MKBN211

Report – Practical 5
Antibiotic susceptibility testing

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ABSTRACT
Antibiotic susceptibility testing methods aid in the rapid beginning of targeted
antibiotic therapy for patients with illnesses and have the potential to extend the life
of current narrow-spectrum antimicrobials. Although conceptually new and rapid
antibiotic susceptibility testing technologies, such as new phenotyping methods,
digital imaging, and genomic approaches, have been described, no single major or
widely accepted technological breakthrough leads the field of rapid antibiotic
susceptibility testing platform development. This could be due to several
impediments to the timely development and implementation of novel and rapid
antibiotic susceptibility testing technologies in healthcare settings. This experiment is
done to see which antibiotic is susceptible to which microorganism. Escherichia coli,
Bacillus cereus, and Staphylococcus aureus are the bacteria used in this
experiment, and Gentamycin (CN 10), Kanamycin (K 30), and Neomycin (N 30)- are
the antibiotics going to be tested. Firstly, the bacteria are spread over the Mueller-
Hinton agar plate and left to dry for 15 minutes. Insert the antibiotics into the plates
and incubate them for 48 hours. Measure the inhibition zone of the antibiotics. After
comparison between the values from the table and the values obtained from the
measurement, it indicates that Bacillus cereus is susceptible to all antibiotics, and
Staphylococcus aureus and Escherichia coli are resistant to all antibiotics. This
testing is important to help one know which antibiotic to use in which bacteria.

INTRODUCTION
Testing the effectiveness of antibiotics in preventing microbe development is a
crucial task for a variety of reasons, the most important of which is the prevention
and treatment of infectious diseases. To effectively protect ourselves from hazardous
bacteria, first learn which medicines and disinfectants can successfully kill or inhibit
those microbes. A Kirby-Bauer test is a type of antimicrobial control testing that uses
the disk diffusion assay method to apply various antibiotic disks to an agar plate to
determine whether different types of microbes are susceptible, resistant, or
intermediate to different types of antibiotics (Mcnutt, 2019). The sensitivity of an
antibiotic is determined by measuring the zone of inhibition surrounding each
antibiotic disk and interpreting the results. The wider the antibiotic's zone of
inhibition, the more sensitive the bacteria are to it (Mcnutt, 2019). In this experiment,
 one is going to be looking at how different antibiotics which are Gentamycin (CN 10),
Kanamycin (K 30), and Neomycin (N 30) affected the growth of the bacteria
Escherichia coli, Bacillus cereus, and Staphylococcus aureus.
AIM
The aim of this experiment is to help choose the antibiotic that will be most effective
against the specific types of microorganisms.

Material and method used.

A small colony of Escherichia coli, Bacillus cereus, and Staphylococcus aureus is
transferred with different droppers for each to a Mueller-Histon agar plate and spread
the bacteria evenly on the plate using a plastic spreader near an alcohol lamp glass.
Let the bacteria dry for 15 minutes and divide the plates into 3 sections. The forceps
need to be sterilized before picking the antibiotic, then place the antibiotic in the
middle of each section. Make sure that the antibiotic disk is in contact with the culture
by gently pressing it down with a sterile loop. Each Mueller-Histon agar plate will
contain Gentamycin, Kanamycin, and Neomycin. Pack the plates and put them in the
incubator for 48 hours. After 48 hours measure the zones of inhibition with a ruler in
millimeters.

RESULTS AND DISCUSSION

Results obtained:

Bacillus cereus Inhibition zone Results

 (mm)
Gentamycin (CN 10) 28 mm S
Kanamycin (K 30) 21 mm S
Neomycin (N 30) 20 mm S

Escherichia coli Inhibition zone Results

(mm)
Gentamycin (CN 10) 0 mm R
Kanamycin (K 30) 0 mm R
Neomycin (N 30) 0 mm R

Staphylococcus Inhibition zone Results

aureus (mm)
Gentamycin (CN 10) 7 mm R
Kanamycin (K 30) 0 mm R
Neomycin (N 30) 9 mm R
Discussion
In this experiment, three bacteria- Bacillus cereus, Staphylococcus aureus,
and Escherichia coli are used to determine the susceptibility of these bacteria
to various antibiotics (neomycin, kanamycin, and gentamycin). Bacillus
cereus had three visible inhibition zones on the culture plate with different
diameters. Based on the measurement obtained in Bacillus cereus, the
inhibition zone for neomycin (N 30) is 20mm, the inhibition zone for
kanamycin (K 30) is 21mm, and the inhibition zone for gentamycin (CN 10) is
28mm. In Escherichia coli, the inhibition zone for neomycin (N 30) is 0mm, the
inhibition zone for kanamycin (K 30) is 0mm, and the inhibition zone for
gentamycin (CN 10) is 0mm. In Staphylococcus aureus, the inhibition zone for
neomycin (N 30) is 9mm, the inhibition zone for kanamycin (K 30) is 0mm,
and the inhibition zone for gentamycin (CN 10) is 7mm. After comparison
between the values from the table and the values obtained from the
measurement, it indicates that Bacillus cereus is susceptible to all antibiotics,
and Staphylococcus aureus and Escherichia coli are resistant to all antibiotics.
The susceptible category means that the bacteria is inhibited by the antibiotic,
and the resistant category means the bacteria is uninhibited. As the distance
from the disc increases, there is a logarithmic decrease in antibiotic
concentration.

Conclusion
During this experiment, it is found that Bacillus cereus is susceptible to all
antibiotics. Staphylococcus aureus and Escherichia coli are resistant to all
antibiotics. When performing this experiment, make sure the testing pH is not
too alkaline or too acidic and be careful of the amount of organisms- it must
not have too light inoculum or too heavy inoculum as this will affect the
inhibition zones and give the wrong interpretation.
References

Mcnutt, D. (2019). Control of microbial growth lab instructions

Khan, Z.A., Siddiqui, M.F. and Park, S., 2019. Current and emerging methods of
antibiotic susceptibility testing. Diagnostics, 9(2), p.49.

Syal, K., Mo, M., Yu, H., Iriya, R., Jing, W., Guodong, S., Wang, S., Grys, T.E.,
Haydel, S.E. and Tao, N., 2017. Current and emerging techniques for antibiotic
susceptibility tests. Theranostics, 7(7), p.1795.