By Jonathan A.
 |
Purpose Hypothesis Experiment Design Materials Procedures Research Report Results Data Conclusion Bibliogaphy |
To conduct this experiment you must make bacterial suspension for each developed week and incubate bacterial suspensions using Mueller Hinton agar plates. Over a time period of five weeks record the zone of inhibition to the test antibiotic, ampicillin.
The results were that the average zone diameter of inhibition to ampicillin in the test E. coli strains met the testing requirements of a less than 13 mm zone size, meaning resistant. Over the five-week period each strain showed more resistance at a different rate. Therefore E. coli has the ability to gain resistance to the antibiotic ampicillin, which may in turn be true for other bacteria and antibiotics.
My results indicate that my hypothesis
should be accepted. The average zone sizes of inhibition to ampicillin
in the test E. coli strains meet the testing requirements of a less
than 13 mm zone size, meaning resistant.
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I became interested in this idea from similar previous projects viewed and relatives in the prescription medicine field. The project rose my interests further when I found that bacterial immunity to antibiotics was on the rise, and becoming a very important issue. When choosing a topic I tried to stay in the microbiology field because it’s really interesting and I find it easier to work on something that I enjoy.
The information gained from this
experiment could be found useful to those who work in the field of microbiology,
those in the medical field, those having contact with antibiotics, including
all antibiotic using citizens, and people who wish to know what they can
do to prevent further growth of antibiotic resistance in bacteria.
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I base my hypothesis on verbal interviews
with a qualified supervisor in the field of microbiology and medical technology;
and information collected about general bacteria, the specific bacteria
genes E. coli, antibiotic resistance, and the antibiotic ampicillin.
An antibiotic is any substance produced by a microorganism, which harms
or kills another microorganism, such as bacteria. However, antibiotics
do not harm viruses. Some bacteria have become resistant to the effects
of different antibiotics, by slowly being exposed to the particular antibiotic.
The resistance occurs when a minimum of one bacteria cell genetically acquires
the ability to destroy the antibiotic. That single cell of bacteria
then divides (possibly at a rate of every 20 minutes) and produces a population
that is no longer affected by that specific antibiotic. For the most
part E. coli is a harmless bacterium that can be found within the intestines
of all humans. E. coli has also been know to have the ability to
exchange genetic information with other organisms gaining some of that
organism’s characteristics. The E. coli strain 0157:H7 is an example
of this action. E. coli strain 0157:H7 was infected with a
bacterial virus and that particular virus had the ability to insert its
own DNA into the bacteria’s chromosome without harming the bacterium.
Ampicillin is a semi-synthetic from of penicillin that has a special feature
that penicillin does not; ampicillin has a resistance to stomach acid,
which penicillin is highly sensitive to. The bacterium E. coli has
also been found to be sensitive to ampicillin.
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The manipulated variable was
the amount of time ampicillin was exposed each week to the eleven strains
of E. coli. All eleven strains of E. coli will be exposed to the
same dose of ampicillin for that same week.
The responding variable will be the
zone sizes around each ampicillin disk located on the agar plates.
To measure the responding variable I record the diameter of the zone size
around the ampicillin disk using calipers. The smaller the zone size
around the ampicillin disk the more resistant the bacteria has become to
the antibiotic. Meaning the ampicillin disk did not kill the
bacterium that was around the antibiotic.
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QUANTITY ITEM DISCRIPTION
Top of Page1 Colorimeter
55 12mm x 75mm test tubes
15 Mueller Hinton Agar Plates
+55 Swabs
N/A Normal Saline at 0.9%
1 35 degree Incubator
1 Calipers
10 BHI Agar Slants
11 Strains of E. coli
55 Ampicillin saturated disks
115.5 ml Tryptic soy broth
11,000 mg Ampicillin dilution (1mg to 1ml)
1 200mg Pipette
+60 Pipette tips
+55 Plastic pipettes
B. Making suspension strains of E.
coli comply with testing needs
1. Gently dab E. coli strain # 1
with sterile cotton swab.
2. Smear E. coli strain #1 cotton
swab on the inside of 12mmx75mm test tube.
3. Add saline to test tube at was
just swabbed with E. coli strain #1 and tightly secure test tube screw
cap.
4. Shake test tube at a reasonable
rate.
5. Place test tube in colorimeter.
6. Test the light transmission of
test tube contents.
7. For the proper amount of E. coli
to saline, the colorimeter should read in the higher red zone (80% light
transmission).
a. If colorimeter reads higher than
80% light transmission add more E. coli, gradually lowering the light transmission
to 80%.
b. If colorimeter reads lower than
80% light transmission add more saline, gradually diluting the E. coli
strain and increasing light transmission to 80%.
8. Once the test tube has met the
80% light requirement repeat steps B. 1-7 to all eleven E. coli strains.
C. Preparation of Mueller Hinton
agar plates
1. Take E. coli suspension strain
#1 and swab a coat of suspension on agar plate.
2. Using the same suspension swab
an overlapping suspension coat over the first coat after the agar plate
has been turned 90 degrees.
3. Once again using the same bacterial
suspension swab another overlapping coat over the previous two, after the
agar plate had been turned another 90 degrees.
4. The agar plate is finally finished
when swabbed three times in three different directions.
5. Now place the ampicillin disks
on the agar plate at equal distances apart.
6. Repeat steps C. 1-5 to all eleven
E. coli suspensions.
D. Making trypitc soy broth (TSB)
solution with suspension and ampicillin dilution
1. To empty test tube add 2.1mL
TSB using pipette.
2. Add 200mg of ampicillin dilution
(1mg to 1mL) to TSB.
3. Add 200mg of E. coli suspension
to TSB+ampicillin solution.
4. Repeat steps E. 1-3 to all eleven
E. coli strains.
5. Incubate all eleven E. coli solutions
for given time period.
E. Incubating
1. Incubate all eleven E. coli suspensions
in Mueller Hinton agar plates for a minimum of 24 hours or longer.
2. After incubation time period
measure the inhibition zones around all ampicillin disks for all eleven
strains of E. coli.
3. Record inhibition zone diameters
using calipers.
What Mueller
Hinton agar plates should look like after incubation.
Disregarding
zone sizes.
II. Week two
A. Partial reconfiguration of previous
week
1. Repeat steps I.A.1-6, I.B.1-8
(with the exception of making bacterial suspensions from previous weeks
TSB solutions), I.C.1-6, I.D.1-4, and I.E. 1-3.
III.Week three
A. Reconfiguration of previous
week
1. Repeat steps I.A.1-6, I.B.1-8
(with the exception of making bacterial suspensions from previous weeks
TSB solutions), I.C.1-6, I.D.1-4, and I.E. 1-3.
IV. Week four
A. Reconfiguration of previous
week
1. Repeat steps I.A.1-6, I.B.1-8
(with the exception of making bacterial suspensions from previous weeks
TSB solutions), I.C.1-6, I.D.1-4, and I.E. 1-3.
V. Week five
A. Reconfiguration of previous
week
1. Repeat steps I.A.1-6, I.B.1-8
(with the exception of making bacterial suspensions from previous weeks
TSB solutions), I.C.1-6, I.D.1-4, and I.E. 1-3.
2. After recording all information
autoclave and dispose all materials used in experiment.
* Should be done before all lab work,
every week.
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INTRODUCTION
MICROBIOLOGY
ESCHERICHIA COLI
Enterotoxigenic E. coli
Diarrhea of infants and adults
in tropical and subtropical climates, especially in developing countries,
strains of enterotoxigenic (ETEC) should be the suspected cause.
In the United States and other developed countries, ETEC diarrhea, sometimes
referred to as “traveler’s diarrhea”, is the most common cause of diarrheal
diseases. The ETEC infection is commonly acquired by consuming infected
food or water. The major contributing factors in the spread and transmission
of the disease include poor hygiene, inadequate sources of drinking water,
and lack of proper sanitation. 106 to 1010 organisms are necessary
to initiate disease in an immunocompetent host. Various protective
mechanisms include stomach acidity have been described as inhibiting colonization
and initiation of disease. Which means those who are suffering from
achlorhydria are at greater risk of inhibiting the disease than those without
achlorhydria. Colonization of ETEC begins near the small intestine.
Once established enterotoxigenic strains of E. coli release into the small
intestine.
Usually the disease caused
by ETEC is characterized by non-bloody, watery diarrhea, nausea, abdominal
cramps, and low-grade fever. To date there is no evidence of mucosal
penetration or invasion. The infirmity may last from one day up to
five days.
Enteroinvasive E. coli
Strains of Enteroinvasive
E. coli (EIEC) are very different from the strains of EPEC and ETEC.
EIEC strains create dysentery, with direct penetration, invasion, and destruction
of the intestinal mucosa. This diarrheal sickness is similar to that
produced by Shigella. The EIEC infections are found in adults and
children.
Clinical infection is characterized
by fever, severe abdominal cramps, malaise, and watery diarrhea (stools
containing pus, mucus, and blood). While EIEC and Shigella have been
discovered to be similar in morphology and in clinical presentation, the
infective dose of EIEC necessary to produce disease in much higher than
that of Shigella.
Enterohemorrhagic E. coli
In 1982, the O157:H7 strain
of E. coli was first recognized during an outbreak of hemorrhagic diarrhea
and colitis. Strain serotype O157:H7 of the enterohemorrhagic E. coli (EHEC)
has since then been associated with hemorrhagic diarrhea, colitis, and
hemolyticuremic syndrome (HUS). HUS is characterized by low platelet
count, hemolytic anemia, and kidney failure.
Illness brought on by EHEC is characterized by watery diarrhea that progresses to bloody diarrhea and cramping abdominal pain, with low-grade fever or no fever at all. The stool of a contaminated person contains no leukcytes, which differentiates it from Shigella dysentery or EIEC strain infection. The infection is potentially fatal to young children and the elderly. Meats, such as undercooked hamburger, unpasteurized milk, and apple cider, have been known to spread the infection.
Cases of O157:H7 in the State
of Washington
Year
Number of Reported Cases
1991
164
1992
300
1993
741
1994
174
1995
140
1996
187
1997
149
1998
144
1999
186
Enteroadherent E. coli
Enteroadherent E. coli, most
recently spoken as of enteroaggregatice E. coli (EAggEC), causes diarrhea
by adhering to the mucosal surface of the intestine. The following
symptoms are the result to a EaggEC infection; watery diarrhea, vomiting
dehydration, and occasionally abdominal pain.
ANTIBIOTICS
AMPICILLIN
The results of the experiment were
that the average zone sizes, at the final episode of testing, of the Escherichia
coli strains used during testing met NCCLS interpretive standards for an
E. coli isolate that produces an ampicillin inhibition zone diameter of
less than 13 mm, classifying tested strains as resistant. Over the
time period allowed the tested strains of E. coli made evident that in
a matter of weeks bacteria can obtain the ability to show signs of resistance
to the specific antibiotic used to terminate them. Although the average
zone size of tested strains met standards as of classifying E. coli resistant
to ampicillin, all E. coli strains tested developed resistance at a varying
rate. Testing also showed individual bacterial mutant colonies within
the more obvious zone of inhibition. This could indicate inoculation
with a mixed culture. However, emergence of resistant mutants of
the test isolate is a more likely reason for this particular growth pattern.
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| WEEK 1 GRAPH/TABLE |
| WEEK 2 GRAPH/TABLE |
| WEEK 3 GRAPH/TABLE |
| WEEK 4 GRAPH/TABLE |
| WEEK 5 GRAPH/TABLE |
The results indicate that this hypothesis should be accepted. The tested E. coli strains had bacterial zone sizes that meet the NCCLS interpretive standards as resistant; an E. coli isolate that produces an ampicillin inhibition zone diameter of less than 13 mm is classified as resistant.
With the outcome of these results of this particular experimentation, I wonder if these certain results would prove true for other bacteria and their specific antibiotic used against them. I also wonder if in the case of using different bacteria and antibiotics it would change the amount of time needed to become resistant whether it be more or less. And furthermore I wonder if it is possible for bacteria strains to become resistant to more than one antibiotic.
These finding could prove to be useful to those in the medical field, microbiologists, and other areas of work related to bacterial resistance and those having contact with antibiotics, including all antibiotic using citizens. Therefore this studies results suggest that when given time and exposure E. coli has the ability to develop a resistance to ampicillin.
If I were to conduct this project
again I would provide a wider variety of bacteria and antibiotics, including
a larger number of test subjects within each group to get sturdier and
reliable results. I would also give bacteria more time to develop
resistance if need.
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Brown, John C. What the Heck is an E. coli. 18 Oct. 1995. Department of Molecular Biosciences. 8 Dec. 2001 <http://people.ku.edu/~jbrown/ecoli.html>.
Brown, John C. What the Heck is Microbiology. 18 Oct. 1995. Department of Molecular Biosciences. 9 Dec. 2001 <http://people.ku.edu/~jbrown/whatmicro.html>.
Clark, Marie. Personal Interview. 12 Dec. 2001.
Ewing WH: Edwards and Ewing’s Identification of Enterobacteriaceae, 4th ed. East Norwalk, CT: Appleton & Lange, 1986, pp 2-3.
Johnson, Eugene M. "Ampicillin." The World Book Encyclopedia. Ed: Dale Jacobs. Chicago: World Book, Inc., 1999. 443.
Selecky, Mary. E. coli. 31 Feb. 1996. Washington Department of Health. 9 Dec. 2001 <http://www.doh.wa.gov/Topics/ecoli.htm>.
Washington J: Laboratory Procedures
in Clinical Microbiology,
2nd ed. New York: Springer-Verlag,
1981, p 181.
