Curcuminonecoli Final Paper Woo

Hoisington Maleszyk 1
Introduction
Humans have been to the moon, conquered all four corners of the atlas,
and established colonies and countries that peoples ancestors could only have
dreamed of. As the list of accomplishments goes on and on, there have been
drawbacks as well. Plagues have been continuous setbacks for the human race
as a whole; the most notable of these is the Black Plague of the 14th century,
where over 75 million succumbed to the vicious infection (The Black Death,
1348). The human race has come a long way in the medical field since then,
however. Although humans as a people are incredibly powerful in all facets, they
cannot fully prevent negative functions of the inner body and, in turn, are infinitely
fearful of microbial parasites.
The purpose of the project was to determine the effect of varying
concentrations of curcumin, once purified from turmeric, on the growth of E. coli.
Curcumin is a very common spice in places like India and other parts of Asia. It is
used as a remedy for many common illnesses, but has not been scientifically
explored in itself. Through the experiment, the aim was to provide information to
the scientific community that may help bring curcumin to the medical field as an
antibacterial medication.
The curcumin is extracted from the turmeric using a Buchner funnel
filtration system and a few different chemicals. The organic turmeric powder in
varying amounts was mixed with Ethylene dichloride (C 2H4Cl2). After this solution
was stirred, the solution, referred to as EDCT, was then carefully poured through
the filter paper in the Buchner funnel atop the Buchner flask. This process
separated the curcumin from the other impurities of the turmeric powder
(Thomas). Isopropyl alcohol was dripped on the now solid EDCT in order to
further purify the curcumin. The was heated in an incubator to fully dry out the
curcumin, and the curcumin was weighed and applied to the E. coli once mixed
in the proper amounts.
In order to test the effect of the different independent variables on the
growth of the E. coli, a colorimeter was used to measure light absorption
(Estapa). A colorimeter is a device that projects light in various wavelengths and
then measures the amount of light that is blocked compared to the light let
through. The ratio that is given explains how much light is blocked, and the light
blocked can be correlated to the amount of bacteria that has grown in the
solution. The three treatments were 0.2 grams of curcumin, 0.5 g, and 0.8 g. The
colorimeter was activated, and results were recorded in the proper data column.
The discrepancy in values was the dependent variable of the experiment, which
were then compared to each other.
The data will be compared through an Analysis of Variance (ANOVA) test
("ANOVA: ANalysis Of VAriance between Groups."). An ANOVA test is perfect for
this collection of data. With the ANOVA test, the means of each different group
are compared to each other. This is more efficient than doing two-sample t-tests,
which would require twelve different statistical tests total.
With the results, the practical applications of curcumin may be discussed.
Curcumin can help drastically in the medical field. It helps make antibacterial
medicines and remedies easy and organic. Globally, this could be a viable
solution for countries that are not wealthy because curcumin is a cheap spice
and is very helpful in preventing sickness. Curcumin is already used in many
Indian dishes because of its cinnamon-like taste. It can be used as a flavoradditive to improve taste of foods that are bland, in addition to the health
benefits. In America, if curcumin were to become more prevalent and readily
consumed, it would strengthen the immune systems of the population and make
the country healthier and stronger overall.
Review of Literature
One of the most notable bacterias of all time is Escherichia coli, (E. coli) a
bacterium that is found in the intestines of healthy people and animals. It is not
typically very dangerous, but there are some strands that are extremely harmful.
The side effects of the harmful strands are vomiting, diarrhea, and abdominal
cramps (E. coli Definition). E. coli exposure occurs through contaminated
water or food, especially undercooked meat or vegetables. From the years 1998
to 2007, 69% of E. coli bacteria stemmed from food sources and 18% from water
sources (MarlerClark). E. coli can be prevented by washing hands before dealing
with food and by always checking food that is about to be eaten to make sure
that it is cooked thoroughly (E. coli Prevention). In the United States, E. coli
annually causes around 73,000 illnesses, and in a study that ran from 19982002, 52% of E. coli illnesses across 49 states were foodbourne, or due to food
contamination (Rangel, Sparling, et al). E. coli is used in this experiment because
of its prevalence and ease of production as well as handling.
Antibacterial medicines are designed to destroy or slow down the growth
of bacteria, whether it is deadly or not (Nordqvist). The plant turmeric, Curcuma
longa, which is a common spice, can be used as an antibacterial if it is in the
proper form. Turmeric can be broken down (Thvar) into a substance called
Curcumin (C21H20O6) through a purification process (Curcumin). Curcumin is the
antibacterial component of the turmeric that can be used to stunt growth and
ultimately control the bacteria. Curcumin is used in many medicines and foods in
the Far East of India and Polynesia (Mercola). It has been found to help promote
the immune system, maintain a healthy digestive system, support a healthy bone
structure, maintain a normal cholesterol level, and promote healthy blood and
liver functions. It can also be used to maintain the levels of many different types
of bacteria.
Curcumin is used in many foods in Southeast Asia and especially India.
Curry is the most popular dish in India and curcumin is one of the main spices
used to flavor and color curry. In India, there are many health problems because
the general population has the sub-par insurance and is lacking resources to
cure many diseases ranging from HIV to the common cold. On average, 35% of
deaths in India are caused by diseases that have a known cure (Sharma). Since
there are cures to these diseases, the people possibly require better nutrition or
supplements to improve their daily living, immune system, and overall health.
Curcumin has health impacts that aide and assist the body in many ways.
Curcumin is an anti-inflammatory and it triggers heat-shock response in the
nerves, which then stimulates the immune system. Also, curcumin can prevent
Alzheimers disease. Alzheimers, caused by a peptide called amyloid beta,
builds up onto the brain and creates deposits, in turn causing memory loss. Since
Indians consume more curcumin than Americans, Indians are 4 times less likely
to contract Alzheimers disease (Benefits of Curcumin). Curcumin also can
eliminate free radicals in the body, which damage and destroy cells and DNA.
Due to its inflammatory properties, researchers at the University of Maryland
recommend to take curcumin supplements daily to relieve symptoms of
osteoarthritis (Ehrlich).
Curcumin is classified as a polyphenol. A polyphenol is a structural class
of organic chemicals characterized by the presence of large multiples of phenol
structures, or anything that has an atomic structure of C6H5. A polyphenol is made
of macromolecules, explaining the carbon and hydrogen. Any polyphenol has a
weight limit for small molecules of 800 Daltons, which makes it possible to rapidly
diffuse across the cell membrane. A Dalton is a unit of measure which is about 10
atoms. Polyphenols are found in the real world every day, typically in dyes in the
production of plastic. The chemical formula for curcumin as stated before is
C21H20O6. It contains multiple phenol structures (Quideau, Deffocux). The natural
phenols give the yellow color to the curcumin. Curcumin is extracted from the
root of Turmeric (Curcuma longa) and because it is a plant extract, curcumin is a
diarylheptanoid. Officially, a diarylheptanoid is a small class of plant secondary
metabolites, which is any organic compound that is not directly involved in the
growth, development, or reproduction of an organism (Fraenkel).
The various properties of curcumin in turmeric make it a safe and effective
method for treating a variety of symptoms and is able to be safely consumed
every day, and it does not taste half bad (its taste resembles ginger). The
accessibility of the compound also makes it cheap and easy to find in any
grocery store.
Problem Statement
Problem:
How can changing the amount of curcumin in a constant amount of
dimethyl sulfoxide, C2H4Cl2, affect the growth of Escherichia coli?
Hypothesis:
If the amount of the curcumin is increased to 0.8 grams, then the
Escherichia coli growth will be inhibited.
Data Measured:
The independent variable in this experiment was the amount of curcumin
diluted in dimethyl sulfoxide. The curcumin, measured in grams (g), had three
different values, based off of the recommended daily serving size of turmeric
(0.2g, 0.5g, 0.8g), the powder the curcumin is extracted from. Absorbance of the
solution in an E. coli broth was measured using a colorimeter and compared to
see how much light was absorbed, ultimately telling the growth of the E. coli. The
difference in percent absorbance was then compared.
In order to assure the results are accurate, numerous controls were used.
There were five controls total (see Experimental Design). Overall, 24 test tubes
were tested. As the collected data is from distinct samples from distinct
populations and many different variables were compared, the data was analyzed
with an Analysis of Variance (ANOVA) statistical test.
Experimental Design
Preparation of Curcumin
Materials:
93.6 g Simply Balanced
Organic Ground Turmeric
1L Ethylene Dichloride,
(C2H4Cl2) 98.97%
500 mL Isopropyl Alcohol,
((CH3)2CHOH) 91%
(3) 50 mL Beaker
Pipette
Watch Glass
Buchner Flask
Scoopula
(2) Hot Mitts
(24) Small Weigh Boats

(9) Large Weigh Boats
Buchner Funnel
(3) Filter Paper
Vacuum Hose
Magnetic Stir
Magnetic Stir Rod
Hot plate
Wax Pencil
108C Incubation Unit
Tongs
Procedures:
1.
Place Buchner funnel in Buchner flask and place filter paper in the funnel.
2.
Add Ethylene Dichloride (EDC) at a ratio of 3ml/g of turmeric powder. Add

15 mL to 5 g of turmeric powder in 150 mL beaker. Complete this 3 times.
Label each beaker (EDCT 1, EDCT2, etc.) using wax pencil.
3.
Place magnetic stir in one of the beakers. Stir solution for 5 minutes on the
hot plate. Repeat for each beaker.
4.
Measure 10 mL of Isopropyl Alcohol (IPA) into 100 mL beaker.
5.
Place vacuum hose on Buchner flask and nearest faucet and turn on the
faucet to begin vacuum seal.
6.
After vacuum seal is created, pour EDCT through filter paper on Buchner
funnel.
7.
Add IPA to Buchner funnel in small amounts using the pipette.
8.
After approximately 3 pipettes are emptied onto the filter paper, remove
filter paper and place on watch glass.
9.
Using the tongs, bring the watch glass over to the 108C incubation unit
and place inside until dry. (Should take approximately 5 minutes.)
10.
Repeat steps 7-10 for each beaker of EDCT. Replace filter paper before
step 7 each time.
11.
Harvest purified curcumin in 50 mL beaker for further experimentation.
E. coli Preparation
Materials:
9.0 g Purified Curcumin
Electronic Balance (0.0001
accuracy)
4.0 g Carolina Dehydrated
Nutrient Broth
(6) Small Weigh Boats
(2) Hot Plates
1 mL Pipette (0.01 mL
accuracy)
5 cm Stirring Magnet
Stirring Magnet Wand
(3) Glass Stirring Rods
Tap Water
500 mL Distilled Water
100 mL Dimethyl Sulfoxide,
(DMSO) 78.13%
Carolina Brand Escherichia
coli bacteria slant
Striker
Bunsen Burner
Vernier Colorimeter
Vernier LabQuest 2.0
(2) 1000 mL Beaker
(1) 1000 mL Erlenmeyer flask
(24) 10 mL Beakers
Foil
Incubator (37C)
TI NSpire Graphing Calculator
(2) Hot Mitts
Tongs
Transfer Loop
White Computer Paper
(24) Kimax Medium Sized
Test Tubes
(24) Kimax Small Sized Test
Tubes
Kimtech Kim Wipes
(2) Test Tube Racks
Tongs
Ethanol Spray Bottle 80%
Hoisington - Maleszyk 10
Broth Preparation
1.
Measure out 500 mL of distilled water using 1000 mL beaker. Pour the 500
mL into the 1000 mL Erlenmeyer flask.
2.
Place a stirring magnet into the Erlenmeyer flask and place the flask onto
a hot
plate. Set the heat dial at high and the stirrer dial at 5.
3.
Measure 4 g of the Dehydrated Nutrient Broth using a weight boat. Pour

the broth into the flask and slowly bring to a boil. Place foil on top of
beaker.
4.
When the broth is boiling, turn off the heat. Remove the stirring magnet
using the magnetic stirring wand.
5.
Using a hot mitt, pour 250 mL of the heated broth into a 500 mL beaker.
6.
Light the Bunsen burner and sterilize the transfer loop. Insert the transfer
loop into the Escherichia coli bacteria slide and scoop out the bacteria
onto the head of the transfer loop.
7.
Insert the transfer loop into the broth and stir the bacteria evenly in the
broth.
8.
Pour the inoculated broth into 12 test tubes. Label tubes accordingly by
each treatment.
9.
Pour the remaining uninoculated broth into the remaining 12 tubes. Label
accordingly.
Application of Curcumin
1.
Weigh 0.2 g of curcumin into 6 separate weigh boats.
2.
Repeat step 1 for 0.5 g variable and 0.8 g variable.
3.
Pour specified amount of curcumin into specified test tube depending on

treatment and broth type for all trials in order previously determined by
randomization.
Incubation of Test Tubes and Recording Data
1.
Label test tubes using tape and wax pencil regarding their treatment.
Place test tubes into rack and place the rack into the 37C incubator.
2.
Incubate the dishes for 24 hours. Remove the test tube rack from the
incubator.
3.
Obtain Vernier Colorimeter and set it to the proper wavelength of 430

nanometers. Allow Colorimeter to heat up for 15 minutes.
4.
Using Eppendorf pipette, transfer solution from test tube to colorimeter

slide. Fill colorimeter slide about full. After transfer is completed, safely
dispose of tip.
5.
Connect colorimeter to LabQuest (See Appendix A for LabQuest setup).
6.
Close colorimeter and begin data collection on LabQuest. Record

absorption rate in data table.
7.
Repeat steps 4 and 5 for each test tube.
Diagram
Figure 1. Method for Slides in Colorimeter

Figure 1 shows how the broth will be transferred from the test tubes into
the colorimeter slide. Using the Eppendorf pipette, transfer the broth from test
tube to colorimeter slide and place into colorimeter. Close the lid of the
colorimeter and begin data collection.
Data and Observations
Table 1
Raw Experimental Data Table
Trial
E. coli with Broth
(+)
Broth (-)
Broth with 0.2 g
Broth with 0.5 g
Broth with 0.8 g

E. coli Broth with
0.2 g
E. coli Broth with
0.5 g
E. coli Broth with
0.8 g
17
3
10
8
14
1
7
18
4
15
2
16
6
5
11
24
21
23
9
22
13
19
20
12
Absorbance Rate
30-Oct
0.321
0.312
0.316
0.253
0.260
0.270
0.929
0.821
0.722
1.256
1.150
1.175
1.373
1.306
1.292
1.023
0.986
1.018
1.187
1.236
1.152
1.446
1.463
1.592
31-Oct
0.425
0.364
0.382
0.265
0.278
0.287
0.842
0.712
0.601
1.172
1.056
1.086
1.293
1.207
1.150
1.527
1.028
1.092
1.111
1.428
1.446
1.355
1.390
1.325
Average
Absorbance Rate
30-Oct
31-Oct
0.316
0.390
0.261
0.277
0.824
0.718
1.194
1.105
1.324
1.217
1.009
1.216
1.192
1.328
1.500
1.357
Table 1 shows the experimental data values that were collected based on
different treatments. The far left column reveals the treatment for each trial. The
treatment details include the materials used. Every trial contained broth, but
E.coli inoculated broth was only used for half, and levels of curcumin varied.
Each trial was randomized, per the non-sequential order on the table. The
average values were taken by adding the three values of each treatment per day
and dividing by 3. The average values are those of which will be used to
calculate the difference between each treatment.
Table 2
Difference of Average Values
No Treatment
0.2gTreatment
0.5gTreatment
0.8gTreatment
Test Tube
1
2
3
1
2
3
1
2
3
1
2
3
Difference in
Absorbance Rate
30-Oct
31-Oct
0.068
0.160
0.052
0.086
0.046
0.095
0.094
0.685
0.165
0.316
0.296
0.491
-0.069
-0.061
0.086
0.372
-0.023
0.360
0.073
0.062
0.157
0.183
0.300
0.175
Average Difference in
Absorbance Rate
30-Oct
31-Oct
0.055
0.114
0.185
0.497
-0.002
0.224
0.177
0.140
Table 2 shows the differences between the absorbance rates of each

respective test tube as well as the averages of these values. The difference was
found by subtracting the E. coli broth by the regular broth for each of the three
test tubes used for each treatment. These are the values that will be used in the
ANOVA analysis test found in the Data Analysis section.
Table 3
Observations Table
E. coli with Broth

(+)
Broth (-)
Broth with 0.2 g
Trial
Observations
17
3
10
8
14
1
7
18
ran out of broth so were evened out E. coli broth test tubes
trial went as planned
strange value compared to other only broth (trial 4)
put in way too much broth, had more broth than all other test
tubes
This was first trial with Researcher 2 stirring the curcumin into
the broth
Other group on same lab table spilled CuCl2 while
researchers were pouring the broth so half was poured at first
and then other half after clean up
beaker was knocked over, pipette fell so broth spilled
some curcumin stuck to weigh boat when pouring into test
tube
4
15
Broth with 0.5 g
Broth with 0.8 g

E. coli Broth with
0.2 g
E. coli Broth with
0.5 g
E. coli Broth with
0.8 g
2
16
6
5
11
24
21
23
9
22
13
19
20
12
Table 3 shows the observations that were recorded throughout data

collection. The most notable observation was in trial 17 when the broth ran out so
it had to be poured into the test tube from other test tubes. This could possibly
affect the growth of the E. coli in the test tubes that poured their broth into it.
Table 4
Curcumin Purification Process Recorded Values
Trial
1
2
3
4
Average:
Incubator Incubator Curcumin Purification

Turmeric
Start Temp End Temp Produced Percentage
Used (g)
(C)
(C)
(g)
(%)
5.0790
5.0717
5.2175
5.2746
5.1607
37
105
116
100
90
95
95
108
104
101
3.2870
2.3710
4.6300
3.6397
3.4819
64.7
46.7
88.7
69.0
67.3
Table 4 contains the values from the curcumin purification process.

Although this data will not be fully analyzed, it is still important to the experiment.
This data was not the values the researchers were searching for, unlike Table 1,
but this part of the experiment was vital to completion. Table 4 shows the amount
of turmeric used for each run or purification, along with the pure curcumin
produced and the percentage of purification, or the amount of curcumin extracted
from the turmeric. Also, the beginning and ending temperatures for the incubator
are included, for they may have had an effect on the amount of curcumin
ultimately produced. The last row of Table 4 is the averages for each column. On
average, approximately 3.5 grams or curcumin was produced per run, and about
5.1 grams or turmeric were used per run. This leads to an average purification
percentage of 67.3%, which is acceptable. This data will not be covered in the
Data Analysis section, because it is not the main experiment.
Table 5
Notable Observations of Curcumin Purification Process
Trial
1
Notable Observations
First run; incubator was not preheated, was turned on when
curcumin wasplaced in; Buchner vacuumstep was completed
while run 2 was stirring
Directlymoved stir fromrun 1to run 2; incubator peaked at
116Cthen declined to 95final temperature; IPA clean up was
required often duringthe first two runs
Used Run 1'sEDCT beaker, washed with water in between;
reddish/brown outside when curcumin wasremoved from
incubator, maybedue to higher temperature; most curcumin
produced; weighed in larger chunks
Use Run 2's EDCT beaker, washed with water in between; only
run that wasalone in incubator for entire duration
Table 5 is the respective observations table for the data Table 4. The
notable observations of each run are included in Table 5. The most important of
these observations are the incubation temperature behaviors, for example how in
trial 2 the incubator peaked at 116C during incubation and then declined to the
end temperature of 95C. This observation is important because it is not reflected
in the data Table 4, but may have had an effect on the amount of curcumin
purified.
Figure 2. Mixing EDCT Solution

Shown in Figure 2 is the mixing of the EDCT solution, or the Ethylene
Dichloride and turmeric. This is the first step in curcumin purification. The hot
plate used the magnetic stir inside of the beaker to mix the solution.
Figure 3. IPA Purification and Vacuum Filtration

Figure 3 showcases the second major part of the curcumin purification
process. The EDCT solution was poured onto the filter paper in the Buchner
funnel attached to the Buchner filtration flask, which was vacuum sealed.
Isopropyl alcohol was then dripped on the (dried) EDCT solid in order to remove
impurities.
Figure 4. Incubation of Curcumin

After the step that took place in Figure 3, the filter paper was moved to a
watch glass. This glass was moved to the incubator and the solid Curcumin was
then incubated to evaporate any remaining liquid, as shown in Figure 4. The
incubator was kept at a temperature of 100C.
Figure 5. Purified Curcumin

After incubation, the purification of curcumin was complete. The final
product of purified curcumin is shown in Figure 5. The solid pile of curcumin was
then crushed, massed, and added to the jar of total purified curcumin.
Figure 6. Test Tubes

Shown in Figure 6 are the test tubes that contained the broth and
inoculated broth solutions with the treatment. They were cleaned and labeled but
not filled at this point. As seen, the clean broth and inoculated broth tubes were
placed in separate racks in order to more quickly and easily distinguish from the
two for experimentation. The tubes in the left rack are the clean tubes without E.
coli and the test tubes in the right rack are the inoculated tubes.
Figure 7. Curcumin Solution Being Mixed

In Figure 7, the curcumin solutions are being made and mixed. These
solutions, once mixed, were then poured into the test tubes containing broth.
They were then incubated for 24 hours.
Figure 8. Test Tubes after Incubation

Shown in Figure 8 are the test tubes after incubation. Although it may be
hard to see from this angle, the E. coli inoculated test tubes were cloudier than
the clean broth test tubes, leading the researchers to believe there was definite
bacterial growth.
Data Analysis and Interpretation
An experiment was conducted testing the effect of varying levels of
curcumin on the growth of E. coli. In the experiment, test tubes containing just
broth and tubes containing just inoculated broth acted as controls, clean broth
with each level of treatment acted as blanks, and all of these were tested against
the inoculated broth tubes containing varying levels of treatments. In order to
improve the precision of the data, each experiment included controls,
randomization, and replication. The controls were used to check the validity of
the data. If the treated tubes were contaminated in any way, the control would
reflect that and allow for a fair comparison. The blanks, or the clean broth tubes
with varying levels of treatment, allowed for a direct comparison to the treatment
tubes, which ultimately gave the researchers the difference in absorbance, the
sought for value for analysis. The order of the trials were randomized using a
calculator program (see Appendix A). Randomization of the trials assures that the
research was a simple random sample, or that each trial had an equal chance of
being selected for each turn. The randomization also assures that the recorded
averages for both populations are unbiased estimators of the population mean
for the respective populations. The absorbance rate data collected was both
continuous, because it has infinite values with connected data points, and
quantitative, because it contains information about specific quantities. The data
that was used in analysis was the average differences of the absorbance rates.
These values are shown below in table 6.
Table 6. Difference of Average Values
No Treatment
0.2gTreatment
0.5gTreatment
0.8gTreatment
Test Tube
1
2
3
1
2
3
1
2
3
1
2
3
Difference in
Absorbance Rate
30-Oct
31-Oct
0.068
0.160
0.052
0.086
0.046
0.095
0.094
0.685
0.165
0.316
0.296
0.491
-0.069
-0.061
0.086
0.372
-0.023
0.360
0.073
0.062
0.157
0.183
0.300
0.175
Average Difference in
Absorbance Rate
30-Oct
31-Oct
0.055
0.114
0.185
0.497
-0.002
0.224
0.177
0.140
Table 6 shows the differences of each test tube along with the averages of
these tubes, depending on each treatment. The difference was found by
subtracting the E. coli broth tubes by the clean broth test tubes. The average was
then found between the three differences of each treatment.
In order to ensure the data can be analyzed, it must be graphed to check
for any trends. This can be found below in Figure 8 and Figure 9.
Figure 9. Dot Plot of Day 1 Data

Figure 9 shows the dot plot for the data of day 1. This shows that the
spread of the data is about 0.2 absorbance units apart. This is a pretty small
spread and analysis can continue with this data.
Figure 10. Dot Plot of Day 2 Data

Figure 10 shows the dot plot for day 2. The spread of the data is about 0.4
absorbance units. This is conclusive evidence that the analysis can continue and
it will be reliable data.
Due to the fact the sample size failed to surpass 30, the Central Limit
Theorem, which guarantees the sampling distribution is normal, was not met.
The normality of the data must, in turn, be checked through normal probability
plots.
Figure 11. Day 1 Normal Probability Plot

In order to insure accurate results, the normality of the data must be
inspected. Although there are not many points to distinguish in Figure 11 it is safe
to say that this data distribution is normal, and therefore the data for Day 1 can
be trusted.
Figure 12. Day 2 Normal Probability Plot

Figure 12 is similar to Figure 11, but for Day 2 instead of Day 1. This data
can also be considered normal, so the normality assumption is met for both days.
The proper statistical test can continue.
An Analysis of Variance (ANOVA) was used for comparison of the various
values. An ANOVA test was appropriate here because the sample means of more
than two populations were compared; in this case, sample means from four
different populations were compared. These populations are the four different
treatments of curcumin; 0.2 g, 0.5 g, 0.8 g, and no treatment. For a sample
ANOVA calculation, see Appendix B. In order for the ANOVA test to be
statistically accurate, three assumptions must be met. The data was collected in
simple random samples, one from each population, so the first assumption was
met. The randomization process can be found in Appendix C. The normal
distribution was assured by the normal probability plots, Figure 11 and Figure 12,
meeting the second assumption. The third assumption is that the populations
have the same standard deviation, , whose value is unknown. In order to check
this, the sample standard deviations can be compared. In order to meet this
assumption, the largest standard deviation cannot be more than two times the
smallest standard deviation. After being checked, it was deemed that they were
similar in value for the first day of data collection (0.011372, 0.102474, 0.079605,
0.114771) and therefore this third assumption was met. For the second day, the
largest sample standard deviation (0.246602) was more than twice as large as
the smallest sample standard deviation (0.040377), and therefore the third
assumption failed to be met. Because of this, the results may be inconclusive
and cannot fully be trusted, but can be investigated nonetheless.
In order to complete an Analysis of Variance test, two hypotheses must
first be determined. These will be known as the null and alternative hypothesis. In
both, represents the population mean for the different populations. o
represents the no treatment population, and x will represent the treatment
groups, with x being instead replaced with the number respective to each
treatment. 1 is the 0.2 g treatment of curcumin, 2 is the 0.5 g treatment, 3 is the
0.8 g treatment, and 4 is the non-treatment of curcumin. The null hypothesis is
that all of these means are equal and that there is no difference in absorbance
rate between the 0.2 g, 0.5 g, 0.8 g, and the non-treatment.
H : 0 = 1 = 2 = 3
o
The hypothesis above is representative of the null. The alternative

hypothesis is different than the null, and states that not all of these means are
equal and that there is a difference between any of the means of the various
treatments. This hypothesis is as follows:
H : not all 0, 1, 2 & 3 are equal
a
The above hypothesis is the alternative. This hypothesis is the alternative

to the null, which is to be accepted if the null is rejected. The alternative states
that not all of the population means are equal. It matters not only how far apart
the means are, but how far apart the means are relative to the variability between
individual observations.
Table 7. ANOVA Values for Day 1

x
Standard Deviation
Plus, Minus
0.055
0.011372
0.185
0.102474
-0.002
0.079605
0.177
0.114771
Table 8. ANOVA Values for Day 2

x
Standard Deviation
Plus, Minus
0.114
0.040377
0.497
0.184582
0.224
0.246602
0.140
0.067668
Shown above in Table 7 and Table 8 is a table of the values needed in

order to complete the Analysis of Variance statistical test. Table 7 and Table 8
include the sample means for each respective sample, the sample size for each,
and the standard deviation of each. Both days are included in the tables.
F=
variation among sample meansbetween each population

MSG
=
populationvariation among individualsall samples of each population MSE
Figure 13. F-Test Equation

Figure 13 shows the equation to determine the F-value to complete the
ANOVA test. The F-value is the variation among sample means between each
population divided by the variation among individuals in all samples of each
population. The F-value is also the mean square group (MSG) divided by the
mean square error (MSE). The equations for each of these can be found in
Appendix B.
Figure 14. Day 1 ANOVA Test Results

Figure 14 shows the results of the ANOVA test for the first day values. The
F-value of 3.37703 along with the p-value of 0.074917 suggests that you must
fail to reject the null hypothesis because the p-value is greater than the accepted
alpha value of .05. There is no evidence that there is a difference between the
population means of the E. coli inoculated test tubes. There is a 7.4917% chance
of acquiring these results by chance alone if the null hypothesis(H ) is true. This
o
test is comparing the differences between E. coli tubes and clean tubes with the
four treatments.
Figure 15. Day 2 ANOVA Test Results

Figure 15 shows the results of the ANOVA test for the second day values.
The F-value of 3.65582 along with the p-value of 0.063311 suggests that you
must fail to reject the null hypothesis because the p-value is greater than the
accepted alpha level of .05. There is no evidence that there is a difference
between the population means of the E. coli inoculated test tubes. There is a
6.3311% chance that results these extreme happen by chance alone if the null
hypothesis (H ) is true.
o
For both days, the null hypothesis was rejected. This means that there is
no evidence that the curcumin had an effect on the growth of the E. coli for each
treatment of curcumin. However, because the assumptions were not fully met for
the second day, results may be inconclusive.
Although the results were not statistically significant, by quickly glancing at
the raw data, the researchers determined that there are trends that may suggest
an effect if further research was conducted. For the first day, it was decided that
the middle treatment of curcumin, 0.5 g, had the greatest effect on inhibiting the
growth of the E. coli because that is the recommended daily serving amount of
curcumin. The absorbance rate of the E. coli tubes was actually less than the
rate in the clean broth tubes, leading the researchers to believe that a minor
broth contamination allowed more bacteria to grow in the clean tube, but the
curcumin prohibited that same growth. On the second day, the third treatment of
curcumin, 0.8 g, appeared to inhibit the growth the most effectively. This is
because the more curcumin over time affected the growth of the E. coli. This
confirms the original hypothesis of the researchers.
Although the middle treatment was more effective the first day, these
results lead the researchers to believe that over time, more curcumin would be
better for prohibiting the growth of bacteria. Interestingly, all of the other tubes
increased in absorbance rate, which means that more E. coli grew during the
second 24 hour period of incubation, which was to be expected. However, the
third treatment group, the 0.8 g curcumin tubes, had a decreased absorbance
rate the second day. Based off these results, the researchers were able to
conclude that the extra curcumin actually killed bacteria over the second
incubation period, instead of simply prohibiting growth.
Conclusion
The intent was to determine whether raising the amount of curcumin in a
test tube, once it was purified from turmeric, could inhibit the growth of
Escherichia coli, E. coli. Three concentrations were of curcumin used. The three
values of this variable were 0.2 grams, 0.5 g, and 0.8 g, the volume of liquid in
which these masses were dissolved was a constant 5 mL. These values were
determined based off of the recommended daily serving of turmeric, the
treatment of 0.5 g. Tubes were filled with broth, half of which were inoculated
while the other half remained clean, and then the curcumin treatment was
applied to the tubes, which were then incubated at 37C for 24 hours before data
collection.
To determine the growth of E. coli, blanks were required. In the
experiment, a blank was a test tube with a treatment, but broth that was never
inoculated. These blanks were important because the curcumin darkened the
color of the test tubes. The E. coli tubes with the same treatment were the same
color as the respective blank, and therefore the only difference was the presence
of the bacteria. In order to test the amount of E. coli growth, a colorimeter was
utilized, an instrument that shines a beam of light through a liquid and measures
absorption. The more E. coli growth, the more light absorbed by the bacteria, and
consequently higher absorption values. The colorimeter was set to a wavelength
of 430 nanometers. This wavelength corresponds to a purple color light, which is
on the opposite end of the visible light spectrum in comparison to orange, the
color of the broth and curcumin solution. The purple and orange combination
allowed the absorption reading to be the most accurate. The values of absorption
were then recorded. There is no SI unit for absorption, it is a coefficient, so it was
simply recorded as the value. The differences between the inoculated tubes and
the blank tubes were then calculated and recorded. After this, an Analysis of
Variance (ANOVA) statistical test was then used to statistically interpret the data.
After the data was analyzed, the researchers rejected the original
hypothesis based on the statistical test. The experiment ultimately showed that
the amount of curcumin did not have a significant effect on E. coli growth.
According to the results of the Analysis of Variance test, there is not sufficient
evidence that raising the amount of curcumin inhibits the growth of E. coli more
effectively. For both days, there was no evidence that the curcumin inhibits the
growth of the E. coli. For day one, there was a 7.5% chance that results this
extreme will happen by chance alone, if the null hypothesis is true. For day two,
there was a 6.3% chance that results this extreme will happen by chance alone,
if the null hypothesis is true. The data was assumed to be normal and results can
be trusted even though there were only four data points. This can be assumed
because of the normality of the probability plots. The Analysis of Variance was
used because multiple sample means were being compared and the researchers
were trying to determine any difference between them.
Through further analysis of the data, the trends in data allowed the
assumption that the curcumin might have actually had an effect on the growth of
the E. coli. The first day of data collection, the middle treatment of 0.5 g of
curcumin seemed to have inhibited growth of E. coli the greatest. Surprisingly,
the absorbance rate of the clean broth tubes was higher than the absorbance
rate of the inoculated tubes, which leads to the belief that a minor contamination
of the clean broth led to some bacteria growth, while the curcumin treatment
prevented such growth in addition to the prevention of the growth of E. coli.
The polyphenols, or a structural class of organic chemicals characterized
by the presence of large multiples of phenol structures, commonly associated
with the structure C6H5, in the curcumin were most active and effective after one
day of incubation in the recommended amount value, most likely because the
highest level of curcumin was so concentrated that the solution was
oversaturated and the polyphenols could not work effectively. In the lowest
treatment, 0.2 g, the lack of curcumin allowed the bacteria to overcome the
presence of the polyphenols and grow regardless of the presence of the
curcumin. This ultimately led to the rejection of the hypothesis, that more
curcumin would be more effective, on the first day of data collection.
The second day of data collection, the highest treatment, 0.8 g of
curcumin, had the greatest effect on the lack of growth of E. coli. All of the other
treatments absorbance rates increased from day one to day two, but the 0.8 g
treatments absorbance value decreased from an average of 1.500 the first day
to 1.357 the second. From this data, it is reasonable to infer that the increased
concentration of curcumin not only prohibited the E. coli from further growth, but
actually killed bacteria over the second incubation period. This can be attributed
to the polyphenols in the curcumin. The increased amount of curcumin allowed
for more polyphenols in the solution by mass, and the increased polyphenols
acted anti-bacterially over time more effectively than the lesser treatments and
were not dissipated as quickly. In the lower treatment groups, the polyphenols
were fully depleted after the first 24 hours of incubation. Therefore, over longer
periods of time, more curcumin is more effective. These results led the
researchers to confirm the original hypothesis that more curcumin is better for
controlling bacteria growth.
The results agree with other works in the field of bacteria. Curcumin is
known to be an antibacterial and stunt the growth of various types of bacteria,
including E. coli. It is mostly used to help promote the immune system by
maintaining the levels of many types of bacteria, including E. coli (Benefits of
Curcumin). This agrees with this work since the more curcumin that was applied
to the E. coli, the less the difference was recorded between the inoculated broth
and clean broth.
There was a negligible amount of problems in the design. One possible
flaw was the E. coli was not allowed enough time to incubate. However, there
was a difference in absorbance values between the inoculated broth and regular
broth tubes that confirmed there was enough time for some bacteria to grow. The
temperature of the incubator that the test tubes were kept in overnight could also
have affected the data. In order to keep a constant temperature, the incubator
must be unopened. Other researchers could have used the incubator,
consequently causing an inconsistent incubator temperature. This would have
stunted the growth of the E. coli in the curcumin solution which would make the
results unreliable. The negative controls exhibited some bacterial growth, more
so than the actual inoculated tubes. Because of this, it was decided that there
was a bacterial contamination in the broth. However, because the same broth
was used for every tube, the contamination would have been constant
throughout every tube, and therefore would not have affected the values. None of
these errors are deemed to be significant in the actual experiment because the
values were of the normal distribution.
In order to further clarify the hypothesis, the scale of the experiment could
be increased. One of the biggest complications, due to time restrictions, was the
lack of data collected. Only 24 samples were used in the experiment, which
allowed for a very small sample size of data to analyze which may have not been
representative of the statistical population. Given more time, many more trials
could have been completed. In addition, more time could have allowed the
analysis of the growth of the E. coli over a longer period of time, and assess the
effects over time as well. Instead of two days of data collection, five could be
beneficial to further research. In other words, a longitudinal study would be
helpful to further clarify the hypothesis.
The benefits of curcumin to modern society are seemingly unending. With
curcumin, antibacterial medicines and remedies are easy and organic. Curcumin,
already very prevalent in the world, can be eaten daily in order to ensure a
strengthened and healthy immune system. It is already used in many areas of
the world as a main dish, which means it is enjoyed by a multitude of cultures
such as India and Polynesia (Mercola), mostly because of its cinnamon-like
taste. Curcumin can be fed to children as a flavoring for things that are normally
flavorless or unpleasant to eat, such as oatmeal, as an added incentive to eat the
necessary nutrients and please the child. Curcumin can also be added to vanillaflavored desserts, such as ice cream, as a flavoring, which could both enhance
the taste of the dessert and provide the consumer with an added health benefit.
Curcumin, once scientifically explored, can offer the world as it is known today
many benefits moving forward. Although curcumin is already prevalent as a food,
research about the pure scientific use is lacking. If curcumin is researched
thoroughly from purely a medical standpoint, maybe eventually producing a daily
vitamin, the benefits to the human race are seemingly unending.
Appendix A: Logger Pro Set Up
The Logger Pro is the tool used to record the absorption over a period of
time.
Materials:
Lab Quest 2
Vernier Colorimeter
Lab Quest Stylus/Pencil
Procedure:
1.
Connect the Vernier Colorimeter into the first input on the top of the Lab
Quest.
2.
Press the power button on the Lab Quest to turn it on. Choose New File
from the File menu.
3.
On the Meter screen, tap Mode using the stylus. Change the data
collection mode to Event Based and change the data collection length to
30 seconds. Select OK.
4.
Start data collection once trial is ready to begin. Complete trial. A level of
absorbance will be displayed on the screen.
5.
Record calculated value into table using graph on the Lab Quest.
Appendix B: Randomization of Trials
The randomization of trials is used to assure validity of data.
Materials:
TI-NSpire Calculator
Procedure:
1.
Turn on TI-NSpire and open a document with a calculator page.
2.
Press Menu, 5: Probability, 4:Random, 2:Integer. This will produce a

command randInt().
3.
Inside of the parenthesis type 1, 24, 24. Press enter.
4.
The twenty-four numbers produced are the corresponding order in which

the trials were ran. Skip over and do not include repeats of trials. Repeat
until each variable tube has a trial assigned. Repeat for each day and
record.
Appendix C: Sample Calculation for ANOVA Test
The ANOVA test was used to check how far apart the sample means are
with how much variation there is between them.
xx =
( n 1x 1 ) + ( n 2 x 2 ) +...+(xx I )
N
Figure 16. xxEquation

Figure 16 shows the equation to find the value for xx. n is the sample size
of each sample. xx1 is the sample mean for each individual treatment. The total
number of observations in all samples is represented by N, which is 12 in this
case.
xx =
( 3.055 ) + ( 3.185 ) + ( 3.002 ) +(3.117 )

12
xx
= 0.10375
n1 (xx 1xx )2+ n2 ( xx2 xx )2 +...+n I ( xx I xx )2

MSG=
I 1
( n 11 )( S 1 )2+ ( n 21 ) ( S 2 )2 +...+( 1)(SI )2

MSE=
N I
Figure 17. MSG and MSE Equations
Figure 17 shows the equations to find the mean square group (MSG) and
the mean square error (MSE). n is the sample size of each sample. xx1 is the
sample mean for each treatment. xxis the number of observations in each sample
times the mean of each sample, similar to weighted means. s is the standard
deviation of each sample. I, in both formulas, is the number of populations, 4 in
this experiment. Finally, N is the total number of observations in all samples,
which is 12 in this case.
MSG=
3(.055.10375) +3 (.185.10375) + 3(.002.10375) +3 (.177.10375)

241
MSG =0.025527
( 31 ) (.011372 )2+ (31 ) (.102474 )2 + ( 31 ) ( .079605 )2+(31)(.114771)2

MSE=
124
MSE = .007535
F=
MSG
MSE
F=.025527 / .007535 = 3.38779

P-value = 0.074917
According to the above calculation, the F-value of 3.38779 along with the
p-value of 0.074917 suggests that you must fail to reject the null hypothesis
because the p-value is greater than the accepted alpha level of .05. There is no
evidence that there is a difference between the population means of the E. coli
inoculated test tubes. There is a 7.4917% chance that results these extreme
happen by chance alone if the null hypothesis (H ) is true.
o
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Hoisington Maleszyk 1

(24) Small Weigh Boats

Add Ethylene Dichloride (EDC) at a ratio of 3ml/g of turmeric powder. Add

Measure 10 mL of Isopropyl Alcohol (IPA) into 100 mL beaker.

Add IPA to Buchner funnel in small amounts using the pipette.

Harvest purified curcumin in 50 mL beaker for further experimentation.

Measure 4 g of the Dehydrated Nutrient Broth using a weight boat. Pour

Weigh 0.2 g of curcumin into 6 separate weigh boats.

Repeat step 1 for 0.5 g variable and 0.8 g variable.

Pour specified amount of curcumin into specified test tube depending on

Incubation of Test Tubes and Recording Data

Obtain Vernier Colorimeter and set it to the proper wavelength of 430

Using Eppendorf pipette, transfer solution from test tube to colorimeter

Connect colorimeter to LabQuest (See Appendix A for LabQuest setup).

Close colorimeter and begin data collection on LabQuest. Record

Repeat steps 4 and 5 for each test tube.

Figure 1. Method for Slides in Colorimeter

Broth with 0.2 g

Broth with 0.5 g

Broth with 0.8 g

Table 2 shows the differences between the absorbance rates of each

E. coli with Broth

Broth with 0.2 g

Broth with 0.8 g

Table 3 shows the observations that were recorded throughout data

Incubator Incubator Curcumin Purification

Table 4 contains the values from the curcumin purification process.

Figure 2. Mixing EDCT Solution

Figure 3. IPA Purification and Vacuum Filtration

Figure 4. Incubation of Curcumin

Figure 5. Purified Curcumin

Figure 6. Test Tubes

Figure 7. Curcumin Solution Being Mixed

Figure 8. Test Tubes after Incubation

Figure 9. Dot Plot of Day 1 Data

Figure 10. Dot Plot of Day 2 Data

Figure 11. Day 1 Normal Probability Plot

Figure 12. Day 2 Normal Probability Plot

The hypothesis above is representative of the null. The alternative

The above hypothesis is the alternative. This hypothesis is the alternative

Table 7. ANOVA Values for Day 1

Table 8. ANOVA Values for Day 2

Shown above in Table 7 and Table 8 is a table of the values needed in

variation among sample meansbetween each population

Figure 13. F-Test Equation

Figure 14. Day 1 ANOVA Test Results

Figure 15. Day 2 ANOVA Test Results

Turn on TI-NSpire and open a document with a calculator page.

Press Menu, 5: Probability, 4:Random, 2:Integer. This will produce a

Inside of the parenthesis type 1, 24, 24. Press enter.

The twenty-four numbers produced are the corresponding order in which

Figure 16. xxEquation

( 3.055 ) + ( 3.185 ) + ( 3.002 ) +(3.117 )

n1 (xx 1xx )2+ n2 ( xx2 xx )2 +...+n I ( xx I xx )2

( n 11 )( S 1 )2+ ( n 21 ) ( S 2 )2 +...+( 1)(SI )2

3(.055.10375) +3 (.185.10375) + 3(.002.10375) +3 (.177.10375)

( 31 ) (.011372 )2+ (31 ) (.102474 )2 + ( 31 ) ( .079605 )2+(31)(.114771)2

F=.025527 / .007535 = 3.38779

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