The Effect of Lemon Juice Concentrate and Temperature on the Growth of Escherichia coli

Emily Eskuri - Meredith Hinz

Macomb Mathematics Science and Technology Center
Biology IDS
Section 9C
Mr. Acre / Mrs. Gravel / Mr. Estapa
28 May 2014

Table of Contents
Introduction..........................................................................................................................1
Problem Statement...............................................................................................................3
Experimental Design............................................................................................................4
Data and Observations.........................................................................................................7
Data Analysis and Interpretation........................................................................................11
Conclusion.........................................................................................................................18
Works Cited........................................................................................................................20

Introduction
What if there was a way to cure cancer? Everyone would want to know about it. It would
be the biggest scientific find of the century. Lemons contain limonin, which is a compound that
fights cancer cells (Gordon). Unfortunately, even though they do contain limonin, it is not
enough to effectively kill cancer cells. However, this does not make lemons useless.
Everyone uses chemicals to clean their houses. People will sometimes even use bleach.
These are harsh chemicals that ruin certain materials if not used correctly. Who wants to take a
chance of ruining their favorite table or cabinet by using a harsh chemical? No one. There is
another alternative to using these chemicals. Lemons! They are everywhere. They are in lemon
water, squeezed to make lemonade, and poured over foods to add flavor. They are very important
to daily life. The citric acid in lemon juice kills bacteria, which means it serves as an
antibacterial. Lemon juice is about 5-6% citric acid by volume. Its concentration of citric acid is
only about 1/3 that of white vinegar, but it has about the same pH of 2 (Acids, Bases, & the PH
Scale). That is because citric acid is a stronger acid than the acetic acid in vinegar (Lemons).
That means that lemon juice citric acid kills more bacteria.
To perform this experiment, there first had to be background knowledge. The purpose of
this experiment was to find the effect of lemon juice concentration and temperature on E. coli.
The reason this topic was picked is, as stated before, lemons contain citric acid which kills
bacteria, so it is important to understand how they affect the growth of bacteria. Citric acid kills
bacteria because it has a low PH that breaks down the cell membranes of bacteria.
The two factors in this experiment were temperature and lemon juice. The reason they
were picked was that they both can have an effect on bacteria. Temperatures below the minimum

(7.8 C) stop bacterial growth but do not kill the organism. However, if the temperature is raised
above the maximum (49 C), bacteria are soon killed (Bacterial E.coli Growth Media).
Escherichia coli grows best at 37 C. It does not grow well at temperatures higher than 42 C
(Bacterial E.coli Growth Media). The temperatures were based on the average persons body
temperature of 38 C. That was the high value. The standard and the low value were picked
because they were within two degrees of the average body temperature.
This project was an original work. The rate of change in E. coli was expected to decline
when more lemon juice and a higher temperature was added.
Scientists have shown that something as simple as lemons can open up thousands of new
pathways to research. How beneficial are lemons? This experiment will go down one of the
many avenues of research.

Problem Statement
Problem Statement
The effect of temperature and lemon juice concentration on Escherichia coli.
Hypothesis
The greater concentration of lemon juice and the higher temperature will increase E. coli
growth.
Data Measured
The independent variables are the temperature and the concentration of the lemon juice.
The temperature will be measured in Celsius. The concentration of the lemon juices will be
measured in milliliters (mL). The dependent variable is the Escherichia coli growth rate. The
growth rate is measured by the change in density over a certain period of time. A two factor DOE

can be used here, with the temperature as one factor, and the amount of lemon juice
concentration as the other factor. 75 trials will be run, with about 6 per day.

Experimental Design
Materials
1 Medium-Sized Lemon

225 mL Water

1 Petri Dish of Escherichia coli

Transfer loop

Incubator (36, 37, 38 degrees Celsius)

Bunsen burner

(5) 9mL Test tubes

Knife

Spectrometer

1 Jar for Lemon Juice Concentrate

Soap

Lemon juicer

Paper Towel

Auger

Eye Dropper

Procedure
Preparing the Lemon:
1.

Get the lemon and clean it off with soap and warm water.

2.

Lay 3 pieces of paper towel around the workspace area.

3.

Take the lemon and the knife and cut the lemon vertically in half.

4.

Take one half of the lemon and place it into the lemon juicer so that the juice will come
out of the holes of the juicer.

5.

Place the jar underneath the juicer so that the lemon juicer.

6.

Squeeze the juicer until no more lemon juice concentrate will come out.

7.

Repeat steps 4-6 for the other lemon half.

Preparing the Petri dish with E. coli:

8.

Take petri dish and label it with last name, date, and starter.

9.

Pour the auger into the Petri dish so that it covers the bottom of the dish.

10.

Take a test tube and put 1 mL of water in it.

11.

Take a swab and swab a test tube of E. coli until it is covered with E. coli.

12.

Take the swab and turn it around in the test tube. The water in the test tube should be
cloudy.

13.

Open the lid of the petri dish and pour in the water from the test tube.

14.

Put the lid back on.

15.

After a few minutes, open the lid and pour out all of the extra liquid.

16.

Close the lid and turn the petri dish upside down.

17.

Place the petri dish into the 38 degree Celsius incubator for 24 hours.

Preparing the test tubes:

18.

Get out the test tubes.

19.

Take each vial and mark them so that each one is labeled as one of these combinations:
Standard, (-,-), (-,+), (+,-), (+,+).

20.

Add 3 mL of water to each test tube.

21.

Turn the Bunsen burner on.

22.

Take the loop and put it over the flame until the loop turns an orange color. That means it
is sterilized.

23.

Take the petri dish and add a swab of E. Coli to each of the test tubes using the loop.

24.

To add a microliter of lemon juice to a test tube, flame the loop and dip the loop into the
lemon juice concentrate once. That is 1 microliter of lemon juice.

25.

For the standard test tube, add 2 microliters of lemon juice then store the tube in an
incubator at a temperature of 37 degrees Celsius.

26.

For the (-,-) test tube, add 1 microliter of lemon juice. Then store the tube in an incubator
at a temperature of 36 degrees Celsius.

27.

For the (-.+) test tube, add 1 microliter of lemon juice. Then store the tube in an incubator
at a temperature of 38 degrees.

28.

For the (+,-) test tube, add 3 microliters of lemon juice. Then store the tube in an
incubator at a temperature of 36 degrees.

29.

For the (+,+) test tube, add 3 microliters of lemon juice. Then store the tube in an
incubator at a temperature of 38 degrees.

Using the Spectrometer:

30.

Prepare the spectrometer. Using the adjustment knob, set the wave length to 500, and the
data to -1.

Reading the Spectrometer:

31.

Put the standard test tube in the spectrometer.

32.

Whatever number it is, add 1 to it because you are subtracting a negative.

33.

Once that is done record the data in a chart.

34.

Repeat steps 11-13 for each test tube and record the data.

35.

Take each test tube and store it back into the correct incubator.

36.

Repeat step 11 again the next day and for the next 15 school days and collect the data.

Diagram
Table 1
Data for Two Runs
(+,+)

Standard

(+,-)

(-,-)

(-,+)

90.4

11.8.2

110.4

128.6

105.8

62.4

50.8

50

54.8

49

50.6

50.2

52.8

31.8

46

48.8

47.4

49.6

47.4

49

47.2

47.4

49.6

50.6

50.4

50

48.6

51.2

48

49.2

45.6

47.2

49.4

42

45.6

56.8

56

56.2

58.8

59.4

53.6

57

56.4

57.4

58.6

10

50.8

51.2

49.8

49.8

50

11

52.2

50.8

50

49.4

49.4

12

75.2

72.8

76.2

78.2

72.4

13

75

76.4

76.4

77.6

77.4

14

76.4

75.4

72.6

75

76.6

15

71.6

76

59.6

77.2

76

Average

59.490909

57.65714

61.47143

60.36364

60.45455

Table 1 shows all of the trials that were taken. It shows how the E. coli grew over a period of 24
hours. Each day the data was collected into the table above.

Data and Observations

Data

Table 2
Two Factors for the Research
Lemon Juice Concentration
(microliters)

Temperature
(degrees Celsius)

Standard
1

Standard
36

37

+
38

Table 2 shows the factors, their standards, and their high and low values for the
experiment. The standard level for the lemon juice was chosen to equal out with the amount of
E. coli used for each test tube. The standard level for the temperature was chosen because that is
average body temperature.

Table 3
Design of Experiment
Rate of E. coli change in lumens
DOE
Standard

(+,+)

(+,-)

(-,-)

(-,+)

90.4

11.8.2

110.4

128.6

105.8

62.4

50.8

50

54.8

49

50.6

50.2

52.8

31.8

46

48.8

47.4

49.6

47.4

49

47.2

47.4

49.6

50.6

50.4

50

48.6

51.2

48

49.2

45.6

47.2

49.4

42

45.6

56.8

56

56.2

58.8

59.4

53.6

57

56.4

57.4

58.6

10

50.8

51.2

49.8

49.8

50

11

52.2

50.8

50

49.4

49.4

12

75.2

72.8

76.2

78.2

72.4

13

75

76.4

76.4

77.6

77.4

14

76.4

75.4

72.6

75

76.6

15

71.6

76

59.6

77.2

76

59.490909

57.65714

61.47143

60.36364

60.45455

Average

Table 3 shows how many DOEs were completed and the exact sequence in which the
trials were performed.

Observations
Table 4
Observations
Date

Observation

March 6th

Trial 1: Tests were run and colonies were higher than expected in test tubes.

March 14th

Trial 6: The (+,+) and (-,+) were moved to the 36 degree incubator on
accident due to another group that moved our test tube rack in there.
Redone with trial 7.

March 7-April 1

Rest of trials varied between 31.8 and 78.2 after the first test was recorded.

Table 4 shows the observations recorded throughout the experiment. Even though there
were some issues that occurred, the experiment stayed on the right path.

Figure 1. Test Tube Trials

Figure 1 shows some of the test tubes that were used for the data collection. The standard
one was not in the picture due to putting it in the spectrometer, but from the left to the right it
goes: (+,+), (+,-), (-,+) and (-,-). The E. coli colonies varied between a few colonies between
each other, but the low standards had higher E. coli colonies.

Data Analysis and Interpretation

Table 5
Factors used in the experiment
Lemon Juice Concentration
(micro liters)
-

Standard
1

Temperature (degrees Celsius)

Standard
36

37

+
38

Table 5 shows the factors that were chosen to see how they affect the growth of E. coli
colonies. The amount of lemon juice concentration (measured in micro liters) and Temperature
(measured in degrees Celsius) were used as the two factors. The table shows the lows, highs, and
standards for each factor. The values for the lemon juice concentrate are: 1 micro liter (low), 2
micro liters (standard), 3 micro liters (high). The values for the temperature are: 36 degrees
Celsius (low), 37 degrees Celsius (standard), 38 degrees Celsius (high). These were chosen
because 37 degrees Celsius is room temperature, and the 36 and 38 degrees were chosen because
they were one degree more or less than the standard.
Table 6
Data for the Fifteen Runs
Growth of E. Coli
DOE

Lemon Juice Concentration (micro liters) Temperature (degrees Celsius)

Standard

Average 59.490909

(+,+)

(+,-)

(-,-)

(-,+)

57.65714

61.47143

60.36364

60.45455

Table 6 shows the change in E. coli colonies measured in lumens. The numbers in this
table represent the results from using a spectrometer. This means that the number represents the
water density. The higher the number is higher there are more bacteria; if the number is lower
there are fewer bacteria. The grand average is 59.9867. To find the grand average, add all of the
averages and divide by four.

Table 7
Data of Standards

62.

50.

48.

47.

50.

45.

Standards
56. 53.

50.

52.

75.

75.

76.

75.6

Table 7 shows how many colonies of E. coli grew under the standards for both lemon
juice concentrate and temperature. The range of standards is 45.6 lumens to 76.4 lumens, making
a range of 30.8 lumens.
Note: 90.4* was an outlier that would change the data and make the range of standards too high,
therefore it was removed from the standards table.

Scatter Plot of Standards

100
80
60
Light Density

40
20
0
0

10

12

14

16

Trial Days

Figure 2. Standards Plot

Figure 2 shows the change in E. coli colonies measured in lumens when given the
standard dose of the factors. The results were as low as 45.6 lumens and as high as 76.4 lumens.
Doubling the range of standards would yield 61.6 lumens.

Table 8
Effect of Lemon Juice Concentrate
Lemon Juice Concentrate
-

60.36364

57.65714

60.45455

61.47143

Avg.
Avg. 59.5643
60.4091

Figure 3. Effect of Lemon Juice Concentrate

Figure 3 shows the effect of lemon juice concentrate. The effect is found from subtracting
the low value, 60.4091 lumens, from the high value, 59.5643 lumens. The effect of lemon juice
concentrate in light density is -0.8448 lumens. This means as the lemon juice increases, the light
density decreases by 0.8448 lumens on average. Table 8 shows the change in E. coli colonies
measured in lumens when lemon juice concentrate was applied. It gives both high and both low
amounts, as well as an average for both. When the high amount of lemon juice concentrate was
applied, an average of 59.5643 lumens was shown. When the low amount of lemon juice was
applied, an average of 60.4091 lumens was shown.

Table 9
Effect of Temperature
Temperature
60.36364
61.47143
Avg.
60.917535

+
57.65714
60.45455
Avg.
59.055845

Figure 4. Effect of Temperature

Figure 4 shows the effect of temperature. The effect of temperature is found by
subtracting the low value 60.917535 lumens, from the high value 59.055845 lumens. The effect
of the Temperature is -1.86169 lumens. This means that as the Temperature increases, the change
in light density decreases by 1.86169 lumens. Table 9 shows the change in light density in the
water when the temperature varied. It gives the high and low amounts, as well as the average for
both of them. When the low amount of temperature was applied, there was an average of
60.917535 lumens. When the high amount of temperature was applied, there was an average of
59.055845 lumens.

Table 10
Interaction Effect
Lemon Juice Concentrate
+
(1
(3
microliter)
microliters)

Temperature

Solid
Segment
Dotted
Segment

+
(38 C)
(36 C)

60.45455

57.65714

60.36364

61.47143

Table 10 is a table comparing the total averages between the Temperature and Lemon
Juice Concentrate when interacting with one another.

62
61
60.45
60.36
60

61.47
Solid Segment (High Temperature)

59
Dotted Segment (Low Temperature)

58

57.66

57
56
1

2
Lemon Juice Concentration

Figure 5. Interaction of Lemon Juice Concentrate and Temperature

Figure 5 shows the interaction effect between lemon juice concentrate. The solid segment
represents the high amount for the temperature and the dotted segment represents the low
amount. The slope of the solid segment is -1.398705 lumens. The slope of the dotted segment is
0.553895 lumens. Knowing these values, the interaction effect can be found. The interaction
effect of these factors is -1.9526 lumens. This is found by subtracting the slope of the dashed
(low) segment from the slope of the solid (high) segment.
It should be noted that when the temperature is held high, on its own (see figure 4), it was
expected to be 59.055845 lumens. In the figure above, 60.45455-57.65714 lumens were recorded
so the average was right in between. Look at the solid segment above which also represents
temperature held high with the interaction of lemon juice concentrate. Notice that when
temperature is low, it was expected to be 60.917535 lumens. In the figure above, the low amount
of temperature was 60.36364-61.47143 so it is in between. The high amount of lemon juice

concentrate yields 61.47143 colonies which is larger than the expected 59.5643 colonies of E.
coli. However, when the temperature is held high, only 57.65714 colonies grew on average.
Further, when lemon juice concentrate is held low, on its own (see figure 3), 60.4091
colonies of E. coli grew on average. Look at the dotted segment above which also represents
lemon juice concentrate held low with the interaction of temperature. Notice that when the
temperature is low, the low amount of lemon juice concentrate yields 60.36364 E. coli colonies,
which is smaller than the average. However when the temperature is high, 60.45455 E. coli
colonies grew on average.
Y= 59.9867+noise
Figure 6. Parsimonious Prediction Equation
Figure 6 shows the parsimonious prediction equation. The difference between this and the
prediction equation is that only the significant effects are used. Nothing is significant so no
effects are included.
Y= 59.9867
Figure 7. Parsimonious Prediction
Figure 7 shows a prediction of 59.9867 colonies. The prediction is only the grand average
because there were no effect variables that were deemed statistically significant.
There was one outlier in the data collection. The first trial was the pre-trial that had an
average of 90.4, so it was taken out due to it affecting the data. The temperature had the biggest
effect on the E. coli and made the rate of change decrease the most. The interaction effect played
a role in how the rate of change was affected.

Conclusion
Overall, the research went well and was a great experience to continue expanding with.
The hypothesis for the experiment was that the greater concentration of lemon juice and the
higher temperature will increase Escherichia coli growth. That hypothesis was accepted. The
high-high factor had the most effect on the rate of change in the E. coli. Escherichia coli were
grown in five test tubes each day. The correct values were added into the test tubes. The test
tubes were then put into the corresponding incubators for 24 hours. The next day they were taken
out of the incubators and put into the spectrometer one-by-one to be observed. The results were
that the high-high value had the most effect on E. coli. This was expected since lemon juice citric
acid kills a lot of bacteria because of its low pH value of 2 (Acids, Bases, & the PH Scale). A low
pH breaks down the cell membrane of bacteria. Lemon juice is also effective on bacteria because
it takes a longer time for bacteria to get used to it and change to become more resistant (Behnke,
Jessica, and Nicholas Johnson). A high temperature kills bacteria also. The 37 degree
temperature was used as the standard factor due to it being body temperature and the optimal
growth of E. coli is 37 degrees Celsius. If 37 degrees was the most favorable temperature to
grow, then the lemon juice would work harder to eliminate. When both of these factors were
combined, they should have had the biggest effect, which was the case. This experiment was
similar an experiment from 2012, The Resistance of Escherichia coli on Lemon Juice and
Baking Soda. It was similar to this experiment because both experiments worked with E. coli
and Lemon Juice concentrate. Although the experiment is similar to this one, not all aspects of
the experiment were kept constant so it resulted in potential variable. This made the experiment
harder to relate to because of its variability. Overall, this experiment was too different from The
Resistance of Escherichia coli on Lemon Juice and Baking Soda to compare results.

There were weaknesses in the design of the experiment that resulted in having different
results. At first the test tubes were not put in their own beaker, so they were moved multiple
times. Since that happened, a few trials had to be done over again. Then the pre-trials were added
into the graph of numbers so the numbers made the values too high. To fix this, the standard
value use in the pretrial was taken out. Another flaw was that the lab was only available four to
five days a week due to weekends and no school during the week so the observations may have
not been accurate because only a few numbers of test tubes were in incubators for multiple days
while most test tubes were in the incubators for only one day.
Some suggestions to give if another person wants to expand on this experiment would be
to raise the high and low ranges to get a bigger difference in the data and analysis. It would
connect the research to the world more because the ranges would be greater. Also, the testing for
the data would give a better understanding if it were recorded in a two sample t test. This
experiment will benefit society because lemons are antibacterials and are very common to use
with foods or beverages, and the food or drink can contain amounts of E. coli due to
contaminated water or raw meat. The lemons can take away some of the E. coli, but some will
still be there. There were some valuable lessons learned when conducting this experiment. A
lesson learned was that when conducting an experiment, there will be mistakes made along the
way, but the key is to be flexible when deciding how to go forward. If there was no flexibility
when conducting experiments, science would not be where it is now. Another lesson learned was
to manage time. The research had 75 trials, expanded over 15 days, or three school weeks. There
were days during the three weeks where there were days off and that threw the schedule off, so it
was difficult to make up the days for the research. Furthermore, the hypothesis was accepted.

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"Bacterial E.coli Growth Media." Bacterial E.coli Growth Media.
Expression Technologies Inc., n.d. Web. <http://www.exptec.com/Expression
%20Technologies/Bacteria%20growth%20media.htm>.
Behnke, Jessica, and Nicholas Johnson. "Macomb Mathematics Science Technology Center."
MMSTC. MMSTC, 2008. Web. 04 Feb.
2014.<http://www.wcs.k12.mi.us/mmstc/students/research/biology/2012/behnkejohnson.asp>.
Gordon. E.C. 12 Surprising Health Benefits of Lemons and Limes. Aviva.ca - Your Health
Superstore. Aviva, 2012. Web.13 May 2014. <http://www.aviva.ca/ article.asp?
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Lemons. Mad Food Science. 20 June 2011. Web. 13 May 2014
<http://www.madfoodscience.com/post/6721642761/lemons>.
"Microbiology." Microbiology. N.p., n.d. Web. 13 May 2014.
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