1. 1
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Tetracycline resistance in Escherichia coli strains collected from North River and Stone
Village Pond
Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, & Loren Smith
Department of Biology, Bridgewater College
Dr. Stephen Baron
December 7, 2021
2. 2
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Table of Contents
Collection of water samples and ColiscanⓇ
filtration
10/18/2021 - Collection of Water Samples pg. 3
10/20/2021 - ColiscanⓇ
Filtration pg. 3
Isolation of Escherichia coli strains
10/25/2021 - Transfer of colonies from Coliscan plates to EMB plates pg. 4-6
11/03/2021 - Transfer incolum from EMB plate to TSA broth pg. 7-8
11/05/2021 - Indole test to confirm E. coli strains pg. 7-8
Determination of Tetracycline sensitive and resistant strains
11/08/2021 - Tetracycline discs diluted and placed on TSA plates containing E. coli pg. 9-11
11/10/2021 - Observe and Measure the zones of inhibition pg. 9-11
11/15/2021- Plasmid Extraction pg.12-13
11/17/2021- Gel electrophoresis of tetracycline resistance plasmids in Escherichia coli strains
pg. 14
References pg. 15
3. 3
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Collection of water samples and ColiscanⓇ
filtration
Dated: 10/18/2021 & 10/20/2021
Purpose:
The purpose of this procedure was to collect water samples from North River and the Stone
Village Pond, to filter these samples at various concentrations to determine the optimum
concentration for colony growth, and to observe the growth of blue and purple Escherichia coli
colonies.
Materials:
The materials used were 2 sterile mason jars, ColiscanⓇ
Membrane Filter Kit, ColiscanⓇ
MF
(Plus) bottle, 50 mm dishes with pads, and sterile 2 mL, 5 mL, and 10mL pipettes.
Methods:
Water samples from North River and the Stone Village pond were collected in sterile mason jars.
Each sample was filtered using a ColiscanⓇ
Membrane Filter kit (2). A filter was placed grid
side up in the funnel and 1.0, 2.0, 4.0, and 10.0 mL of each sample was diluted in 100 mL of
deionized water. The diluted sample was then filtered into the apparatus using a syringe plunger.
The filters were then placed into 50 mm dishes containing pads that were saturated with 2 mL of
ColiscanⓇ
MF (Plus). The 50 mm dishes were incubated at 37℃for 24 hours. After incubation,
the plates were refrigerated at 4℃ until further use.
Results:
After incubation, the coliscan plates were observed for blue and purple colonies, which indicated
Escherichia coli. The plates with water filtered from North River contained more blue and purple
colonies than the plates with water filtered from the Stone Village Pond. There were also more
purple and blue colonies found on the 4 mL and 10 mL plates.
Conclusion:
The water samples were filtered at differing dilutions in order to confirm the presence of
Escherichia coli (E.Coli) in each water sample.
4. 4
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Isolation of Escherichia coli strains
Dated: 10/25/2021
Purpose:
The purpose of this procedure was to transfer blue and purple colonies from the coliscan plates to
eosin methylene blue (EMB) plates to isolate Escherichia coli strains.
Materials:
The materials used were coliscan plates with Escherichia coli strains, Eosin methylene blue
(EMB) plates, toothpicks, and a Bunsen burner.
Methods:
Fourteen blue and purple colonies were transferred to individual EMB plates using a toothpick.
Plate J was streaked with a confirmed strain of E. coli as a positive control. The plates were
incubated at 37°C for 48 hours. After observation, subcultures from each plate were restreaked
on to EMB plates to obtain a pure culture.
Results:
After incubation, the growth of colonies with a green sheen was observed in plates A, B, C, D, E
F, G, H, I, J, K, L, M, N, and O.
5. 5
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Figure 1. Initial Eosin Methylene Blue (EMB) plates with isolated Escherichia coli that were
transferred from coliscan plates. Lactose fermentation (+ E. coli) is indicated by the culture’s
production of a green sheen. Plates O and F were re-streaked to obtain a pure culture.
6. 6
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Conclusion: After inspecting the Eosin methylene blue (EMB) agar plates showing green
colonies, some needed to be re-streaked till they showed green colonies.
7. 7
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Indole test to confirm Escherichia coli
11/03/2021 & 11/05/2021
Purpose:
The purpose was to transfer inoculum from the EMB plates to trypticase soy (TSA) broth for
further isolation of Escherichia coli strains.
Materials:
The materials used were Eosin Methylene Blue (EMB) plates with E.Coli colonies, Tubes
containing trypticase soy broth (TSB) and Kovacs Reagent.
Methods:
A subculture was taken from Each EMB plate and transferred to a tube containing trypticase soy
broth (TSB). The broth cultures were incubated at 37℃ for 48 hours. Indole tests were performed
to confirm that the strains were Escherichia coli species. 1 mL of each TSB culture was placed
into sterile tubes and 2 drops of Kovacs reagent was added.
Results:
After adding the Kovacs reagent to broths A, B, C, D, E, F, G, H, I, K, L, M, N, and O it showed
all broths were Escherichia coli positive which was indicated when the broth turned red. Strain J
was a known sample of E. coli, however, it did not turn red. This was due to the strain being
older, so the test was inaccurate. Strain J was retested with a reactivated culture and indicated
positive results.
8. 8
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Figure 2. Indole test results to confirm Escherichia coli species. TSA broth tubes were inoculated
from colonies on EMB plates A, B, C, D, E, F, G, H, I, J, K, L, M, N, and O that produced a
green color. The tubes were incubated at 37℃for 48 hours. Tube J is a positive control and
contains a known sample of Escherichia coli, which is supposed to be indole positive (not
indicated in figure). Strain J was reactivated and tested to obtain a positive indole test result.
Conclusion:
While the EMB plates were a heavy indicator that the strains were Escherichia coli, an indole
test is confirmatory. Enterobacter and Klebsiella also grow on EMB media but are indole
negative. Based on the results, the fifteen isolated strains are confirmed to be Escherichia coli.
9. 9
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
11/08/2021 & 11/10/2021
Determination of Tetracycline sensitive and resistant strains
Purpose:
The purpose of this procedure was to determine the sensitivity that each strain had when
exposed to Tetracycline.
Materials:
The materials used were 100μL of a 50 μg/μL concentration of Tetracycline, 900 μL of sterile
ethanol, TSA plates, TSB tubes with Escherichia coli strains, and paper disc.
Methods:
A 10-fold dilution of Tetracycline was prepared using 100μL of a 50 μg/μL concentration of
Tetracycline and 900 μL of sterile ethanol. TSA plates, labeled A, B, C, D, E, F, G, H, I, J, K, L,
M, N, and O were streaked with E. coli from the previously incubated TSB tubes. 7 discs with
different amounts of tetracycline were placed on each TSA plate. Going clockwise after that
there was 0.5µL, 1.0µL, 2.5µL, 5.0µL, 7.5µL and then like stated before 10µL at the top. Using
just 10µL of ethanol as the control, was placed into the middle of the TSA plate. Incubated at
37℃for 48 hours. After observing the zones of inhibition around the discs, we measured the
growth in mm, by taking both sides and taking the average of the two numbers and subtracting
6mm which is the measurement of each disc.
Results:
After incubation, the growth of zones of inhibition around the discs was observed in plates A, B,
C, D, E, F, G, H, I, J, K, L, M, N, and O. Plates showed different growth zones of inhibition
which indicated the sensitivity or resistance to the tetracycline.
Figure 3. E. coli resistance with Tetracycline diluted concentration.
10. 10
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Strain 2.5 μg 5 μg 12.5 μg 25 μg 37.5 μg 50 μg Average
F 8.25 13.50 0.00 13.00 13.75 6.00 9.08
B 0.00 11.75 10.75 12.00 13.75 13.00 10.21
J 6.50 9.50 11.00 11.25 12.25 12.50 10.50
D 0.00 10.00 12.75 15.25 13.50 14.00 10.92
I 6.25 10.50 13.50 10.75 12.25 12.25 10.92
A 3.00 11.50 12.50 13.00 13.00 13.00 11.00
N 9.25 10.50 12.75 13.75 11.00 12.75 11.67
H 8.25 11.50 11.75 13.00 13.75 14.00 12.04
E 9.75 12.25 12.25 14.00 11.50 13.75 12.25
L 11.25 12.75 10.75 11.75 14.00 14.50 12.50
K 8.25 13.50 11.75 15.00 14.25 13.75 12.75
G 13.00 13.50 13.50 13.50 14.25 11.50 13.21
M 12.50 13.00 13.50 14.25 15.00 14.25 13.75
C 9.00 6.25 14.75 18.00 19.00 18.50 14.25
O 16.25 13.00 13.50 16.25 17.00 15.25 15.21
Figure 4: The grand averages of the zones of inhibition, and the measurements of the zone of
inhibition which was subtracted by 6 mm to account for the size of the disc.
11. 11
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Figure 5. The grand averages of the zones of inhibition (mm) in Escherichia coli strains A, B, C,
D, E, F, G, H, I, J, K, L, M, N, and O when exposed to various tetracycline concentrations.
Conclusion:
After observing the growth zones of inhibition around the discs, zones were measured, and
graphed for further studies. TSA plates were incubated.
12. 12
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
11/15/2021
Plasmid Extraction
Purpose:
The purpose was to isolate plasmids from Escherichia coli strains with no chloroform present
Materials:
The materials used were 100µL of resuspension buffer (50 mM glucose/10 mM EDTA/10 mM
Tris-Cl), 200 µL lysis solution (0.2 M NaOH/1% sodium dodecyl sulfate [SDS], 150 µl
chloroform, 150 µL 7.5 M ammonium acetate, 200 µL precipitation solution (30%polyethylene
glycol 8000/1.5 M NaCl), Indole broth with Escherichia coli strains and microcentrifuge tubes.
Methods:
From the broth culture, pelleted 1.5 mL of cells then centrifuging and removing supernatant,
secondly, we vortexed 100µL of resuspension buffer (50 mM glucose/10 mM EDTA/10 mM
Tris-Cl) then added in 200 µL lysis solution (0.2 M NaOH/1% sodium dodecyl sulfate [SDS])
then inversion and Incubated at room temperature for 5 minutes then thirdly we added in 150 µL
7.5 M ammonium acetate and 150 µl chloroform. Mixed by inversion, and chilled on ice for 10
minutes and then centrifuged for 10 minutes after that step we transferred the supernatant to 200
µL precipitation solution (30%polyethylene glycol 8000/1.5 M NaCl). Mixed by inversion and
then chilled on ice for 15 minutes and lastly we centrifuged to get pellet DNA then removed the
supernatant, then added in TE to each tube (1). After this the microcentrifuge tubes were ready
for the gel electrophoresis.
Results:
The only thing present in our microcentrifuge tubes were plasmids. No chloroform present in the
microcentrifuge tubes.
Figure 6. Each microcentrifuge tubes with isolated plasmid of Escherichia coli strains
13. 13
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Conclusion:
Isolation of plasmids in Escherichia coli strains was successful and ready for dye and gel
electrophoresis
14. 14
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
Gel Electrophoresis of DNA plasmids
Purpose:
The purpose was to observe for plasmid and chromosomal DNA, Gel electrophoresis of
tetracycline resistance plasmids in Escherichia coli strains
Materials:
The materials used were microcentrifuge tubes with plasmid, Gel loading dye, Gel Doc EZ
Imager and Agarose 0.7%.
Methods:
After this, the microcentrifuge tubes were ready for the gel electrophoresis. Gel loading dye was
added to each tube. The plasmid was inserted into the gel. The gel electrophoresis was run for
about an hour and then observed for plasmid and chromosomal DNA.
Results:
After observing it was concluded that some plasmid and chromosomal DNA was shown as
shown in Figure 7. It was also concluded that the results may have turned out more clear if it was
left in the gel a little longer.
Figure 7. Gel electrophoresis of large plasmid DNA in Escherichia coli strains. The gel was run
on (0.7%) agarose for about an hour. The DNA ladder is located in lane 12 and 17. Plasmid
DNA was found in lanes A, B, C, I, L, N, O.
Conclusion:
After observation, it was concluded that large plasmid DNA was present in strains A, B, C, I, L,
N, and O.
15. 15
Signed: Sarah Autry, Anissa Boyers, Daniela Espinoza-Hernandez, Loren Smith
References
1) Heringa, S., Monroe, J., & Herrick, J. (2007, October 24). A simple, rapid method for
extracting large plasmid DNA from bacteria. Nature Precedings. From
https://doi.org/10.1038/npre.2007.1249.1.
2) (n.d.). (2021, September 15). Resources, Micrology Labs. Micrology Labs. From
https://www.micrologylabs.com/resources-2/.
