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Spicy Hot: The Effect of Jalapeño Peppers on Bacterial Inhibition

Rebecca Barresi

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Abstract:

The main purpose of this experiment is to prove that a specific concentration of the

jalapeño pepper (containing capsaicin) inhibits bacterial growth of E. coli. If proven, further

steps can be taken in order to eventually produce an organic bactericide for agricultural means.

In this experiment, various concentrations (25%, 50%, 75%, 100%) of the jalapeño pepper will

be used in order to determine the ideal amount and if it were to actually inhibit the growth of E.

coli. Predictions for this experiment state that the 100% concentration will inhibit the most E.

coli.

In order to complete this experiment, the basic bacterial inhibition procedure is followed.

An E. coli broth is spread onto agar, onto which inhibition disks that have been soaking in the

various concentrations are placed using forceps. The plates were then placed into the incubator at

37ºC for two weeks. Data was recorded on the third, fifth, and eleventh day in order to ensure the

effects are constant for a relatively long time.

The results of this experiment have proven that the hypothesis was supported; the 100%

concentration of jalapeño pepper inhibited the most amount of bacteria. In some plates, there was

some source of contamination, as there was an unknown bacterium that grew. Further testing

should be completed in order to validate these results. Once sure the results are accurate, the next

step in developing the bactericide should be completed by testing on an actual pathogenic plant

bacterium, as this is only the preliminary test.

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Introduction:

Bacterial inhibition is an important technique in both the agricultural and medicinal field

nowadays. In agriculture, farmers need to prevent the infestation of plant diseases, such as leaf

blights or even crown gall tumors, causing cancer in plants. Most of the bacteria that infect plants

tend to be bacillus, or rod, shaped. In the medicinal field, facilities, such as hospitals, need to

maintain a sterile environment so various pathogenic bacteria will not further harm the patient,

causing them to become sicker.

The purpose of this experiment was to determine if jalapeño peppers, containing

capsaicin, would have the natural trait to inhibit bacteria. Capsaicin is a component in most

peppers that attribute to their spiciness. If the hypothesis was supported with E. coli, it would

then be tested on plant bacteria in order to apply to the agricultural field. Therefore, rather than

using bactericides containing harmful chemicals, there would be an eco-friendly, organic

alternative. Jalapeño peppers have been previously shown to inhibit bacterial growth. In fact, it is

encouraged to consume the peppers in order to decrease the risk of getting a bacterial infection in

the body or to prevent bacterial diseases (WebMD, 2009). Furthermore, capsaicin has been used

to inhibit the growth of cancer cells in the human body, as it is an antioxidant. The capsaicin

inhibited the ability of the dihydrotestosterone to activate the PSA promoter and enhancer (Mori,

A., Lehmann, S., O’Kelly, J., Kumagai, T., Desmond, J., Pervan, M., McBride, W., Kizaki, M. &

Koeffler, H.P., 2006). Therefore, since it works to inhibit growth in human cells, it should also

be able to inhibit the growth of bacterial cells, since these are far less complex.

Additionally, capsaicin has been previously shown to inhibit the growth of

Colletotrichum capsici, a fungus that causes fruit rot in plants (Kraikruan, Sangchote, &

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Sukprakarn, 2008, p. 1). Therefore, it has been proven to inhibit the growth of fungi. This

sparked curiosity on whether or not it would also inhibit bacterial diseases in plants.

E. coli was used as a model organism in this experiment in lieu of an actual plant

bacterium. Most plant bacteria, as well as E. coli, are rod shaped. Therefore, the results gathered

from the E. coli should be similar to those if tested on a plant pathogen. A similar experiment

was conducted to this, where capsaicin was used to inhibit E. coli. The results from this

experiment showed that it slowed down the growth of the E. coli, however it did not fully hinder

the growth (Molina-Torres, Garcia-Chavez, & Ramirez-Chavez, 1999). This may be due to the

amount of capsaicin used or if a low concentration was used in the experiment. Due to this

uncertainty, there will be multiple levels of concentration in this experiment to ensure that these

results were not solely due to a lack of different amounts of the independent variable.

One related experiment tested the effect of capsaicin on bacteria swabbed from around

the house. The results of this experiment supported the hypothesis that the bacterial growth

would be inhibited; however, it was not fully reduced. It was only visible that there were fewer

colonies than the control (Foo, 2007). Though the experiment is similar to the one that will be

conducted, the results cannot necessarily be compared to those of this experiment. This is due to

the fact that these results were mostly quantitative, which is, for the most part, unreliable and

subjective to opinion.

All in all, the purpose of this experiment is to determine which concentration of capsaicin

from the jalapeño pepper inhibited the most amount of E. coli. Predictions state that the highest

concentration of 100% capsaicin will inhibit the most growth. This experiment was chosen due

to interest in microbiology and due to its application to both the medicinal and agricultural fields.

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If the hypothesis is supported, then further testing can be completed to determine whether or not

the organic bactericide actually works on pathogenic plant bacteria.

Materials:

The materials used in this experiment include the following: LB Agar base, LB Broth

base, E. coli, forceps, micropipettes (p-20, p-200, and p-1000), corresponding micropipette tips,

disposable inoculating loops, inhibition disks, media bottles, weigh boats, microcentrifuge tubes,

microcentrifuge tube rack, distilled water, tabletop balance, autoclave, incubator, parafilm, petri

dishes, 10% bleach, jalapeño peppers, mortar and pestle, hotplate, magnetic stirrers, scoopula,

70% ethanol, flame stick, falcon tubes, scalpels, tabletop balance, and beakers.

Methods:

In order to complete this experiment, all of the materials were first gathered and the

working area was sterilized with 70% ethanol. The first step was making the LB agar and LB

broth. In order to make 250 ml of LB agar, 8.75 grams of LB agar base was measured out using

the table top balance. See figure 1.1 for the calculations for this amount. This measured amount

was then added to the beaker, which was filled up with distilled water until it measured 250 ml.

The magnetic stirrer was added to the beaker that was placed on the hotplate, which was set to

about 250ºC and a stirring power of about 150 rotations per minute. One the agar began to boil, it

was removed and poured into the media bottle to be autoclaved.

The LB broth was made by adding 1.25 grams of LB broth base to a clean beaker. See

figure 1.1 for the calculations for this amount. The beaker was then filled up to the 50 ml mark

with distilled water, and a magnetic stirrer was added to the mixture. It was heated on the hot

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plate on high power until it turned clear and was removed before it began to boil. It was then

poured into a media bottle to be autoclaved.

Once both the agar and broth were autoclaved, the plates were poured and the bacterial

broth was prepared. The E. coli broth was made by first pouring the broth into a falcon tube. A

colony of E. coli was taken from a streak plate using an inoculating loop. The colony was added

to the broth in the falcon tube by stirring the inoculating loop until it was visible that no part of

the colony was left on the inoculating loop. The broth was placed in an incubator at 37ºC for 24

hours to allow the E. coli to grow. Following this step, the plates were poured. This was done by

first heating up the cooled, hardened agar in the microwave. This step was extremely important

since precautions needed to be taken to prevent the agar from boiling over and spilling in the

microwave. The cap of the media bottle was loosened to relieve any pressure buildup, and the

bottle was removed from the microwave about every ten seconds and swirled to prevent the

newly liquefied agar from rising above the hardened agar and over spilling. Once the agar was

completely melted, the lip of the media bottle was flame sterilized to prevent the agar from being

contaminated while pouring. The agar was then poured into the petri dishes until a quarter-sized

area was left (this area would fill itself). The plates then cooled for 24 hours until the E. coli

broth was ready to be added on.

The last preparatory step for this experiment was creating the concentrations of the

jalapeño pepper, the positive control, and the negative control. In order to prepare the pepper

concentrations, the jalapeño pepper was cut up into pieces using the scalpel. It was then placed

into the mortar and pestle, along with 50 µl of distilled water in order to easily acquire the liquid

from the mashed pepper. The peppers were then mashed and the concentrations were made using

the liquid from the peppers and distilled water. See figure 1.2 for the amounts of each used. In

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order to make the positive control, 1000 µl of 10% bleach was placed into a microcentrifuge

tube. The negative control was made by adding 1000 µl of distilled water to a microcentrifuge

tube. The concentrations were made in the microcentrifuge tubes; after created, five inhibition

disks were added to the solutions and sat overnight to soak.

After 24 hours, the actual experimentation was ready to begin. First, each plate was

labeled with initials, date, type of agar, and was split into four equal quadrants labeled with what

solution was in that quadrant. Then, 1 ml (1000 µl) of the E. coli broth was added to each plate.

The broth was then spread throughout the agar by using a sterile inoculating loop for each plate

and was left to absorb for fifteen minutes. After the fifteen minutes, the inhibition disks that were

left absorbing overnight were placed into its corresponding quadrant using flame-sterilized

forceps. See figure 1.3 for the set-up of the plates. Once this was complete, the plates were

parafilmed closed and placed in the incubator at 37ºC for two weeks. Results were recorded on

the third, fifth, and eleventh day. This procedure was based off of lab 6D in the Biotech Lab

Skills class Lab Manual.

In this experiment, there were five replicates per trial. There were six trials total: the

positive control, the negative control, 25% pepper, 50% pepper, 75% pepper, and 100% pepper.

The positive control was 10% bleach, as it has shown to have bacterial inhibition by being used

to kill bacteria after completing previous labs. The negative control was distilled water since it

has not shown bacterial inhibition properties in previous labs. The dependent variable in this

experiment is the amount of E. coli growth inhibited, while the independent variable is the

amount of jalapeño pepper used. Some constants in this experiment include: the temperature

during the incubation period, the incubator environment, the type of agar/plates, amount of

bacteria on plates, amount of concentrations, type of inhibition disks, and the amount of time per

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trial. While gathering data, the range of inhibition was measured in millimeters with the use of a

ruler and then recorded in a data table. This data was processed by completing a t-test and

finding the mean, mode, range, and standard deviation for the range of inhibition. Pictures were

also taken at the end of the experiment to show quantitative results.

Results:

When this lab concluded, all concentrations, besides the negative control, showed some

extent of bacterial inhibition. Around the positive control, there was a large circle of inhibition,

but there was no ring evident around the negative control. This means that there was no error

with contamination, and the results for each trial should be valid. Generally, the 25%

concentration, 50% concentration, and 75% concentration had similar amounts of bacterial

inhibition throughout the gathering of the results, while the 100% concentration had the most

amounts.

The data was gathered over the course of two weeks in order to ensure the pepper would

maintain the amount of bacteria inhibited so it would not grow back. However, in certain

replicates, there was some E. coli that grew back. In the quadrants containing 25% pepper

concentrations and 75% pepper concentrations, there was some initial bacterial inhibition.

However at the five day mark, some bacteria began to grow back at around a 0.01 mm amount,

which later became more evident when measured after eleven days. Also, in the trial containing a

75% pepper concentration, an unidentifiable bacterium grew where the E.coli originally was.

This only occurred in about two replicates. The quantitative results are Figures 1.4-2.6 attached.

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Data Tables:

Table 1.1: Day 3

Plate Positive Control Negative Control 25% 50% 75% 100%

A 0 __ 0.8 0.7 0.8 __

B __ __ 0.9 0.9 0.9 0.8

C 1.7 0 __ 0.8 0.7 __

D __ __ 0 1.1 0.9 1

E 1.5 0 0 __ __ 0.8

F 1.5 0 __ 1.2 __ 1.1

G 1.5 0 0.9 __ __ 0.9

H 1.8 0 0.8 1 __ __

I 1.5 0 1 1.2 __ __

J 1.9 0 0 __ 1 __

K 1.5 0 0.9 __ 1 __

L 1.5 0 __ __ 1 0.7

Table 1.2: Day 5

Plate Positive Control Negative Control 25% 50% 75% 100%

A 2.6 __ 0.9 0.8 0.8 __

B __ __ 1 1 1.1 0.9

C 1.6 0 __ 0.9 1.1 __

D __ __ 0.6 0.6 0.7 0.9

E 1.5 0 1 __ __ 1

F 1.6 0 __ 1.3 __ 0.9

G 1.6 0 0.7 __ __ 0.9

H 1.5 0 0.9 0.8 __ __

I 1.8 0 0.9 0.9 __ __

J 1.6 0 1.3 __ 0.9 __

K 1.4 0 1.1 __ 1.4 __

L 1.4 0 __ __ 0.8 1

Table 1.3: Day 11

Plate Positive Control Negative Control 25% 50% 75% 100%

A 3 __ 0.9 0.9 0.9 __

B __ __ 0.9 0.9 1.1 0.9

C 1.6 0 __ 0.9 1.1 __

D __ __ 0.6 0.6 0.7 0.9

E 1.6 0 1 __ __ 1

F 1.6 0 __ 1.1 __ 1

G 1.2 0 0.9 __ __ 0.8

H 1.5 0 1 1.2 __ __

I 1.9 0 0.9 1.1 __ __

J 1.6 0 1.3 __ 0 __

K 1.5 0 1.1 __ 0 __

L 1.6 0 __ __ 0 1

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Table 1.4: Statistical Analysis

Analysis (Overall)

Positive Control

(mm)

Negative Control

(mm)

25%

(mm)

50%

(mm)

75%

(mm)

100%

(mm)

Mean 1.6 0 0.83 0.95 0.8 0.92

Median 1.6 0 0.9 0.9 0.9 0.9

Mode 1.5 0 0.9 0.9 0.9 0.9

Range 3 0 1.3 0.7 1.4 0.4

Standard Deviation 0.46 0 0.34 0.2 0.38 0.1

For the statistical analysis of the mean of each trial, it was proven that the positive control

had the highest average, as it should. Following that, the 50% concentration and 100%

concentration had the second most and third most mean accordingly. This was done in order to

determine the average amount of inhibition in order to easily compare results. However, these

averages may be affected by any outliers, but they are relatively reliable.

The median shows the middle amount of bacteria inhibited. Therefore, one is able to

determine the general range of the inhibited bacterial growth.

The range would show how consistent the data set is. The lower the range, the more

consistent the data is, as it would be numbers that are closer together. Therefore, the 100%

concentration had the most consistent data, followed by the 50% concentration, then 25%

concentration, and finally the 75% concentration. Another way to determine the consistency of

the data set would be the mode. This would be the measurement that appears the most, which

would make the amount more valid. It also accounts for any error. Showing that since the

measurements occurred more than once, either the error was consistent or there were no major

errors made.

Lastly, the standard deviation analysis shows the relation of data. It shows the

consistency of the data and the similarity of the numbers as well. Additionally, it helps explain if

any errors were made and how influential those errors were to the data. The most related data set

was those of the 100% concentration, closely followed by the 50% concentration. Therefore, it

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can be determined that while preparing these trials, few errors were made and the data is reliable.

However, the 25% and 75% had a slightly higher deviation. This might be due to the fact of an

error in the procedure or contamination. However, the rate of standard deviation was relatively

low, which means that the data is applicable.

Table 1.5: Averages Throughout the Experiment

Positive Control Negative Control 25% 50% 75% 100%

Day Three 1.44 0 0.58 0.99 0.90 0.88

Day Five 1.66 0 0.93 0.90 0.97 0.93

Day Eleven 1.71 0 0.96 0.96 0.54 0.93

Graph 1.1:

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

A B C D E F G H I J K L

Ran

ge o

f In

hib

itio

n (

mm

)

Plate

Results After 3 Days

Positive Control

Negative Control

25%

50%

75%

100%

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Graph 1.2:

Graph 1.3:

0

0.5

1

1.5

2

2.5

3

A B C D E F G H I J K L

Ran

ge o

f In

hib

itio

n (

mm

)

Plate

Results After 5 Days

Positive Control

Negative Control

25%

50%

75%

100%

0

0.5

1

1.5

2

2.5

3

3.5

A B C D E F G H I J K L

Ran

ge o

f In

hib

itio

n (

mm

)

Plate

Results After 11 Days

Positive Control

Negative Control

25%

50%

75%

100%

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Graph 1.4:

Discussion:

At the end of this experiment, it was concluded that the 100% pepper concentration had

the best bacterial inhibiting qualities. This was evident by examining the average amount of

inhibition over the third, fifth, and eleventh day. By the third day, all trials, except for the

negative control, have shown some sort of inhibition.

By the fifth day, some trials continued to inhibit more bacteria, some remained constant,

and some decreased. This decrease may have been due to the weakness of the capsaicin and the

bacteria may have become resistant to the small amount. This occurred in all trials, however

some were more than others. For example, it occurred mostly in the 50% concentration and 75%

concentration. However, it was also evident in the 25% concentration and 100% concentration,

but in a lesser amount. Visibly, there were very minor changes in the ring of inhibition on the

fifth day. Therefore, it is possible that measurements may have been incorrectly taken, whether it

be through mechanical errors in the ruler itself or through human error.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Positive Control

Negative Control

25% 50% 75% 100%

Ave

rage

Ran

ge o

f In

hib

itio

n (

mm

)

Trial

Changes in the Average Amount of Inhibition Throughout the Experiment

Day Three

Day Five

Day Eleven

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By the eleventh day, it was clear which trials had more bacterial inhibiting qualities

compared to others. For example, the 50% concentration had multiple cases where E. coli had

grown back. Additionally, in one replicate of the 50% concentration, there was an abnormal and

unrecognizable bacterial colony that grew towards the side of the plate. This may have been due

to contamination when pouring the agar or contamination of the peppers. The peppers were not

washed prior to cutting and crushing in the mortar and pestle. As a result, there may have been

some bacteria on the skin of the pepper from when it was picked up in the store or even when cut

with the scalpel. On some replicates in the 75% concentration, there was a mysterious bacterium

that grew back in the space where the inhibited E. coli originally was. The reason for this

contamination is most likely the same reason as before: contamination of plates or of the pepper

themselves. In the 25% concentration trials, the 50% concentration trials, and the 75%

concentration trials, it was most evident that some E. coli had grown back. Though some E. coli

had grown back in the 100% concentration trials, the growth was extremely minimal, at less than

a 0.01 mm difference.

As for the research question, it can be determined that the 100% jalapeño pepper

concentration had the most effect on inhibiting the growth of E. coli. Therefore, the hypothesis

can be supported. Even though all trials have shown some type of bacteria being grown back, the

least amount was in the 100% concentration. Additionally, in Graph 1.4, it is evident that as the

course of the experiment went on, the only other trials besides the positive control that had a

continuous increase in the average range of inhibition was the 25% and 100% concentration trial.

All of the other trials either decreased or decreased then increased again. However, due to the

fact that the 25% trial had some growth of an unknown bacterium, it was determined that the

100% concentration was the most ideal.

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In order to ensure the reliability of these results, this experiment should be completed one

more time. This will help eliminate any unwanted data due to contamination and prove these

results to be accurate and consistent. It will also confirm that there were no human or mechanical

errors made due to any time constraints. Time was a major problem in this experiment, as the

days of the open lab were limited. Therefore, there was no room for uncertainty when

completing the procedure. This allows for much room for error. Such errors may include:

contamination, inaccurate measuring of materials, inaccurate data recording, lack of proper

supplies, incorrectly calibrated machines, and so on. If there were more time to ensure the

proper use of all equipment and complete understanding of the procedure, then the results would

be more legitimate.

Overall, this lab has proved that capsaicin has some property of bacterial inhibition.

However, the extent of this property is still unclear until the lab is redone to confirm results. If

the results of the lab when redone were to still support the hypothesis, then further measures

would be taken in order to apply it to agriculture.

In order to further the experiment in the agricultural field, then a pathogenic plant

bacterium would next be grown and tested on with the 100% concentration of the jalapeño

pepper. Additionally, the peppers effect on the Wisconsin Fast Plant would be determined to see

if the pepper solution would either kill the plant or make it thrive even more. Such methods to

determine the effect on the plant would be to either spray the solution directly on the leaves or to

put the solution in the soil for the plants’ roots to uptake. If both methods were to kill the plant,

then it may be possible to genetically engineer the plant to produce capsaicin on its own. After

determining the best method to apply it to the plant and if it were to actually inhibit bacterial

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growth, the final step would be to test on crops, first in the laboratory followed by the use of

actual farmers.

In conclusion, if this expanded experiment were to prove that capsaicin inhibits the

growth of pathogenic plant bacteria, it would be greatly influential in agriculture. Farmers would

be able to use organic means to save crops that would ordinary die from plant diseases. This

allows both the farmer and the economy to thrive, since there would be more organic crops on

the market at a cheaper price.

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References:

Foo, F. (2007). Capsaicin and microbial relations. Retrieved from

http://www.usc.edu/CSSF/History/2007/Projects/S1413.pdf

Kraikruan, W., Sanchote, S. & Sukprakarn, S. (2008). Effect of capsaicin on germination of

Colletotrichum capsici conidia. Retrieved from

http://www.thaiscience.info/journals/Article/Effect%20of%20capsaicin%20on%20germi

nation%20of%20colletotrichum%20capsici%20conidia.pdf

N.A., (2009). Capsaicin-topic overview. WebMD. Retrieved from http://www.webmd.com/pain-

management/tc/capsaicin-topic-overview?page=2

Molina-Torres, J., Garcia-Chavez, A., & Ramirez-Chavez, E. (March 24 1999). Antimicrobial

properties of alkamides present in flavouring plants traditionally used in Mesoamerica:

affinin and capsaicin, Journal of Ethnopharmacology, 64 (3). Retrieved from

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T8D-3W37VVB-

6&_user=10&_coverDate=03/31/1999&_rdoc=1&_fmt=high&_orig=search&_origin=se

arch&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0

&_userid=10&md5=7285adfb0f88ba5e9c0f9b6521d1335e&searchtype=a

Mori, A., Lehmann, S., O’Kelly, J., Kumagai, T., Desmond, J., Pervan, M., McBride, W.,

Kizaki, M. & Koeffler, H.P. (2006). Capsaicin, a component of red peppers, inhibits the

growth of androgen-independent, p53 mutant prostate cancer cells.Cancer Research, 66.

Retrieved from http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T8D-

3W37VVB-

6&_user=10&_coverDate=03/31/1999&_rdoc=1&_fmt=high&_orig=search&_origin=se

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arch&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0

&_userid=10&md5=7285adfb0f88ba5e9c0f9b6521d1335e&searchtype=a

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Figures

Figure 1.1

Calculations for Agar Calculations for Broth

35g/1000ml=x g/250ml

8750g/ml=1000x g/ml

8.75 g= x

25g/ 1000ml= x g/ 50 ml

1250 g/ml= 1000x g/ml

1.25 g= x

Figure 1.2

Concentration Amount of Jalapeño Pepper Amount of Distilled Water

25% 250 µl 750 µl

50% 500 µl 500 µl

75% 750 µl 250 µl

100% 1000 µl 0 µl

Figure 1.3

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Figure 1.4

Figure 1.6

Figure 1.8

Figure 2.1

Figure 1.5

Figure 1.7

Figure 1.9

Figure 2.2

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Figure 2.3

Figure 2.5

Figure 2.4

Figure 2.6