibition. The medication with the largest zone of inhibition will be the

medication most effective for killing Staphylococcus epidermis and Staphylococcus aureus that cause

acne.


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3 MATERIALS

o Bunsen Burner

o “Sparker”

o 1 Tube of Escherichia coli broth

o 2 Plates of Tryptic Soy Agar

2.8 g of TSA

70 mL of DI water

250 mL Erlenmeyer flask

Hot plate

2 magnets

Heat glove

o Cotton Swab

o Test tube rack

o 8 un-impregnated paper disks

o 10 mL of DI water (For use as control on both plates)

o Grease Pencil/ Marker for labelling

o Tweezers

o 3 vials for putting medications into

4 PROCEDURAL METHOD

4.1 SCOPE The following procedure is to be implemented in a lab capable of containing bacteria such as

Staphylococcus aureus, and Staphylococcus epidermis, where aseptic technique is to be used at all

times. The procedure will take 48 hours to complete, at which time the TSA plates are to be disposed of

in the proper safety hazardous bag.

4.2 SUMMARY 140mL of Tryptic Soy Agar (enough to make 2 bottles, and approximately 6 plates), will be prepared and

4 plates will be poured. The 3 acne medications and DI water will be transferred into sterilized vials.

Next lab period, 2 TSA plates will be streaked with Staphylococcus aureus, and 2 will be streaked with

Staphylococcus epidermis. Using aseptic technique, each paper disc will be soaked in its designated acne

medication and will then be placed in its appropriate half of the TSA plate. The plates will then be left for

48 hours to allow for growth. After 48 hours is up, the plates will be taken out and the zone of inhibition

will be measured for each medication. Observations and results will be recorded. TSA plates will be

discarded into proper waste bag, and excess bacteria tubes will be placed in “graveyard” (ie; discard

area).


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4.3 DEFINITIONS TSA: Tryptic Soy Agar

Paper Discs: Small paper discs used in the Kirby-Bauer Method as described on Page 231 of the Lab

Manual

DI water: Deionized water

Autoclave: Machine that sterilizes items typically used in a lab setting

PPE: Personal Protective Equipment

Aseptic Technique: Technique followed to ensure no contamination in the lab area

4.4 HEALTH AND SAFETY WARNINGS If this procedure is to be improperly followed, it could result in a Staphylococcus infection. It is very

important to always follow aseptic technique, and to wear PPE at all times.

If at any time bacteria is dropped or spilled, the supervisor is to be alerted immediately, to prevent

further contamination.

Long hair or loose pieces of clothing/jewelry is to be tied back and/or left outside of the lab, as an open

flame will be present in this lab.

4.5 CAUTIONS Be sure to label all test tubes, and agar plates properly to ensure validation of results. Sterilizing cotton

with alcohol solution which is made available during lab period, and sterilizing the lip of test tube with

Bunsen burner flame is vital in this experiment to keep risk of contamination as low as possible.

4.6 INTERFERENCES If by chance the medicated paper disks are placed in the wrong quarter of the plate, this could cause for

inaccuracy in the test. To ensure this does not happen, proper labelling and lab etiquette is necessary

for this lab. One must remain focused on task at hand to ensure accuracy throughout.

4.7 PERSONNEL QUALIFICATIONS/REQUIREMENTS Person and/or persons complete this procedure are to be certified in WHMIS and have a general

knowledge of lab safety. It is also required that the person(s) have a good understanding of the Kirby-

Bauer paper disk method, and the safety precautions/risks of working with Staphylococcus aureus, and

Staphylococcus epidermis


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4.8 EQUIPMENT AND SUPPLIES o Bunsen Burner

o “Sparker”

o Tube of Staphylococcus aureus broth

o Tube of Staphylococcus epidermis broth

o 4 Plates of Tryptic Soy Agar

5.6 g of TSA

140 mL of DI water

250 mL Erlenmeyer flask

Hot plate

2 magnets

Heat glove

o Cotton Swab

o Test tube rack

o 8 un-impregnated paper disks

o 10 mL of DI water

o Grease Pencil/ Marker for labelling

o Tweezers

o 4 vials for putting medications into

4.9 PROCEDURE

4.9.1 Preparation of Tryptic Soy Agar (see Appendix A)

4.9.2 Medication Prep

1. Acquire 4 empty vials

2. Autoclave/sterilize vials to eliminate risk of contamination

3. Fill the first vial with 10 mL of DI water

4. Repeat this step for the next 3 medications

4.9.3 Inoculation of plates

1. Set up Bunsen burner

2. Place 4 plates around Bunsen burner, along with test tube rack, and cotton swab

3. Using aseptic technique, open the Staphylococcus aureus test tube, and flame the lip of the tube

4. Dip the cotton swab inside, and squeeze along the side to prevent dripping on work area.

5. Flame lip of test tube

6. Close test tube

7. Lift lid off of first TSA plate, and streak the plate

8. Do the same for the second plate

9. Repeat for Staphylococcus epidermis test plates


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4.9.4 Paper Disk Method

1. Label each plate as follows

a. Name, bacteria, date

b. Split plate into 4 quarters

c. Label each quarter: DI water, Simple, Cucumber, Proactive

2. Impregnating Disks and Plates

a. Place plates around the Bunsen burner

b. Dip each plate into the appropriate medication using tweezers, and place this plate in

the center of the assigned quarter of each plate (Will be 2 for each medication; one on

each plate) See Appendix B.

c. Close lids and place in cubby at room temperature for 48 hours

4.9.5 Analyze and Measure Zones of Inhibition

1. After 48 hours, remove the plates from respective cubby

2. Observe the growth in each quarter of the plates

3. Measure the zone of inhibition for each medication

4. Record findings

5. Dispose of plates in biohazard bag

4.9.6 Conclusion

1. Complete Final report of findings and conclusion

2. Get an A++

4.10 QUALITY CONTROL & ASSURANCE A control of DI water was included in each infected TSA plate to ensure no contamination on the plate

was present, and be as accurate as possible.

Aseptic Technique was to be followed at all times throughout this procedure.


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5 TIME LINE

Thurs, Feb 19 Tues, March 3 Thurs, March 5 Tues, March 10

Thurs, March 12

Preparation of Tryptic Soy Agar

Preparing glassware for autoclave

Pouring of Agar Plates

Streak 2 plates with Staphylococcus aureus

Streak 2 plates with Staphylococcus epidermis

Place 2 paper discs on each plate, as designated previously. (Each bacteria will be exposed to each medication via paper disc diffusion)

Leave plates for 48 hours

Analyse plates

Measure zones of inhibition on each acne medication

6 RESULTS

Water Control Proactive Toner Simple Natural Cucumber pH 6.5 3.5 4.0 5.5

Zone of Inhibition(S. aureus)

0.2 mm 11 mm 8 mm 0 mm

Zone of Inhibition (S. ep)

0.2 mm 18mm 9 mm 0 mm

See Appendix C and D for Zone of Inhibition

See Appendix E for pH level concerns


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7 DISCUSSION

7.1 OBSERVATIONS Proactive Revitalizing Toner (See appendix F for ingredients) has the most acidic pH, and also had the

largest zone of inhibition. The Natural Cucumber Toner (See Appendix G for ingredients) not only did

not fight against either bacteria, but it actually promoted growth. The Simple toner (See appendix H for

ingredients) also worked quite well and was very similar to Proactive in terms of pH and zone of

inhibition size. The water control, which should not have created any zone of inhibition actually created

a very small one, and acted as a better inhibitor than the Natural Cucumber toner.

7.2 HOW TO IMPROVE This experiment could have been improved by making duplicates of each plate to ensure accuracy of the

plates. The extra growth where the cucumber toner paper disc was placed could have been isolated and

gram stained to see what the growth actually was, and determine how the contamination occurred.

8 CONCLUSION

This experiment went relatively well. The plates grew both bacteria successfully and the paper disc

diffusion method went as planned. Proactive is the best toner of the 3 medications tested, and

successfully inhibits and destroys all Staphylococcus aureus, and Staphylococcus epidermis bacteria.


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9 APPENDIX

9.1 APPENDIX A

Tryptic Soy Agar Preparation:

1. Measure 2.8 g of TSA powder

2. Measure 70 mL of DI water

3. Pour into 250mL Erlenmeyer flask

4. Place smaller magnet of the two inside the flask

5. Place flask on hot plate and turn heat and stirrer on halfway

6. Wait until agar starts to creep up the sides of the flask

7. Remove flask from hot plate, and put on bench

8. Once cooled, pour agar into bottles

9. Autoclave bottle for 10-15 minutes at 121®C at 15 psi

10. Cool and store at room temperature

11. Boil to liquefy

12. Cool to 50®C in water bath for 10 minutes

13. Pour 2 agar plates

14. Allow time to solidify

9.2 APPENDIX B

Plate Inoculation:


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9.3 APPENDIX C

Staphylococcus aureus:

9.4 APPENDIX D

Staphylococcus epidermis:

9.5 APPENDIX E

pH Level Concerns:

When looking for a new skin care product, one with a low pH is recommended (slightly acidic). Products

that are of a high pH level tend to counteract with the skin’s essential oils and has an adverse effect on

the skin, such as dryness and redness. The typical pH of human skin is 5.5, and if a skin care product that

is above 5.5 is introduced to the skin, the skin will reactive negatively as it is trying to reach an

equilibrium.


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9.6 APPENDIX F

Proactive Revitalizing Toner:

Ingredients: water (aqua), glycolic acid, hamamelis virginiana (witch hazel) extract, anthemis nobilis

flower extract, rosa canina fruit extract, aloe barbadensis leaf juice, sodium PCA, panthenol, glycerin,

propylene glycol, allantoin, polysorbate 20, hydroxyethylcellulose, sodium hydroxide, benzophenone-4,

tetrasodium EDTA, imidazolidinyl urea, methylparaben, propylparaben, fragrance (parfum), blue 1 (CI

42090), yellow 5 (CI 19140).

9.7 APPENDIX G

Natural Cucumber Toner

½ of a large organic cucumber blended with ½ cup of tap water

Solution was left to drip until entire solution was pure cucumber water, without solids

9.8 APPENDIX H

Simple Toner

Aqua, Sorbitol, Sodium PCA, Allantoin, Disodium Laureth Sulfosuccinate Methylparaben, Propylene

Glycol, Hamamelis Virginiana Distillate, 2 Bromo-2-Nitropropane-1,3-Diol Chamomilla Recutita Extract,

Phenoxyethanol, Potassium Sorbate, Butylparaben, Ethylparaben Propylparaben, Isobutylparaben.


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10 REFERENCES

http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_p019.shtml#procedure

Microbiological Applications, Benson’s Thirteenth Edition: 2015, Brown S., Smith H.

http://www.international.boots.com/en/Simple-Kind-To-Skin-Soothing-Facial-Toner-200ml_1523/

"Simple Kind To Skin Soothing Facial Toner 200ml." Boots. N.p., n.d. Web. 10 Mar. 2015.

http://www.health24.com/Medical/Skin/Caring-for-your-skin/What-is-skin-pH-20120721

"What Is Skin PH?" Health24. N.p., 19 Jan. 2005. Web. 10 Mar. 2015.

http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_p019.shtml#procedure

http://www.international.boots.com/en/Simple-Kind-To-Skin-Soothing-Facial-Toner-200ml_1523/

http://www.health24.com/Medical/Skin/Caring-for-your-skin/What-is-skin-pH-20120721

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Which Acne Medication Works Best