Comparing the effect of different antimicrobials on the growth of E.coli

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Comparing the effect of different antimicrobials on the susceptibility of Escherichia coli by measuring the diameter of the zone of inhibition

Biology Laboratory Report IV

Standard Level


Comparing the effect of different antimicrobials on the susceptibility of Escherichia coli by measuring the diameter of the zones of inhibition

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.”

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.”

“Ampicillin belongs to a class of antibiotics called penicillins that are used for treating bacterial infections.” 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.”

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

“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.”

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.

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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 ...

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