UNIVERSITY OF NAMIBIA

SCHOOL OF MEDICINE

MODULE: MICROBIOLOGY I

TITLE: ANTIBIOTIC SENSITIVITY TESTING

NAME: FYSAL ELMARAKBY

STUDENT NO.: 201606983

EXPERIMENT: 4

DATE OF EXPERIMENT: 16 AUGUST 2017

GROUP: 1

DUE DATE: 23 AUGUST 2017

INTRODUCTION
Infectious diseases have become less common as causes of death and disability in regions of
improved sanitation in the world in addition to adequate supplies of antibiotics. Antibiotics are
molecules that kill, or stop the growth of bacteria. Those that kill bacteria are called bactericidal
and those that stop the growth of bacteria are called bacteriostatic. Antibiotics are used for
treatment or prevention of bacterial infection (Encyclopedia of children, 2017). They are created
through bacterial and fungal metabolism in a lab setting. The first true antibiotic was discovered
by Alexander Fleming in 1928 and this was called penicillin. From this a day a whole new
revolution of antibiotics were then created e.g. Amoxicillin, Azithromycin, Oxacillin, Cefuroxime
etc. Antibiotics are categorized into broad spectrum and narrow spectrum classes. The narrow
spectrum antibiotics are those that imply activity against some limited spectrum of bacteria. Broad
spectrum antibiotics act over a range of bacteria. This antibiotics perform selective toxicity
meaning they treat disease by acting on the bacteria and not affecting the human cells.

The determination of a bacteria’s susceptibility to a specific antibiotic is crucial in determining the

appropriate treatment for a disease and this is known as Antibiotic sensitivity testing (WebMD,
2017). Bacterial resistance to antibiotics increasingly hinders treatment of life threatening illnesses
and therefore this test is crucial because misuse and overuse of antibiotics are crucial in the
development of antibiotic-resistant bacteria (Gilchrist et al. 2007). The two most common methods
of antibiotic sensitivity testing are the Kirby-Bauer Antibiotic Disk Diffusion test and the
Minimum Inhibitory Concentration (MIC) Method. For the sake of the experiment, the Kirby-
Bauer Antibiotic Disk Diffusion test was used. This method is also called the agar diffusion method
and the procedure followed is simply done when a filter disk impregnated with an antibiotic is
applied to the surface of an agar plate containing the organism to be tested. Bacteria are either
susceptible (sensitive), intermediate or resistant (insensitive) to certain antibiotics.

AIMS
 To know examples of different antibiotics used in clinical practice
 To understand and utilize the Kirby-Bauer Antibiotic Disk Diffusion test for sensitivity
testing of different antibiotics to Escherichia coli
 To know how to use a Vernier Caliper to measure the diameter of zone of inhibition and
analyzing and record the results produced by the test.
  To select the most effective antibiotic against Escherichia coli

MATERIALS
 Culture plate of Escherichia coli and of Staphylococcus aureus
 Two Mueller-Hinton agar plates
 Sterile cotton-tipped applicators
 Vials containing antibiotic disks of the following: Amoxicillin (AML), Erythromycin (E),
Oxacillin (Ox), Penicillin (P), Cefuroxime (CXM), Ceftazidine (CAZ), Gentamycin
(CN), Ciprofloxacin (CIP) and Imipenem (IPM).
 Insulin syringes
 McFarland standard

METHODS

Students worked in pairs. Each pair of students obtained two Mueller-Hinton agar plates and they
were labelled with the initials of each student so each pair does not lose their culture in the midst
of all other plates during incubation. The sterile cotton-tipped applicators was used to obtain a
small amount of the culture they would love to use, either from Escherichia coli or from
Staphylococcus aureus. The organism was then spread on each of the two plates until evenly spread
over the whole agar plate. The syringe was then used to transfer one disk of each antibiotic from
the vial to the agar plate. 5 antibiotic disks were transferred to one agar plate and 4 to the other
plate. In total 9 antibiotics were used. The disks were placed in a way in which they were well
spaced to avoid overlapping of the zones of inhibition of the different antibiotics. The plates were
incubated at 35°C for 24 hours. The plates were then studied and results were obtained from them
by measuring the zones of inhibition (area around the antibiotic disk that is clear) of each antibiotic
by a Vernier caliper. An evaluation table was used to determine whether the bacteria was resistant,
intermediate or susceptible.

RESULTS
Table showing the inhibition zones and sensitivity of different antibiotics to E. coli
ANTIBIOTIC INHIBITION ZONE OF Resistant, Sensitive or
E. coli (mm) Intermediate
Amoxicillin (AML) 18 Resistant
Erythromycin (E) 9 Resistant
Oxacillin (Ox) insensitive Resistant
Penicillin (P) insensitive Resistant
Cefuroxime (CXM) 6 Resistant
Ceftazidine (CAZ) 31 Sensitive (Susceptible)
Gentamycin (CN) 23 Intermediate (Inconclusive)
Ciprofloxacin (CIP) 37 Sensitive (Susceptible)
Imipenem (IPM) 35 Sensitive (Susceptible)

PLATE 1: E. COLI (AML, CIP, OX, CN and E)

PLATE 2: E.COLI (CAZ, IPM, P and CXM)

DISCUSSION

Escherichia coli bacteria is highly susceptible to three of the antibiotics used in our experiment
according to the results obtained. Ciprofloxacin, Ceftazidine and Imipenem produced a large zone
of inhibition during the experiment which were 37mm, 31mm and 35mm respectively. This
indicated that E. coli is highly sensitive to this antibiotics. The following three antibiotics would
be successful in treating a patient that is infected by this bacteria. These antibiotics can easily
diffuse into the bacteria and produces the effects that lead to its inactivation.

However Gentamycin is an antibiotic that falls into the incapable category with an inhibition zone
of 23mm. This E. coli bacteria is not so sensitive to the antibiotic. The effect that this antibiotic
will produce will be intermediate meaning that some bacteria in the population will die due to their
sensitivity to the antibiotic while other cells will survive due to their resistive properties to the
antibiotic. Gentamycin is a type of aminoglycoside that is used to treat several types of bacterial
infections mostly gram negative bacteria such as Pseudomonas, Proteus, E. coli and gram positive
Staphylococcus.

The other 5 antibiotics Amoxicillin (18mm), Erythromycin (9mm), Penicillin (0mm), Cefuroxime
(6mm) and Oxacillin (0mm) fall in the resistant group due to the low inhibition zone produced.
This indicates that the bacteria are growing normally even in the presence of the antibiotic. Some
antibiotics like Penicillin and Oxacillin are completely resistant to by E. coli producing a zero
effect. Penicillin cannot penetrate the membrane of gram negative bacteria such as E. coli because
E.coli produces few porins.

Factors that could have influenced the bacterial susceptibility test include: moisture on the plate
and the pH of the Mueller-Hinton agar which should be between 7.2-7.4. If it is too low certain
drugs will appear to lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other
agents may appear to have excessive activity (e.g., tetracycline). If the pH is too high, the opposite
effects can be expected.

CONCLUSION
The overall experiment was a success. The Kirby-Bauer Antibiotic Disk Diffusion test was
successfully used to test the susceptibility of E. coli to a wide variety of antibiotics. The zone of
inhibition was clearly understood, identified and measured. Future recommendations include
placing the antibiotic discs further apart so as to prevent or minimize overlapping of inhibition
zones. However the different antibiotics where identified for their different susceptibility and this
allowed us to identify the best antibiotic against Escherichia coli bacteria.

QUESTIONS
1. Why does the Kirby-Bauer procedure require that the concentration of the bacteria
be the same, the stage of growth constant, the growth medium the same and the
concentration or amount of drug in each disk constant?

-The bacteria should be the same for all the antibiotics, this is because antibiotics may have
different effects toward different bacteria. This effects differ due to resistance mechanisms
developed by certain bacteria. If the criteria should be different then it would not be possible to
determine the drug that is most effective. The study conducted is too see the effectiveness of
different antibiotics to a specific bacteria and not the study of the effects of an antibiotic to different
bacteria. This is also done to maintain consistency regardless of which lab performs the test and it
makes this a fair test.

-The growth medium and stage of growth should be kept constant to maintain a constant
environment so that the results produced are due to the anti-microbial agent action and not as a
result of environmental change. Failure of the antibiotic to produce any effect should be attributed
to the resistance of the bacteria and not environmental inactivation.

-The aim of the experiment is to identify the strongest antibiotic therefore the drug concentrations
should be constant in each disk to allow efficacy measure and not how quantity influences effect.
A weak antibiotic may produce a stronger effect than a stronger drug if it is used in higher
concentrations therefore the amount of drug should be constant.

2. A pure culture was inoculated onto a Muller-Hinton agar plate. The Kirby-Bauer
procedure performed. One of the drugs tested showed a large zone of inhibition but
also had small colonies growing within this zone. Further testing showed that these
colonies were not the results of contamination. Why would these colonies be present
within this zone of inhibition?
Because the strain of the test organism that has become antibiotic resistant. These colonies
became resistant to the antibiotic during the incubation period through the emergence of
mutation and this allowed them to survive in the zone of inhibition.
3. How to address the antibiotics that are classified as inconclusive to assist the
patient?
A drug that is inconclusive is neither sensitive nor resistant to by the microbe. It is not sufficient
to give a large effect but has somewhat an intermediate effect. If a physician encounters such an
antibiotic than multi drug combination therapy can be used whereby one or more drugs can be
added to the antibiotic and this can produce the positive effect of synergism whereby the effect
produced would be greater than the additive effects of the drugs. However this may also have
negative effects such as increased antimicrobial resistance, adverse effects, and costs. The
antibiotic could also be administered in higher concentration to produce greater effect.
REFERENCES
Antibiotics. (n.d.). Retrieved August 20, 2017, from
http://www.healthofchildren.com/A/Antibiotics.html

Antibiotic Sensitivity Test - Topic Overview. (n.d.). Retrieved August 20, 2017, from
http://www.webmd.com/allergies/tc/antibiotic-sensitivity-test-topic-overview

Gilchrist MJ, Greko C, Wallinga DB, Beran GW, Riley DG, Thorne PS. 2007. The potential role
of concentrated animal feeding operations in infectious disease epidemics and antibiotic
resistance. Environ Health Perspect 115:313-316.