Burgin - Rupert 1

Introduction
Every Lysol commercial says that their product kills 99.9% of germs, but can that 0.1%
mutate and become more resistant? (Lysol) If enough Lysol is not used, can a super bacteria be
created that is immune to the active ingredient? Some sources say that this is possible and that it
is happening right now. The intention of this experiment was to find the answer to these
questions, and to find out if these sources are correct.
Lysol is a brand name of cleaning and disinfecting products distributed by Reckitt
Benckiser. The line includes solutions for hard and soft surfaces, air treatment, and handwashing.
The active ingredient in most Lysol products is benzalkonium chloride, as it is extremely
efficient in killing bacteria.
The two factors that were chosen for this experiment were benzalkonium chloride and
temperature. Lysol Power and Fresh Multi-Surface Cleaner, which is the variety of Lysol that
was used for the experiment, is 1% to 2% benzalkonium chloride which is the active ingredient.
This variety of Lysol was chosen because it is the choice cleaner of many people to clean their
bathrooms, kitchens, and other places where bacteria grow the most. Benzalkonium chloride kills
bacteria by breaking down their cell membranes (Freeman). This causes leakage of the cells
internal material, which kills the bacteria. The standard temperature, 37 Celsius, was chosen
because that is the optimal temperature for E. coli to grow. When temperature gets higher, it
starts to alter the E. coli cells wall and membrane, and damage the internal structures, such as
proteins or and nucleic acid (Port). When the temperature gets lower, the E. coli cells
metabolism slows down which results in the number of proteins synthesized dramatically
reducing. it also results in cold induced inactivation of parts need by the cell to function properly
and live.

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Trials were conducted on Escherichia coli (or E. coli) with varying amounts of
benzalkonium chloride and temperature. A small disk was soaked in a Lysol-water solution for
three minutes and placed in a pre-prepared Petri dish containing E. coli. In the Petri dish, a zone
of inhibition would form around the disk where E. coli would not grow. The zone would then be
measured and compared to previous and future generations. The E. coli for the next trial was
taken from the previous Petri dish. Over the research period, the zone of inhibition was intended
to shrink. This is because the E. coli was growing resistance to the benzalkonium chloride, and
was able to grow further into the area where the Lysol was spreading. The E. coli was still only
able to grow so close to the disk though, because the closer to the disk it got the stronger the
Lysol solution would be that it would have to endure.
Antibiotic resistance occurs when bacteria change in some way that reduces or eliminates
the effectiveness of drugs, chemicals, or other agents designed to cure or prevent infections. The
bacteria survive and continue to multiply, which can cause more harm. Bacteria can do this using
several different tactics. Some bacteria develop the ability to neutralize the antibiotic before it
can do harm, others can rapidly pump the antibiotic out, and still others can change the antibiotic
attack site so it cannot affect the function of the bacteria (Antibiotic Resistance: Questions).
The purpose of this experiment is to add to information that is already known in science.
After all, the purpose of science research is not just to make huge discoveries, but to do research
that aids other scientists in their studies.

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Problem Statement
Problem:
The effect of exposing Escherichia coli to Benzalkonium Chloride over a period of time.
Hypothesis:
The zone of inhibition around a paper disk in a dish of E. coli will shrink after being
exposed to small amounts of benzalkonium chloride.
Data Measured:
The independent variables are the amount of benzalkonium chloride used, measured in
milliliters (ml), and the temperature, measured in degrees celsius. The dependent variable is the
diameter of the zone of inhibition around the disk soaked in benzalkonium chloride. The
averages of the effects of both the temperature and amount of benzalkonium chloride will be
compared using a two factored Design of Experiment (DOE). The researchers will use a standard
control group to compare the effects of benzalkonium chloride on E. coli.

Experimental Design
Materials:

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Petri dishes (25)
Test tubes (5)
1L beaker
1 ml eye dropper
Escherichia coli
Stirring magnet
1L beaker
2.54 cm masking tape
Forceps

Incubator (3)
Antibiotic disks (50)
30.43 cm ruler
1525 ml distilled water
24.5 cm stirring hot plate
Agar powder
15 ml Lysol Power and Fresh Multi-Surface cleaner
Transfer loop
100 ml beaker (3)

Procedure:
Agar preparation:
1.

Add 1 ml of water to a 1 L beaker

2.

Place on a stirring hot plate, and place the hotplate on high

3.

Add a stirring magnet to the 1 liter of water, and place the setting on #4

4.

Slowing add 2.3 grams of agar powder to the flask be careful not to get it on the sides
(a full batch 100 ml or 1 L will fill 50 Petri dishes)

5.

Allow the solution to turn from a cloudy liquid to clear like apple juice (the temperature
will be nearly 100 Celsius)

6.

Remove from the heat and allow to cool before it is poured.

7.

Adjust amounts to fit needs. (ie. 11.5 grams agar in 500ml of water for a half
batch about 25 plates)

Agar pouring:
8.
When pouring the agar into your petri dishes do not open the cover or expose the inside
of the
petri dish to the air.
9.

Always open and close the lid without placing the lid on the desktop.

10.

Open lid just enough to pour in the agar

11.
12.

Pour just enough to cover the bottom of the dish with a thin layer of agar (1-3 mm thick)
Allow to cool without moving the petri dishes (leave at their location on the
desk). Agar that gets on the lid of the petri dish will ruin the pour and must
be repeated.
13.
When cool bacteria may be added. After adding invert the plates and place them in the
correct
incubator for your specific amount of time.

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Testing:
14.

Prepare 5 Petri dishes using correct agar preparation (steps 1-13). Label them (+,+), (+,-),
(-,+), (-,-), and standard.

16.

Sterilize the tweezers by dipping them in boiling water.

17.

Prepare and label the following mixtures in a 100 ml beaker:

a.
Labeled 0.5% - 0.5 ml of Lysol is mixed with 99.5 ml water
b.
c.

Labeled 1% - 1 ml of Lysol is mixed with 99 ml water

Labeled 1.5% - 1.5 ml of Lysol is mixed with 98.5 ml water

18.
Soak four paper disks in a 0.5% Lysol-water solution. Soak two disks in a 1%
Lysolwater
solution. Soak four disks in a 1.5% Lysol-water solution. All disks should be
soaking for 3
minutes.
19.

Each of the five Petri dishes should have 2 disks in them. Insert two disks from the 1.5%
solution into both the (+,+) and (+,-) dishes. Insert 2 disks from the 1% solution
into the
standard dish. Insert 2 disks from the 0.5% solution into both the (-,+) and
the (-,-) mixtures.
20.

Put the (+,+) and (+,-) Petri dishes in the 38 incubator, the standard Petri dishes in the
37 incubator, and the (-,+) and the (-,-) Petri dishes in the 36 incubator.

21.

After 24 hours, there will be a circular area around the antibiotic disk called the zone of
inhibition where there will be no bacterial growth. Measure the diameter
of this area record it in
a data table. (If the zone of inhibition is uneven, measure 3
axes and average them to get the
diameter.)
22.
23.

Label 5 test tubes (+,+), (+,-), (-,+), (-,-), and standard.

Put 1 mL water in each of the test tubes.

24.

Sterilize the transfer loop by running it over a flame.

25.
Collect the surviving bacteria along the border of the inhibition zone of each Petri dish by
using
the transfer loop and scraping off some of the bacteria around the zone of
inhibition.
26.

Drop the loop in its corresponding test tube with the 1 ml of water.

27.
Petri

Prepare another Petri dish (steps 1-13), and pour the contents of the test tube into the new
dishes.

28.

Repeat steps 1-27 five times.

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Data and Observations

Data:
Table 1
Design of Experiment Values

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Table 1 shows the factors that were chosen to see how they affect the growth of
Escherichia coli. 1% was chosen as the standard amount of Lysol because Lysol contains 1% of
benzalkonium chloride, the active ingredient. The high and low amounts were chosen because
they are both 0.5% less than the standard measurement. 37 C was chosen as the standard
temperature because it is the temperature where E. coli grows best, and and the high and low
temperatures were chosen because they are both one degree away from the standard.
Table 2
Data for the First Three Runs

Table 2 shows the results of the experiment for three of the five runs that were performed.
Each run had two measurements per group (high-high, low-low, etc.). It also shows that the same
sequence was performed each time.
Table 3
Data for the Fourth and Fifth Runs

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Table 3 shows the results of the experiment for the last two of the five runs that were
performed. Each run had two measurements per group (high-high, low-low, etc.).
Observations:
Table 4
Observations
Date

Observations

3/19/2014

The E. coli grew, leaving an uneven diameter

around the disk.

3/20/2014

The average diameter shrunk slightly.

3/21/2014

The average diameter shrunk slightly.

2/26/2014

The average diameter shrunk greatly.

Date
3/27/2014

Observations
The average diameter grew greatly (on some
of the groups) showing a possible human
error.

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Table 4 shows the observations for each run. Five days were taken to do this experiment.
The observations include how the zones of inhibition were changing over time.

Figure 1. Disinfecting the Transfer Loop

Figure 1 shows the transfer loop being disinfected. This is vital to the experiment because
it stops the spread of unwanted bacteria into the Petri dishes. The loop is sterilized by moving it
over an open flame.
When putting the E. coli mixture into the Petri dish, there was some extra liquid making
it impossible to invert the Petri dish when putting it in the incubator. The researchers found it
helpful to dump the excess water before putting the Petri dishes in the incubator.

Burgin - Rupert 10

Figure 2. Disinfecting the Scraper Loop

Figure 2 shows the disks that were soaked in Lysol being put in the Petri dishes. This was
one of the most important parts of the experiment because the disks stay in one spot. If they did
not stay in the same spot the Lysol would be everywhere and it would not be possible to find the
diameter of the zone of inhibition. It also helps by showing where the center of the zone is, and

Burgin - Rupert 11
where the ruler should pass through when finding the diameter.

Figure 3. Prepared Petri Dishes

Figure 3 shows the Petri dishes fully prepared before they were put in the designated
incubators.
The zones of inhibition were not symmetrical, so to find the diameter three diameters
were measured and were averaged. Over time the some zones shrunk, showing that the
experiment was set up right and was working. On the other hand though, some of the zones grew
showing possible human errors.

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Figure 4. Petri Dishes in the Incubator

Figure 4 shows the Petri dishes used in this experiment (among other experiments) in the
incubator. This particular incubator is 37 C, and the zones of inhibition for the standard were
usually observed to be around 25 mm in diameter.

Data Analysis and Interpretation

Experiment:
The Effect of Benzalkonium Chloride and Temperature on Escherichia coli
Hypothesis:
Over a five day experiment, the zone of inhibition of a Petri dish with E. coli will shrink.
Response Variable:
Zone of inhibition (millimeters)

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Predictor Variables:
Amount of benzalkonium chloride (% solution) and temperature (Celsius)
Table 5
Factors Used in Experiment

Table 5 shows the factors that were chosen to see how they affect the growth of
Escherichia coli. The two factors were the amount of benzalkonium chloride (the active
ingredient in Lysol) and the temperature in which the E. coli was stored. The values for the
Lysol-water solution are 0.50% Lysol (low), 1.00% Lysol (standard), and 1.50% Lysol (high).
The values for the temperature are 36 C (low), 37 C (standard), and 38 C (high).

Table 6
Average Zone of Inhibition Data

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Table 6 shows the results for the zones of inhibition for each combination and each run. It
also shows the average of each combination with the factors. The grand average is the average of
all eight averages. In this case, the grand average is 20 1/4 mm.

Figure 5. Plot of Standards

This figure shows the measurements of the ten trials that were given the standard dose of
both factors. The range of standards is 13 2/3 mm, so doubling the range of standards gives 27 1/3
mm. This means that any effects would have to have an effect value greater than 27 1/3 or less
than -27 1/3 to be considered statistically significant. All of the standards are in the 20 mm to 35
mm range and do not have a very significant downward trend, which is not what was expected.
The standards did start off as good controls, as the values of the standards in the first run were
smaller than those of the (+,+) trials and larger than those of the (-,-) trials, which was expected.
It should be noted that because the zone of inhibition was intended to shrink over time, the range
of standards was intended to be large. The proper design for a DOE has standards that are
supposed to stay consistent over time, so in this case, none of the effects were outside of the
large range of standards.

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Table 7
Effect of Benzalkonium Chloride

Figure 6. Effect of Benzalkonium Chloride

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Table 7 shows the average zones of inhibition when benzalkonium chloride was applied.
This table shows the high and low amounts, and their averages. When the low amount (0.5%)
was applied, the average size of the zone of inhibition was 18 49/60 mm. When the high amount
(1.5%) was applied, the average size of the zone of inhibition was 21 41/60 mm. Figure 6 shows
the effect that benzalkonium chloride has on E. coli. When the low average (18 49/60) is subtracted
from the high average (21 41/60), the effect of benzalkonium chloride is found to be 2 13/15. This
means that as the amount of benzalkonium chloride increases, the zone of inhibition grows by 2
13

/15 millimeters. It was noticed that as benzalkonium chloride went from the low amount to the

high amount, the zone of inhibition increased dramatically. This means that benzalkonium
chloride does have a significant effect on E. coli.
Table 8
Effect of Temperature

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Figure 7. Effect of Temperature

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Table 8 shows the average zones of inhibition when the temperature was changed. This
table shows the high and low amounts, and their averages. When the low temperature (36C) was
applied, the average size of the zone of inhibition was 19 1/15 mm. When the high temperature
(38C) was applied, the average size of the zone of inhibition was 21 13/30 mm. Figure 7 shows
the effect that temperature has on E. coli. When the low average (19 1/15) is subtracted from the
high average
(21 13/30), the effect of temperature is found to be 2 11/30. This means that as the temperature
increases, the zone of inhibition grows by 2 11/30 millimeters. Temperature does not have a
statistically significant effect on E. coli, though it was noticed that as the temperature went from
low to high, the zone of inhibition got significantly larger. This was especially noticeable when
the amount of benzalkonium chloride was held low. The significant change in size occurred
because it is harder for E. coli to grow in higher temperatures.
Table 9
Interaction Effect

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Figure 8. Interaction Effect

Table 9 and figure 8 show the interaction between benzalkonium chloride and
temperature. In figure 8, the solid line segment represents the high amount of benzalkonium
chloride, and the dotted segment represents the low amount of benzalkonium chloride. The
interaction effect is found by subtracting the slope of the low amount from the slope of the high
amount. Because the solid segment has a slope of 9/20, and the dotted segment has a slope of 1

Burgin - Rupert 20
11

/12, the interaction effect is -1 7/15. This interaction is not considered statistically significant, but

there is a noticeable interaction between the two factors. Notice in figure 6, the low amount of
benzalkonium chloride produced a zone of inhibition that was 18 49/60 mm. When the low amount
of temperature was applied, the zone of inhibition shrunk noticeably, and as the temperature went
from low to high the zone of inhibition grew noticeably. This is because higher temperatures and
higher amounts of antibacterial substances have more effect in altering the membranes of
bacteria. This causes cell leakage, and more cells are killed. Similarly, when lower temperatures
and lower amounts of antibacterial substances are applied, they have a smaller effect on the
membranes and structures in cells, so that is when the zones of inhibition were observed to
shrink.

Figure 9. Dot Plot of Effects

Figure 9 displays the effect values of benzalkonium chloride (B), temperature (T), and
their interaction (BT). These values can be compared to double the range of standards to see if
they are statistically significant. In this case, double the range of standards has an absolute value
of 27 1/3. None of the effect values are outside of double the range of standards, so none of the
effects can be considered statistically significant. Though neither of the factors can be considered
mathematically significant, they did have an effect on each other. Notice (figure 8) how the zone

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of inhibition grows significantly in both cases as the temperature goes from low to high. Also,
the zone of inhibition grows significantly as the amount of benzalkonium chloride goes from low
(dotted line) to high (dashed line). The insignificant effects were not due to design experimental
design errors or human errors, but due to the fact that the range of standards was intended to be
large.

Figure 10. Parsimonious Prediction Equation

Figure 10 shows the parsimonious prediction equation. Only the significant effects are
used in this equation. This parsimonious prediction equation has only the grand average and
noise, as none of the effects were statistically significant.

Figure 11. Parsimonious Prediction

Figure 11 shows the parsimonious prediction equation being used to predict an average of
20 1/4 mm. This means that if one were to do this experiment again, with no changes the average
measurement for the zone of inhibition would be 20 1/4 mm, or the grand average. Interpolated
data, or data that is within two already known values, is used for parsimonious prediction
equations because extrapolated data, or data that is outside of the already known values (noise
for example), does not produce accurate predictions. Because none of the effects were considered
statistically significant, no effect values are seen here being plugged into the equation. If there

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were significant effects, interpolated values could be plugged in to make more accurate
predictions.

Conclusion
At the beginning of this experiment, it was hypothesized that when exposed to
benzalkonium chloride, the zone of inhibition around a Lysol-soaked disk would shrink. The
hypothesis was accepted with all of the zones of inhibition except the low-high group, which
grew 6 mm. The other results are as follows: high-high shrunk 7 mm, high-low shrunk 5
mm, standard-standard shrunk mm, and low-low shrunk 1 mm.
Benzalkonium chloride is a cleaning agent that is used by many people. It kills bacteria
by breaking down the cell membranes. This causes leakage of the cells internal material, which
kills the bacteria. A cell cannot continue living after its internal material has been exposed to the
extracellular environment. Benzalkonium chloride also effectively kills cellular enzymes, which
speeds up the process of killing the cells (Freeman).

Burgin - Rupert 23
Temperature is the other factor that was used in this experiment. 37 Celsius is the most
efficient temperature for Escherichia coli to grow and replicate. This is because E. coli lives in
the intestines of human beings, and 37 C is the average body temperature of humans. Under
these conditions, E. coli can double in quantity every 20 minutes. When higher temperatures are
used, such as in the 38C trials, the membranes of the cells start to break down. This causes the
internal material of the cell, such as the cytoplasm, proteins, and nucleic acid to fail (Port).
The hypothesis of this experiment was accepted because of the E. colis ability to develop
resistance to antibacterial substances. Antibiotic resistance occurs when bacteria change in some
way that reduces or eliminates the effectiveness of drugs, chemicals, or other agents designed to
cure or prevent infections. The bacteria survive and continue to multiply causing more harm.
Bacteria does this using several different tactics. Some bacteria develop the ability to neutralize
the antibiotic before it can do harm, others can rapidly pump the antibiotic out, and still others
can change the antibiotic attack site so it cannot affect the function of the bacteria (Antibiotic
Resistance: Questions).
The purpose of the experiment was to determine how much Lysol one should use when
cleaning their house. Using an amount too small can result in the remaining bacteria developing
a resistance to the cleaner, and using too much cleaner can be unsafe. The researchers tried to
determine how much Lysol should be used to kill the bacteria, without using too much. Most of
the results supported the researchers hypothesis. This is because as time went on and generations
multiplied, the E. coli built up a resistance to the benzalkonium chloride making it harder to kill,
resulting in more E. coli growing. It is possible that if the experiment was continued the bacteria
would have eventually became completely resistant, or immune to the Lysol.

Burgin - Rupert 24
It is generally thought that widespread use has indeed selected for antiseptic resistance as
found with antibiotics. Currently suggested antiseptic resistance mechanisms uncannily resemble
those conferring antibiotic resistance. Generally, these mechanisms may originate as either an
intrinsic (natural) or acquired property (by genetic mutation or horizontal gene transfer) of the
microorganism. In the case of rapidly growing microorganisms, beneficial mutations are selected
for then stabilized in a growing population resulting in acquired resistance (Antiseptic
Efficacy).
Other research in this field includes an experiment done by Michael Cox, a biochemistry
professor at the University of Wisconsin-Madison, and John R. Battista, a professor of biological
science at Louisiana State University. They used a radioactive isotope to kill 99% of a population
of E. coli. The remaining bacteria was then grown out and exposed to the radioactive isotope
again and again. The final generation of bacteria could survive 1000 times the radiation that
could kill a human. Their research, along with the research conducted in this experiment, can
prove useful in cleaning radioactive waste sites and helping cancer patients that are undergoing
radiation therapy (Study: E. Coli Bacteria Can Build Resistance Quickly, Even To Ionizing
Radiation).
There were a few design flaws that could be fixed pretty easily. Less water should be
added to the test tubes when the new Petri dishes are being prepared to prevent the loss of
bacteria when the excess water is dumped out. The percentage of Lysol should be lessened by 0.5
as well, to prevent the inhibition zones from overlapping and making it difficult to measure. Also
the scraper loop and distilled water should be allowed to cool for a set amount of time (such as
one minute) before bacteria is added to either item to prevent the bacteria from being killed by
the heat. No noticeable errors were made while performing the procedure.

Burgin - Rupert 25
The public will benefit from our research because it shows that if enough of the cleaner
that is being used, the bacteria can become resistant, and over time can become immune to the
cleaner. This makes it harder, then impossible to kill when the cleaner is applied. New cleaners
must then be used with a different active ingredient causing the people using the inactive cleaner
to buy a new one. The research will prevent this from happening by showing how much cleaner
should be used without creating resistance in the bacteria. This research will benefit not only
professionals, but average people who clean their homes.
The lessons learned during this experiment are important ones. It was shown that videos
and pictures need to be taken of the procedure, and results. This helps prove that the research was
done by the people presenting, and it also helps the audience understand the research being done
better. It was also proven that too little cleaner can be a bad thing in the long run. This was
shown by the shrinking of the zones of inhibition throughout the experiment.

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Burgin - Rupert 26
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Burgin - Rupert 27
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