Comparing the Effect of Different Antimicrobials on the Growth of Escherichia Coli

Background information

“Antimicrobial is the name for a chemical that either kills or prevents the growth of microbes (‘bugs’ or ‘germs’) such as bacteria, viruses, fungi or protozoa. Some antimicrobials are produced by bugs themselves (e.g. penicillin is produced by the penicillium mould), others are designed in the laboratory. Different bugs are susceptible to different antimicrobials e.g. the penicillium mould is not killed by the penicillin it produces, but some bacteria are susceptible to penicillin.”[1]

This experiment is based on the Kirby-Bauer Antimicrobial Susceptibility Test. “The Kirby Bauer test is a qualitative assay whereby discs of paper are impregnated with a single concentration of different antibiotics. The discs are placed on the surface of an agar plate that has been inoculated with test bacteria. During incubation, the antibiotics diffuse outward from the discs creating a concentration gradient. After 18-24 hours, the zone diameter (zone of inhibition) is measured and reference tables are used to determine if the bacteria are Sensitive (S), Intermediate (I) or Resistant (R) to the antimicrobial drugs.”[2]

“Ampicillin belongs to a class of antibiotics called penicillins that are used for treating bacterial infections.”[3] Penicillins “stop bacteria from multiplying by preventing bacteria from forming the walls that surround them. The walls are necessary to protect bacteria from their environment and to keep the contents of the bacterial cell together. Bacteria cannot survive without a cell wall. Penicillins are most effective when bacteria are actively multiplying and forming cell walls. Ampicillin is effective against many bacteria including H. influenzae, N. gonorrhoea, E. coli, Salmonella, and Shigella, streptococci and certain strains of staphylococci.”[4]

Kanamycin is “a water-soluble broad-spectrum antibiotic obtained from the soil bacterium Streptomyces kanamyceticus.”[5] It “acts by inhibiting the synthesis of protein in susceptible organisms.”[6]

“Tetracycline is an antibiotic with a broad spectrum, that is, it is active against many different bacteria. It is effective against Hemophilus influenzae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Neisseria gonorrhoeae, and many others. Tetracycline prevents growth of bacteria by preventing the bacteria to manufacture proteins that they need to survive.”[7]

Research question

Does a higher concentration of an antimicrobial have a stronger inhibiting effect on Escherichia coli? Which of the antimicrobials has the strongest inhibiting effect on the growth of the bacterial culture of Escherichia coli? Due to the fact that the concentrations of the antibiotics and the other two antimicrobials were given in non-corresponding units a valid comparison can be made only between the three antibiotics and between the two other antimicrobials separately. In order to answer the research question the diameters of the zones of inhibition created onto the bacterial culture of Escherichia coli by equivalent concentrations of different antimicrobials has to be compared.

Hypothesis

A higher concentration of an antimicrobial has a stronger inhibiting effect on the growth of the bacterial culture of Escherichia coli. If a lower concentration of one antimicrobial creates a zone of inhibition in the bacterial culture of Escherichia coli which has the same diameter as the zone of inhibition created by a higher concentration of another antimicrobial, then this first antimicrobial has a stronger inhibiting effect on the specific bacteria.

Variables

Independent variable

The different antimicrobials in different concentrations. Five different substances were used in this experiment. The antimicrobials: ampicillin (Amp) in concentrations of 100, 125, 150, 300, 500 and 1000 µg/ml; kanamycin (Km) is concentrations 100, 125, 150, 300, 500 and 1000 µg/ml; tetracycline (tet) in concentrations 50, 100, 125, 150, 300 and 500 µg/ml; a disinfectant Sekusept in concentrations 0.5%, 1%, 2.5%, 5% and 10%; and a stain remover Vanish in concentrations 5%, 10%, 20%, 50% and 100%. The size of the area of chemical infiltration around the disc is dependent on the solubility and molecular size of the substance.

Dependent variable

The diameter of the zone of inhibition that formed around the filter paper discs. The zones of inhibition become visible as the chemical in the discs dissolves into the culture medium (depending on the solubility and molecular size of the substance). The organism Escherichia coli that was placed on the agar was susceptible to the chemicals and therefore did not grow around the disc, forming the zones of inhibition. If comparing the diameters of the zones of inhibition created by the same concentrations of different antimicrobials, it is possible to assess the strength of their inhibiting effect of the microbial culture of Escherichia coli.

Controlled variables

The composition of the antimicrobials.

The concentrations of the antimicrobials. These were determined by the tutor.

Materials

Two petri dishes containing the Luria Bertani (LB) culture medium, prepared by our tutor;

A marker for labelling the petri dishes with the name of the substance that would be tested in it and 5 or 6 dots (depending on the substance they chose) to mark the places the filter paper discs impregnated with the chemical would be placed;

A bunsen burner for sterilizing the environment of the experiment;

A 1:1 dilution of an overnight culture of Escherichia coli and sterile saline solution; prepared by the tutor;

A sterile cotton-stick for planting the organism (Escherichia coli) onto the culture medium;

The saline solutions of different concentrations of different chemicals as the independent variable of the experiment. Five different substances were used in this experiment. The antimicrobials: ampicillin (Amp) in concentrations of 100, 125, 150, 300, 500 and 1000 µg/ml; kanamycin (Km) is concentrations 100, 125, 150, 300, 500 and 1000 µg/ml; tetracycline (tet) in concentrations 50, 100, 125, 150, 300 and 500 µg/ml; a disinfectant Sekusept in concentrations 0.5%, 1%, 2.5%, 5% and 10%; and a stain remover Vanish in concentrations 5%, 10%, 20%, 50% and 100%. The solutions of the substances were prepared by the tutor;

The filter paper discs impregnated in different concentrations of the different microbial growth inhibiting substances. These were prepared by the tutor;

Forceps for placing the discs onto the surface of the agar;

An incubator with a temperature 37 degrees Celsius where the zones of inhibition would become visible through microbial growth;

A ruler (range 0-30 cm, basic unit 1 mm, uncertainty of measurement 0.5 mm) for measuring the diameters of the zones of inhibition formed around the discs on the culture medium.

Method

Each student received two petri dishes containing the Luria Bertani culture medium (prepared by our tutor) and used a marker to label the dishes with the name of the substance that would be tested in it and 5 or 6 dots (depending on the substance they chose) to mark the places the filter paper discs impregnated with the chemical would be placed.

A bunsen burner was ignited to ensure a sterile environment for the experiment. All of the procedures were carried out as close to the bunsen burner as possible in order to avoid any contamination.

A sterile cotton-stick was dipped into the broth culture of the organism (50% overnight culture of Escherichia coli and 50% sterile saline solution; prepared by the tutor) and gently squeezed against the inside of the tube in order to remove excess fluid.

The cotton-stick was then used to streak a petri dish containing the Luria Bertani culture medium in order to spread the organism as a lawn of growth onto the culture medium. The procedure was repeated for the second petri dish.

The two plates were allowed to dry for five minutes.

During the time the plates were drying the tutor used sterile forceps to place 5 or 6 (depending on the substance) filter paper discs impregnated with different concentrations of different chemicals onto a paper towel and allowed then to dry for about five minutes.

Forceps were sterilized in the flame of the bunsen burner and used to place the discs onto their specific place on the surface of the agar. The discs were gently pressed into the agar in order to prevent them form falling off when the dishes were inverted.

The inverted plates were incubated at 37 degrees Celsius for 24 hours.

Using a ruler (range 0-30 cm, basic unit 1 mm, uncertainty of measurement 0.5 mm) the zones of inhibition for all substances and for all concentrations were measured.

The produced data was processed.

Data

The data obtained form the experiment was processed so that an average could be found. An average diameter was found for the zones of inhibition of each concentration of each antibiotic, so the comparison between the antibiotics would be valid. An average is found by adding together all of the elements (all diameters of the zones of inhibition of a specific concentration of a specific substance) and dividing the sum by the number of all elements. The formula for finding arithmetic average is .

As seen from Table 1, different antibiotics have a different strength of inhibiting the growth of the bacterial culture of Escherichia coli. It also clearly portrays the assumption that the concentration of the substance is in a linear relationship with the diameter of the corresponding zone of inhibition. This means that when the concentration of the antibiotic is increased, its inhibiting effect on the bacterial culture of Escherichia coli is also increased, resulting in a longer diameter of the zone of inhibition. Even though the concentrations of the antibiotics represented in the given table are not directly correspondent, it can still be observed that the most efficient antibiotic inhibiting the growth of the bacterial culture of Escherichia coli is kanamycin. Second in efficiency is tetracycline. Ampicillin showed significantly smaller efficiency, only inhibiting the growth of the bacterial culture of Escherichia coli at the concentration of 1000 µg/ml. This shows that the bacteria Escherichia coli is not susceptible to ampicillin. For graphical representation see Graph 1.

In Table 2 an average diameter was found for the zones of inhibition of each concentration of each antimicrobial, so the comparison between the antimicrobials would be valid. An average is found by adding together all of the elements (all diameters of the zones of inhibition of a specific concentration of a specific substance) and dividing the sum by the number of all elements.

Just as Table 1, Table 2 also shows that when the concentration of the antimicrobial is increased, its inhibiting effect on the bacterial culture of Escherichia coli is also increased, resulting in a longer diameter of the zone of inhibition. This means that there is a linear relationship between the concentration of the antimicrobial and the diameter of the corresponding zone of inhibition. As the concentrations of the antimicrobials of the two substances represented in Table 2 are to a large extent incompatible, comparison is possible only at the concentrations of 5% and 10% of the substances. The table suggests that at the concentration 5% (5% of the antimicrobial, 95% of saline solution) the diameter of the zone of inhibition created by Vanish was 7.3 mm and by Sekusept 17.9 mm (uncertainty of measurement 0.5 mm). At the concentration 10% this was 11.8 mm for Vanish and 19.8 mm for Sekusept. For graphical representation of this see Graph 3. This suggests that Sekusept has a much stronger inhibiting effect of the growth of the bacterial culture of Escherichia coli.

Graph 1: The average diameters of the zones of inhibition created by the antibiotics Kanamycin, Ampicillin and Tetracycline in the bacterial culture of Escherichia coli in millimetres (± 0.5 mm) based on Table 1

As seen on Graph 2, the most efficient antibiotic for inhibiting the growth of the bacterial culture of Escherichia coli is kanamycin. Second most efficient is tetracycline. Ampicillin has almost no inhibiting effect on the bacteria as it only created a zone of inhibition with an insignificant diameter at the concentration of 1000 µg/ml.

Graph 2: The average diameters of the zones of inhibition created by the antimicrobials Vanish (stain remover) and Sekusept (disinfectant) in the bacterial culture of Escherichia coli in millimetres (± 0.5 mm) based on Table 2

Even though these two substances can only be compared at the concentrations of 5% and 10%, it becomes obvious from Graph 3 that the disinfectant Sekusept is significantly more efficient in inhibiting the growth of the bacterial culture of Escherichia coli than the stain remover Vanish.

Conclusion

The results of the experiment show, as seen on both graphs, that the concentration of the substance is in a linear relationship with the diameter of the corresponding zone of inhibition. This means that when the concentration of the antibiotic is increased, its inhibiting effect on the bacterial culture of Escherichia coli is also increased, resulting in a longer diameter of the zone of inhibition.

Unfortunately, the data about the concentrations of the substances was given so that the results of the antibiotics (kanamycin, ampicillin and tetracycline) could not be compared to the results of the rest of the antimicrobials (disinfectant Sekusept and stain remover Vanish) used in this experiment. Nevertheless, it was possible to draw some valid conclusions when comparing the three antibiotics and the two other antimicrobials separately.

Based on the data in Table 1, represented visually on Graph 1, it can be concluded that the most efficient antibiotic for inhibiting the growth of the bacterial culture of Escherichia coli, was kanamycin. Second most efficient was tetracycline. Ampicillin had almost no inhibiting effect on the bacteria.

Out of the other two antimicrobials the disinfectant Sekusept was considerably more efficient in inhibiting the growth of the bacterial culture of Escherichia coli as can be seen from Graph 2, based on Table 2.

Evaluation

This experiment had some drawbacks which could easily be avoided in the future.

The most significant obstacle in the given experiment was that the concentrations of the antibiotics and the other two antimicrobials were given in non-corresponding units, therefore a valid comparison could be made only between the three antibiotics (given in µg/ml in saline solution) and between the two other antimicrobials (% of saline solution) separately. This rises the question of the purpose of adding a disinfectant and a stain remover into the experiment alongside with the three antibiotics as no valid comparisons could be made between all of the five substances. In order to avoid this error in the future, all of the concentrations of the substances should be given in equivalent, or at least in comparable units.

Another complication arose when some of the zones of inhibition on the petri dishes had merged together and were therefore slightly more difficult to measure. However, this probably did not significantly confound the results as the diameter of the zone of inhibition could be measured from another angel so that the value obtained was still valid. Regardless, for avoiding this potential obstacle in the future, the impregnated filter paper discs should be placed onto the bacterial culture so that the zones of inhibition would not merge. This can be done either by using larger petri dishes, placing a smaller number of impregnated filter paper discs on one petri dish, or reducing the concentrations of the substances the filter paper discs were impregnated with.

References:

[1] City eHealth Research Centre. “What are Antimicrobials?” Bugs and Drugs on the Web. National Electronic Library of Infection Antimicrobial Resistance Website, 7 January 2004. Web. 17 May 2011.

< http://www.neli.org.uk/arfaqs.nsf/c142977c99209f3680256c91003fdf4a/

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[2] Sockett, D. C., Valley, A. “Antimicrobial Susceptibility Testing,” 14 April 2006. Web. 20 May 2011. <http://www.wvdl.wisc.edu/PDF/WVDL.Info.Antimicrobial_Susceptibility_Testing_at_WVDL.pdf>

[3] Ogbru, O. “Ampicillin, Omnipen, Polycillin, Principen.” MedicineNet.com. Web. 6 March 2012. <http://www.medicinenet.com/ampicillin/article.htm>

[4] Ibid.

[5] The American Heritage Medical Dictionary. “Kanamycin.” The Free Dictionary by Farlex: Medical Dictionary. Web. 6 March 2012. <http://medical-dictionary.thefreedictionary.com/kanamycin>

[6] Mosby’s Dental Dictionary. “Kanamycin.” The Free Dictionary by Farlex: Medical Dictionary. Web. 6 March 2012. <http://medical-dictionary.thefreedictionary.com/kanamycin>

[7] Ogbru, O. “Tetracycline, Sumycin.” MedicineNet.com. Web. 6 March 2012. <http://www.medicinenet.com/ampicillin/article.htm>