Table Of Contents

Acknowledgements....2
Purpose and Hypothesis....3
Review of Literature.4
Materials....8
Procedures..9
Variables...13
Results..14
Data Analysis...18
Experimental Error.19
Statistical Analysis...20
Conclusion....21
Reference List..23

Acknowledgements
Ms. Petrakis, thank you for urging me to go into STEM when I was unaware of all that it
implied. Without your recommendation, I wouldnt have been able to create this experiment that
now holds a special place in my heart.

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To the ever evolving world of makeup, thank you for always keeping me on my toes and
inspiring me daily.
I would also like to thank Ms. Camel for the constant feedback, help and motivation to
improve this project. I dont think it wouldve been as unique and innovative as it was without
your presence.
And to my ever uplifting and courteous family and friends, your support never goes
unnoticed.

Thank you all so very much.

Purpose and Hypothesis

The overall purpose of this experiment is to find an efficient, natural and accessible
alternative formulation when compared to the average lipstick. The underlying purpose of this
experiment is to find an effective alternative to an average preservative with use of natural
antimicrobials/antibacterials.

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Hypothesis: If a lipstick contains natural ingredients and natural antimicrobials/antibacterials that

provide multifunctional properties, then the lipstick will be just as efficient when compared to an
average lipstick.

Rationale: Honey will perform most efficiently since it is able to fight bacteria like Escherichia
coli. Turmeric will perform closely when compared to honey, but not as efficiently, since it isnt
able to fight bacteria like Escherichia coli. Cinnamon will be least effective since it is only an
antibacterial and not an antimicrobial like the cinnamon and turmeric.

Review of Literature
Preservatives, metals, and chemicals added to elongate the life of lipstick and enhance its
formulation tend to be dangerous to the one wearing it. Fortunately, instead of continuing the
fabrication of harmful products, many of which have been correlated to health irregularities,
many companies are formulating lipsticks with less harmful ingredients by taking an overall
more natural approach. However, obvious disparities still exist, and oftentimes the ingredients
consist of a mix of natural and industrial products. If it is possible to eliminate all unnatural

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ingredients in exchange for those that are natural, accessible, and beneficial, then lipsticks would
be less harmful to the consumer.
Formulations of lipsticks tended to be poisonous. The popular carmine color (red)
pigment was extracted from the bodies of cochineal insects, initially in Egypt. While other
pigments found in Mesopotamia was extracted from crushed gems] were not (Metz-LonginetteGahring, 2003). Some cultures continue to use these techniques today, although many stray from
them due to regulations of pigment.
Due to the Federal Food, Drug and Cosmetic Act, color additives must undergo a series
of specific tests in order to indicate their harmlessness (Winter, R. 2009). If the color additive is
approved with regards to the tests, it is then listed in a regulation. Also known as a listing
regulation, the regulation specifies what its made of, how it can be used, and its limitations
(Winter, R. 2009). Specifically for lead, it is limited at 0.04 parts per million. Parts per million is
the unit for chemical concentration. 1 ppm= 0.0001%. Lead, a color additive, varies in color
based on how it is being used in a product. The FDA, though permitting its use in cosmetics, is
now questioning whether to further require an upper limit of lead within lipsticks to protect
welfare and health of customers (U.S. Food and Drug Administration). Based on tests with 400
different lipsticks, lead ranged to be 0.026 parts per million (ppm) to 7.19 ppm. (p>l). The
detection limit, lowest quantity of substance that can be distinguished from the absence of that
substance within stated limit, is set at 0.04 ppm. The average of the 400 lipsticks held at 1.11
ppm of lead.
Also under the Federal Food, Drug and Cosmetic Act, other ingredients are not required
to go through FDA approval. It is entrusted within the cosmetic company that no ingredient will
impose harm on others (U.S. Food and Drug Administration). Preservatives, being of the

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entrusted ingredients, are used to prevent microbial contamination within aqueous cosmetics. Its
principles include: Asepsis (keeping microorganisms out of the process stream), removal of
microorganisms, and retarding growth or killing microorganisms. These principles are utilized
most successfully when in self-preserving products. Products as such contain low pH,
multifunctional material that have antimicrobial activity and combinations of these. Though it
creates a safer use of cosmetics, preservatives arent free of allergenic potential (Kabara, J. J., &
Orth, D. S., 1997).
Even if a product is not in use, it will still grow bacteria. The time it takes to grow
bacteria is solely based on the preservative in the product. Yet, even if the product has an
effective preservative in its ingredients, the preservative itself cant keep the product stable
anywhere longer than 12 months. On average, a lipstick will become full of bacteria with use
after about 6 months (Harper, J. A.). A product purchased at a store isnt likely to have just been
created; it might have been on the shelf for over a year, therefore the product is already spoiled
before use (Harper, J. A.).
Within a lipstick, often one will find similar ingredients. One of those is wax. Yellow
beeswax comes in a form of a flakey faint honey color. Its primarily used as a thickener or
emollient (able to soften or relax), yet it still has emulsifying properties, which means it is able to
combine oil and water into a smooth emulsion. Its components include: esters, free wax acids
and hydrocarbons. Jojoba wax comes in liquid wax form and acts like a moisturizer and
emollient. It can also be used as a anti-wrinkle agent since it does not form a film. Containing
vitamins A and E, shea butter is able to moisturize and boost collagen. Shea butter is an ivory
colored natural vegetable fat (Ingredient Glossary). Vitamin F is able to protect and heal, as well.
Oils are also known to be found in lipsticks. Argan oil, a honey colored liquid with hints of

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hazelnut flavor, is both healing and anti-aging (Benefits of Argan Oil Healing Liquid Gold
From Morocco). Sunflower oil, slightly amber in color, is an emollient and contains emulsifying
properties. Each ingredient containing a benefit of its own combines material that is multifunctioning leading to a self preserving product.
A substance containing antibacterial properties is similar to a substance containing
antimicrobial properties except when regarding the microorganisms they act upon. If it is able to
fight fungi, bacteria and some viruses, it is antimicrobial. If able to stunt the growth of bacteria,
it is antibacterial. Honey is a natural antimicrobial due to its effectiveness against several human
pathogens including Escherichia coli (E. coli), Enterobacter aerogenes, Salmonella typhimurium,
and S. aureus. Its antibacterial compounds can be found in the high fructose concentration,
hydrogen peroxide and low pH (Mandal, M. D., & Mandal, S). A natural spice in Eastern
cultures, turmeric powder is known to kill bacteria and viruses. Specifically Hepatitis C Virus,
Vibrio Vulnificus Infection, and Human Papillomavirus Cancer Cells (Frock, B). The active
component, curcumin, has been tested to prove that it contains antimicrobial properties.
Cinnamon, a caramel powder, has potential for natural food antibacterial application like the two
other natural antimicrobials. It has compounds likely to contribute to its antibacterial properties
like E)-cinnamaldehyde and proanthocyanidins (Agric, J).
Lip makeup, initially created through cultural rituals throughout certain areas of the
world, slowly became a statement of femininity and a common accessory to all within the
cosmetic industry worldwide. But as techniques have developed, makeup technology has in turn
advanced in shelf life and wear. Although this could be beneficial to some degree, the list of
ingredients involved could hold a greater potential risk. With use of more natural ingredients, a
natural antimicrobial could likely act as a natural preservative for an organically natural lipstick

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formulation due to its antibacterial properties. Consuming these natural ingredients also wont
impose a greater threat when compared to regular lipstick ingredients.

Materials
(Bacteria)

Escherichia coli K-12 Nutrient Broth

Incubator
Autoclave bag
Autoclave
Sterile loops

4 grams nutrient agar

150 mL distilled water
20 petri dishes
Erlenmeyer flask
Microwave
Heat protective gloves

3 grams yellow beeswax

0.0500 grams organic cinnamon powder
0.0500 grams organic turmeric powder
0.5000 grams raw organic honey

(Agar)

(Lipstick)

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5 grams raw organic shea butter

3 grams jojoba oil
2 grams argan oil
2 grams sunflower oil
550 mL water
0.450 grams Mac Lipstick Ruby Woo
0.450 grams Burts Bees Lipstick Russet River
Beakers (5 50 mL beakers, 1 400 mL, 1 500 mL beaker)
Hot plate
Refrigerator
Whisk
1 15 mL High Clarity Polypropylene Conical Tube
Blank discs
Sterile cotton swabs
Tweezers

Procedures
(For bacteria)
1. Before working with bacterial cultures, wash your hands with soap and water,
ensure that the work area is draft free, and wipe the work surface with 70% alcohol or
similar disinfectant
2. Transfer broth cultures to fresh broth using a sterile pipet or loop or streak onto
agar using a sterile inoculating loop. For faster growth, you can incubate most cultures at
25 to 30 C (77 to 86 F).
3. After making the transfers, clean the work area with disinfectant and wash your
hands again.
(For lipstick)
1. Mass 5.00 grams of shea butter, 3.00 grams beeswax, 3.00 grams jojoba oil, 2.00
grams sunflower oil and 2.00 grams argan oil.
2. Using a double broiler, melt the beeswax and shea butter in 400 grams of water.
3. Once melted, add jojoba, sunflower and argan oil. Whisk mixture together with a
mini whisk until fully combined.
4. Pour mixture into test tube in order to put into a refrigerator.

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(For agar plates)

1. Mass 4.00 grams of nutrient agar.
2. Measure 150 mL distilled water.
3. Pour both nutrient agar and distilled water into an erlenmeyer flask.
4. Using a microwave, set 30 second increments to heat nutrient agar and water.
When 30 seconds is over, stir then repeat until fully dissolved.
5. Autoclave mixture at 121C for 15 minutes.
6. Let agar cool for 10 minutes before pouring agar into 20 small petri dishes. When
pouring, keep lid as closed as possible. Pour agar until it fully covers the bottom of the
petri dish.
7. When agar is cool and stiff, put petri dishes into the refrigerator.
8. Repeat steps 1-8 for more agar plates.
(For added variables)
1. Wear gloves to eliminate bacteria transfer.
2. Shake tube of bacteria to ensure bacteria is fully distributed.

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3. With a sterile loop, coat 5 petri dishes at once using a zig zag technique. Once
coated, cap the top of the dish and let dry for about 5 to 10 minutes.
4. While dishes are drying, mass 0.45 grams of lipstick.
5. Once dried, coat 5 blank discs evenly with 0.45 grams lipstick. Use tweezers to
hold blank disc in place while using a sterile cotton swab to distribute the lipstick evenly
on each blank disc.
6. Place blank disc in the center of each petri dish. Push blank disk lightly upon agar
to secure its place.
7. Place petri dishes in an incubator at 37C. Let stand for 48 hours before collecting
data.
8. Collect data for trial and discard to be disposed of.
9. .Mass 0.0500 grams cinnamon and 0.45 grams lipstick. Mix together in beaker
using sterile cotton swab.
10. Repeat steps 3, 5-7 with cinnamon mixture.
11. Mass 0.0500 grams turmeric and 0.45 grams lipstick. Mix together in beaker
using sterile cotton swab.
12. Repeat steps 3, 5-7 with turmeric mixture.
13. Mass 0.500 grams honey and 0.45 grams lipstick. Mix together in beaker using
sterile cotton swab.
14. Repeat steps 3, 5-7 with honey mixture.
15. Repeat steps 3-14 for a second trial.
16. Collect data for trial and discard to be disposed of.
(For customer based lipsticks)
1. Wear gloves to eliminate bacteria transfer.
2. Shake tube of bacteria to ensure bacteria is fully distributed.
3. With a sterile loop, coat 5 petri dishes at once using a zig zag technique. Once
coated, cap the top of the dish and let dry for about 5 to 10 minutes.
4. While bacteria is drying, mass 0.45 grams MAC lipstick.
5. With sterile cotton swab, coat 5 blank discs with a sterile cotton swab. Keep each
blank disc in place with a tweezer while coating.
6. Place blank disc in the center of each petri dish. Push blank disk lightly upon agar
to secure its place.

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7. Place petri dishes in an incubator at 37C. Let stand for 48 hours before collecting
data.
8. Collect data for trial and discard to be disposed of.
9. Mass 0.45 grams Burts Bees lipstick.
10. Repeat steps 1-3, and 5-8.
(For disposal of bacteria)
1. Clean work area with disinfectant and wash hands
2. Put used petri dishes in a autoclave bags.
3. Sterilize the bag with the petri dishes contents.
4. Once bag has cooled down enough to handle, throw bag and its contents in trash.

Variables
Independent variables: Cinnamon, turmeric, honey.
Dependent variables: Escherichia coli K-12, zone of inhibition.
Controlled variables: Trial time (48 Hours), temperature (37C), created lipstick, amount of
independent variables added.
Control: Lipstick with no variable, MAC lipstick and Burts Bees lipstick.

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Results
Lipstick Tested

Trial 1

Trial 2

Control

Cinnamon

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Honey

Turmeric

MAC

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Burts Bees

Table 1 visually organizes the two trials done with the lipstick on its own, the lipstick with either
independent variables (cinnamon, turmeric or honey) and two popular lipsticks. For all lipsticks, no
definitive zone of inhibition was created.

Image 1 shows a disc fully coated with naturally created lipstick and turmeric.

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Image 2 provides a close-up of the petri dish coated with E.Coli K-12 and disc covered with naturally
created lipstick and honey set in middle.

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Data Analysis
Hypothesis (refer back to page 2): The results of testing a lipstick combating bacteria was
consistent in all trials with or without the independent variables when compared to the
commercial lipsticks. Though the results did not fully prove that adding a natural
antimicrobial/antibacterial was beneficial to the lipstick as a whole, it did nonetheless show a
lipstick created with natural ingredients was just as efficient as a commercial lipstick. For both
trials, the efficiency of the lipstick to work as a commercial product was maintained in terms of
consistency, long-term sustainability and long-term film. Therefore the hypothesis proved to be
successful.

Rationale (refer back to page 2): The results demonstrated that not one independent
variable was more efficient than the other. Though honey had properties that named it able to
fight Escherichia coli, its inability to create a zone of inhibition proved otherwise. Similarly, the
inability for both turmeric and cinnamon to create a zone of inhibition proves their antimicrobial
and antibacterial properties are ineffective. Therefore, not one independent variable was more
effective than the other, even when comparing their antibacterial or antimicrobial properties.

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Experimental Error
Many occurrences of experimental error were likely if the lab wasnt handled with
enough precaution. While creating agar plates, bacteria could have entered while plates were
being poured, therefore making the plates wouldnt have been fully sterile. Thus, excess bacteria
could have been harbored during the experiment.
While creating the lipstick for the experiment, tools used might have not been fully
disinfected. This would then create excess bacteria in the base of the experiment, leading to
inaccurate results.
The discs used for lipstick coating likely caught bacteria in the air before being coated.
With lipstick overtop of the coating, it creates a weaker lipstick formula that will not efficiently
combat the bacteria.
Though the incubator is effective when keeping the temperature of the incubator
consistent, inflections in temperature could have occurred and stunted the bacterias growth.
Thus, the lipstick couldnt have fought the bacteria fully under inconsistent conditions.
Each trial was set to be done in 48 hours. Though that was enough time to create a zone
of inhibition, the amount of time limited one to see what would happen beyond creating a zone
of inhibition. The lipstick could have created fungi with bacteria surrounding it or kept itself
intact.
Being that none of the lipsticks and added variables created a zone of inhibition, it is
likely that Escherichia coli K-12 was too harsh of a bacteria to test lipsticks with. Better results

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would have been found if a bacteria common to lipsticks was tested with instead of the E. Coli
itself.
Statistical Analysis
Because numerical values were not available for this experimentation, statistical analysis
was done through the means of statistical visual analysis. In regards to pigmentation, both trials
proved that the pigmentation remained the same as it would with the two control products, the
MAC lipstick and the Burts Bees lipstick.
Viscosity rates, consistency, dry rate, and overall feel of the substance were recorded as
well, and these results either exceeded or matched up exactly with those of the control group,
thus proving their statistical significance.
Finally, statistical analysis occurred through analyzation of the zone of inhibition. The
zone of inhibition was used to test the way the lipsticks reacted to the bacteria brought into its
environment. All test and control groups were tested in order to see if the substances would
create a zone where the bacteria would not touch/interact with the lipstick, because its chemical
properties and materials meant that the bacteria would be fought off. The statistical test was a
visual test here as well; no machinery was needed, as the human eye was able to see if zones
were created within the petri dishes. Over the period of 48 hours, areas within the petri dish were
observed. However, since there was no zone of inhibition created within the the control groups as
well as the test groups, the test lipsticks worked just as well as the current commercial ones, thus
proving their statistical significance.

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Conclusion
The fundamental purpose of this experiment was to find an alternative formulation of
lipstick that was effective, natural and accessible when compared to a commercial lipstick. This
was to be done by creating a lipstick with naturally curated items, like beeswax, shea butter and
oils, and testing to see how it will combat Escherichia coli K-12 in a petri dish within 48 hours.
To ensure the results of the lipstick would compare the that of a commercial lipstick, popular
consumer based lipsticks, MAC and Burts Bees, were also tested with the E. Coli. Results
proved that both created and commercial lipsticks didnt hold up against the bacteria, thus
making it evident that the natural lipstick is just as effective as the commercial lipstick.
The underlying purpose of this experiment was to test which natural
antimicrobial/antibacterial added to the natural lipstick would make the lipstick more effective at
combating the bacteria. This would then create a natural preservative within the lipstick as
opposed to the common chemical preservatives found in regular lipsticks. The results proved that
even with an addition of cinnamon, turmeric or honey wouldnt change the result of the lipsticks
effectiveness against E. Coli. Thus, no variable was more suitable than the other.
It is evident that natural lipsticks will be just as effective and more beneficial and
accessible to the average consumer since it is created by naturally found items. The lipstick also
wouldnt be overpriced since the ingredients are inexpensive on their own. The time and effort
taken to create the lipsticks will also be cut in half since the tactics of creating chemicals are
unnecessary. Hence why this lipstick formulation altogether is well-rounded and more beneficial
to both the modern consumer in search of a simple product.

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Daily, consumers are making more of an effort to know exactly what they are purchasing
and why. They are becoming aware of what is being put into and on their body for themselves
and those around them. Consumers have already begun taking a stance against makeup
containing harsh chemicals and searching for products with natural ingredients. With use of this
project, it appeals to the modern consumer and takes a new and innovative approach to organic
cosmetology. Furthermore, the technology created with this project can be used to drive the
makeup industry forward, thus mitigating harmful effects of makeup such as melanoma and
various cancers.

Reference List

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Agric, J. (2007, July 15). Result Filters. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/17567030
Benefits of Argan Oil Healing Liquid Gold From Morocco. (2011). Retrieved from
http://undergroundhealthreporter.com/benefits-of-argan-oil-healing/
Frock, B. (2015, June 13). Antimicrobial Activity of Turmeric. Retrieved from
http://www.livestrong.com/article/141168-antimicrobial-activity-turmeric/
Harper, J. A. (2013, August 07). How long before a lipstick grows mold and bacteria? Not as
long as you think once you consider... - The Red Apple Lipstick Blog. Retrieved from
http://blog.redapplelipstick.com/how-long-before-a-lipstick-grows-mold-and-bacteria-not
-as-long-as-you-think-once-you-consider/
Ingredient Glossary. (2016). Retrieved from
http://www.truenatural.com/glossary/entry/Shea_Butter/
Kabara, J. J., & Orth, D. S. (1997). Preservative-Free and Self-Preserving Cosmetics and Drugs.
Retrieved from
https://books.google.com/books?hl=en&lr=&id=qHgB7ZFLOC4C&oi=fnd&pg=PA1&d
q=harmful%2Bpreservatives%2Bin%2Bcosmetics&ots=niyJlfliJV&sig=lE-UAd6EIB6_
m84jszCjYsOzeqU#v=onepage&q=harmful%20preservatives%20in%20cosmetics&f=fal
se
Mandal, M. D., & Mandal, S. (2011, April). Honey: Its medicinal property and antibacterial
activity. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609166/
Metz-Longinette-Gahring, 2003. Ask Candace: World Loyality Lipstick. The Origin of The
Lipstick. Retrieved October 5th, 2015, from: http://www.askcandace.com/Lipstick.pdf
U.S. Food and Drug Administration. (2011, December 5). Retrieved from
http://www.fda.gov/Cosmetics/ProductsIngredients/Products/ucm137224.htm
U.S. Food and Drug Administration. (2014, March 26). Retrieved from
http://www.fda.gov/Cosmetics/ProductsIngredients/PotentialContaminants/ucm388820.ht
m
Winter, R. (2009). A Consumer's Dictionary of Cosmetic Ingredients, 7th Edition. Retrieved

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November, 2015, from

https://books.google.com/books?hl=en&lr=&id=3hLmyma2-MsC&oi=fnd&pg=PA7&dq
=bacteria%2Bfound%2Bin%2Bcosmetics%2B&ots=DyFSQbBVqK&sig=fSfZba284Ha
VWV0YLzLZzP09gk0#v=onepage&q=bacteria%20found%20in%20cosmetics&f=false

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