1. 4/12/15
Development of Antibiotic Resistant in E. coli Against Antiseptics
By: Jill Engeli
Introduction
We are in constant contact with microbes in our society. This is a scenario that can be
good or bad. Some microbes are key for our survival while others are detrimental; some do not
harm us until they leave their natural environment while others will not harm us at all. For
example, Escheria coli is found in the intestines of humans and animals, but when it is ingested
from undercooked meat or contaminated water, it can make a human or animal very sick. E. coli
can also be found in our kitchens and bathrooms where it could also make us sick. It can be
found on kitchen counters that have not been properly disinfected after cutting or handling raw
meat or in sinks after someone has washed fruits and vegetables. In bathrooms, it can mostly be
found in the toilet bowl. It is commonly found in the toilet bowl because E. coli can be carried in
feces and some of the E. coli will still be present in the toilet bowl after flushing. When people
flush the toilet they do not realize that polluted water vapors erupts out of the toilet bowl and it
can take several hours for these particles to finally settle (Shaw, 2008). The problem is that the
contaminated particles are not visible and can settle anywhere in the bathroom. If they settle on a
toothbrush and then the toothbrush is used, it can make the person sick as they are
unintentionally ingesting E. coli particles.
Methods and Materials
Materials:
 12 1.5 mL microcentrifuge tubes
 150 mL beaker
 20 Sterilized test tubes
 60 nutrient agar plates
 240 sterile discs
 Biotrue® multi-purpose solution
 Bunsen burner
 Distilled water
 Escheria coli culture
 Gloves
 Hole puncher
 Incubator
 Inoculating loop
 Lysol® Power & Fresh Toilet Bowl Cleaner
 Microcentrifuge tube rack
 Permanent marker
 Ruler
 Scrubbing Bubbles® multi-surface bathroom cleaner
 Sterile container

2.  Sterilized tweezers
This study was conducted in the bacteriology lab in Boebel Hall at UW-
Platteville. The Scrubbing Bubbles, Lysol and Biotrue were all purchased from Wal-Mart
in Platteville, WI. All of the products were checked to make sure they were not expired
and that the seals were not broken or tampered with. Then 1 mL was taken from each of
the antiseptics and pipetted into three 1.5 mL microcentrifuge tubes for the 100%
dilution. Another 1.5 mL microcentrifuge tube was obtained and 1 mL of distilled water
was pipetted into the tube; this is used for the control. Nine more 1.5 mL microcentrifuge
tubes were gathered and 0.5 mL of distilled water was pipetted into each tube. These
tubes were labeled with a code for each antiseptic (see Table 2) and then 75%, 50% and
25%, respectively. 0.5 mL was pipetted from each 100% dilution to each 75% tube and
mixed thoroughly for each antiseptic. Then 0.5 mL was pipetted from each 75% tube to
each 50% tube and mixed. Finally, 0.5 mL was pipetted from each 50% tube to each 25%
tube and mixed.
All dilutions were placed in a microcentrifuge rack and placed in a refrigerator at 4°C.
The dilutions were centrifuged when they were taken out to be used during the
experiment.
The sterile discs were prepared by using grade C filter paper. A hole punch was
used to punch out the discs from the filter paper. The discs were then placed into a tray
that was lined with aluminum foil. Another layer of aluminum foil was placed over the
discs to make sure they were fully covered. The pan was put into an oven that was set at
300° for 30 minutes to sterilize the discs. Once the discs were sterilized they remained in
the pan with the aluminum foil on them and kept in a refrigerator to ensure they remained
sterile.
Sterilized petri dishes were gathered and split into four different sections using a
permanent marker. Each section was labeled with the type of dilution that would be
placed in it and then the code of antiseptic on the plate, what round, what replicate and
the day the plates were inoculated. This experiment was done in triplicate. Nutrient agar
was poured into ten of the labeled petri dishes and allowed to cool and set for 10-15
minutes. Once the agar was set, the plates were inoculated with E. coli aseptically. Using
a sterile tweezers, a sterile disc was picked up and dipped into one of the antiseptic
dilutions and then placed in the corresponding section on an inoculated plate. For
example, a sterile disc dipped in the 75% dilution of Lysol will be placed in the Round 1
plate labeled Lysol and in the section labeled 75%. This process was done until all ten
inoculated plates contained four sterile discs dipped in the corresponding antiseptic
dilutions or distilled water. All the discs were checked in each plate to ensure there was
proper contact with the agar and then allowed to sit for 15 minutes. The plates were
inverted and stored overnight in an incubator set at 37°C. The next day, zone of
inhibitions were measured and recorded for each section on all of the inoculated plates.
Next, 20 sterile test tubes were filled with 10 mL of distilled water and labeled as
“Control”, “100% Lysol,” “75% Lysol, “50% Lysol,” “25% Lysol,” etc. The colonies
that remained around the sterile discs were picked up by using a sterilized inoculating
loop. The colonies were then put into the corresponding test tube and shaken. Once all
the colonies were picked and put into the test tubes, an inoculating loop was used to plate
these colonies onto their corresponding sections on 10 other nutrient agar plates. The
sterile discs were placed on the plates by using the same process as previously described.

3. Once all the discs were soaked in an antiseptic dilution and placed in their corresponding
places on the plates, the plates were inverted and stored as previously described. This
same process was done three more times for a total of five rounds of selection. Zone of
inhibition was measured and recorded for all five rounds. All material was autoclaved
and disposed of once the experiment ended.
Data and Results
Biotrue Multi-purpose Contact Lens Solution (CL)
Biotrue was susceptible to E. coli in all of the dilutions in the first two rounds. In
the third round the 100% and 75% dilutions had zone of inhibitions of 9 and 7 mm,
respectively (See Table 1). In the fourth round the 100% and 75% had zone of inhibitions
of 11 mm and 8 mm. In the final round of selection, only the 100% dilution had a zone of
inhibition which was 8 mm. No zone of inhibitions were observed in the 50% or 25%
dilutions of CL. E. coli was almost resistant to every dilution of CL in each round expect
for the 100% dilution in the fourth round. A zone of inhibition of 11 mm is intermediate
so the E. coli was neither resistant nor susceptible to CL.
Lysol Power & Fresh Toilet Bowl Cleaner (Lysol)
In every round for each dilution, Lysol had significant zone of inhibitions (Table
1). The zone of inhibitions slightly decreased each round for the 100% dilution. In the
first round of the 100% the zone of inhibition was 30 mm and by the fifth round it had
decreased to 26 mm. For the 75% dilution, the zone of inhibition remained the same (20
mm) until the fifth round, in which it was 17 mm. The 50% dilution zone of inhibition
slightly fluctuated. In Round 1, the zone of inhibition was 17 mm, and by Round 5 it was
only 15 mm. The 25% dilution had a significant change in zone of inhibition from Round
1 to Round 2 as it went from 9 mm to 17 mm (Table 1). After Round 2, the zone of
inhibitions slightly decreased. E. coli was susceptible to Lysol in all five rounds at 100%
and 75%. In the 50% dilution, E. coli was susceptible in rounds 1, 2 and 3. It was neither
resistant nor susceptible to Lysol in rounds 4 and 5 of the 50% dilution. In the 25%
dilution, E. coli was susceptible in rounds 2 and 3, resistant in round 1, and neither
susceptible nor resistant in rounds 4 and 5 (Table 1).
Scrubbing Bubbles (SB)
Scrubbing Bubbles only had zone of inhibitions in rounds 2-5 of the 100%
solution. No zone of inhibitions were observed in the 75%, 50% and 25% dilutions
(Table 1). E. coli was neither susceptible nor resistant to SB in the 100% solutions in
rounds 2-5. It was resistant in every round for the 75%, 50% and 25% dilutions.
Discussion
Out of all the antiseptics tested in this experiment, Lysol was the only one to have
significant zone of inhibitions at all dilutions and rounds of selection while Scrubbing Bubbles
had the least amount. These results were not what was expected as all of these antiseptics are
guaranteed by the manufacturers to kill 99.9% of germs. It was expected that E. coli would be

4. susceptible to all three of the antiseptics at each dilution during each round of selection. Due to
these results, the hypothesis cannot be fully supported.
Tables and Figures
Table 1: Average Zone of Inhibition of E. coli using Antiseptics
* = diameter of zone of inhibition was measured in millimeters (mm).Size of sterile discs = 6 mm. If ZI was <10mm, the bacteria
was resistant to the antiseptic; ZI of 11-15 mm was intermediate; ZI of >16 mm, the bacteria was susceptible to the antiseptic.
The experiment was done in triplicate form.
Table 2: Codes used on petri dishes and dilutions to identify each antiseptic and its
characteristics
Code Antiseptic Manufacturer Active Ingredient(s)
CL Biotrue® Multi-purpose
Contact Lens Solution
Bausch + Lomb Hyaluronan
Lysol Lysol® Power & Fresh
Toilet Bowl Cleaner
Reckitt Benckiser LLC Alkyl dimethyl benzyl
ammonium chloride
(0.49%), Octyl decyl
dimethyl ammonium
chloride (0.37%), Didecyl
dimethyl ammonium
chloride (0.22%), Dioctyl
dimethyl ammonium
chloride (0.14%)

5. SB Scrubbing Bubbles® S.C. Johnson & Son Lactic Acid (1.77%)
.
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