Figures 1.1 : Escherichia coli 1.

2 : Staphylococcus aureus
 Figure 1.3: Electron micrograph of Staphylococcus aureus

The Staphylococci

Staphylococci (staph) are Gram-positive spherical bacteria that occur in

microscopic clusters resembling grapes. Bacteriological culture of the nose
and skin of normal humans invariably yields staphylococci. In 1884, Rosenbach
described the two pigmented colony types of staphylococci and proposed the
appropriate nomenclature: Staphylococcus aureus (yellow) and
Staphylococcus albus (white). The latter species is now named Staphylococcus
epidermidis. Although more than 20 species of Staphylococcus are described
in Bergey’s Manual (2001), only Staphylococcus aureus and Staphylococcus
epidermidis are significant in their interactions with humans. S. aureus
colonizes mainly the nasal passages, but it may be found regularly in most
other anatomical locales, including the skin, oral cavity and gastrointestinal
tract. S epidermidis is an inhabitant of the skin.

Taxonomically, the genus Staphylococcus is in the Bacterial family

Staphylococcaceae, which includes three lesser known genera, Gamella,
Macrococcus and Salinicoccus. The best-known of its nearby phylogenetic
relatives are the members of the genus Bacillus in the family Bacillaceae, which
is on the same level as the family Staphylococcaceae. The Listeriaceae are also
a nearby family.

Staphylococcus aureus forms a fairly large yellow colony on rich medium; S.

epidermidis has a relatively small white colony. S. aureus is often hemolytic on
blood agar; S. epidermidis is non hemolytic. Staphylococci are facultative
anaerobes that grow by aerobic respiration or by fermentation that yields
principally lactic acid. The bacteria are catalase-positive and oxidase-negative.
S. aureus can grow at a temperature range of 15 to 45 degrees and at NaCl
concentrations as high as 15 percent. Nearly all strains of S. aureus produce
the enzyme coagulase: nearly all strains of S. epidermidis lack this enzyme. S.
aureus should always be considered a potential pathogen; most strains of S.
epidermidis are non-pathogenic and may even play a protective role in
humans as normal flora. Staphylococcus epidermidis may be a pathogen in
the hospital environment.

Staphylococci are perfectly spherical cells about 1 micrometer in diameter. The

staphylococci grow in clusters because the cells divide successively in three
perpendicular planes with the sister cells remaining attached to one another
following each successive division. Since the exact point of attachment of
sister cells may not be within the divisional plane and the cells may change
position slightly while remaining attached, the result is formation of an
irregular cluster of cells.

The shape and configuration of the Gram-positive cocci helps to distinguish

staphylococci from streptococci. Streptococci are slightly oblong cells that
usually grow in chains because they divide in one plane only, similar to a
bacillus. Without a microscope, the catalase test is important in distinguishing
streptococci (catalase-negative) from staphylococci, which are vigorous
catalase-producers. The test is performed by adding 3% hydrogen peroxide to
a colony on an agar plate or slant. Catalase-positive cultures produce O2 and
bubble at once. The test should not be done on blood agar because blood
itself contains catalase.

Problem statement: Which antibiotic is most effective on bacteria?

Hypothesis: Different antibiotics have different effect on bacteria. Ampicillin is

the most effective antibiotic against Escherichia Coli and Staphylococcus
aureus compared to other antibiotics.

Variables:
Manipulated Variable: Types of antibiotics and types of bacteria.

Responding Variable: The diameter of clear zone around the paper discs.

Fixed Variable: Surrounding temperature, humidity, light intensity, size of

paper discs.

Apparatus: Agar plate, Bunsen burner, marker pen, autoclaved forceps.

Materials: Bacteria E. coli, bacteria S. aureus, bench spray of disinfectant, 1%

Virkon, soap and dettol, paper towels, antibiotic- impregnated paper disc,
adhesive tape.
Figure 1.4: Gram stain of Staphylococcus aureus in pustular exudate

Figure 1.5: Staphylococcus aureus

Procedure:
 Hands are washed with dettol handwash. Disinfectant spray is sprayed
thoroughly to the working area. Paper towels are then used to wipe the
working area.
 Two sterile Petri dishes are labeled correctly. One is filled with the
bacteria S. aureus and another one with E. coli. The label is pasted at the
side of the Petri dishes.
 The apparatus needed: bottle containing sterile nutrient agar, cultures
and sterile Petri dishes labeled correctly.
 200ml of E. coli bacteria culture is pipetted into a sterile Petri dish
beside a burning Bunsen burner.
 Molten agar is poured into the Petri dish until the bottom of the Petri
dish is covered by the agar.
 The Petri dish is then covered and gently pushed back and forth and in
all four directions to mix the bacteria well with the agar.
 The agar is then allowed to set.
 Steps 4 to7 are repeated for S. aureus.
 The 2 Petri dishes containing the agar lawn are allowed to set.
 One paper disc is placed in a solution of antibiotics named Ampicillin
using sterile forceps.
 The paper disc is then soaked into Petri dish containing the agar.
 Steps 10- 11 are repeated for antibiotics Tetracyclin, Carbenicillin, and
sterilized distilled water.
 The Petri dish is closed and the bottom of the Petri dish is labeled to
identify the position of each paper disc.
 The agar plates are then left in 30.0 0C incubator for 24 hours.
 Hands are washed thoroughly again after working with the bacteria
culture.
 After 24 hours, the agar plates are observed with the Petri dishes closed.
The diameter of the clear region around the paper discs are measured
and recorded. The results are recorded in a table

Precautions:
The bacteria must be pipetted into the agar before the agar is set so that the
bacteria can mix well with it.

During observation, the lid of the Petri dishes must not be lifted up as the
bacteria are harmful to our healths.

Result
Antibiotics

Diameter of the clear zone, cm

Escherichia coli
Staphylococcus aureus
Ampicillin
3.2

3.0

Tetracycline
2.3

2.7

Carbenicillin
1.3

2.8

Distilled water
0.5

0.5

Discussions:
Analysis of data

From the result, it is found that the largest inhibition zone or clear region is
formed around the paper disc soaked in Ampicillin for E. coli bacteria lawn,
followed closely by Tetracyclin and Carbenicillin. Ampicillin’s paper disc caused
the largest area of inhibition zone in E. coli. This showed that Ampicillin is
most effective in inhibiting the growth of the E. coli. Meanwhile, Carbenicillin’s
paper disc which caused the smallest area of inhibition zone in E. coli showed
that it is the weakest antibiotic against E. coli.

Similarly, Ampicillin is also the most effective antibiotic against bacteria

Staphylococcus aureus as the area of inhibition zone around the paper disc
soaked with Ampicillin solution is the greatest, which has a diameter of 3.0 cm.
Followed by that is the antibiotic Carbenicillin and the least effective antibiotic
is Tetracyclin which has a slightly smaller diameter of clear zone than
Cabenicillin, which is 2.7cm.

Therefore, it can be concluded that Ampicillin is the most effective antibiotic

against both type of bacteria and being the broad spectrum antibiotic while
the effectiveness of Tetracyclin and Carbenicillin towards both bacteria varied.
This showed that different antibiotic has different effect on different bacteria
as well.

The inhibition zones are all circular. If it is not circular, it is sensible that the
diameter should be measured by using two points which are furthest from
each other within the clear region. The diameter of the inhibition zones is
affected by the strength of antimicrobial properties of the antibiotics towards
different bacteria. It is important not to always choose the antibiotic with the
largest inhibition zone to treat the patients as some other factors should be
considered as well such as the side effect caused by the antibiotics, the
conditions of the patients and the risk of that particular antibiotics.

Control

In this experiment, the control used is the sterilized distilled water. Paper discs
soaked in sterilized distilled water are also put in two of the Petri dishes. This is
to show that the sterilized distilled water has no effect on the bacteria. This
enables us to compare the results for paper discs with the antibiotics and
those with the distilled water to show that the formation of the inhibition zone
or the clear region is due to the antibiotics but not because of the presence of
water. In this case, clear region cannot be seen around the paper disc soaked
in sterilized distilled water in both Petri dishes. Therefore, the presence of clear
region around other paper discs must be due to the antimicrobial property of
the antibiotics.

Variables

Three different antibiotics are used in this experiment to manipulate the types
of antibiotics and to compare the effectiveness of each antibiotic to inhibit the
growth of the bacteria. The antibiotics used are Tetracycline, Carbenicillin and
Ampicillin. Two different types of bacteria, E. coli and S. aureus are
manipulated by putting them in different Petri dishes with the agar medium.
This enables us to identify the varying antimicrobial properties of the same
antibiotics on different types of bacteria.

The responding variable in this experiment is the diameter of the clear region
around the paper discs after 24 hours. The greater the diameter of the clear
zone around the paper discs, that means the more effective the antibiotic
inhibiting the growth of the bacteria. The diameter of the clear zone can be
measured from one point of the circular clear region to another point through
the centre point by using a ruler.

The amount of different bacteria cultures used must be the same by pipetting
equal amount of the two bacteria, which is 200 ml into the agar medium. The
temperature, humidity and light intensity must also be kept constant
throughout the experiment. All these factors may affect the rate of growth of
the bacteria. This can be done by placing the two agar plates into an incubator
at 30 0 C. The size of the paper discs should also be kept constant. Paper discs
which are larger will absorb more antibiotics and may lead to a greater
diameter of clear zone compared to the smaller paper discs in the same
bacteria culture. This is done by preparing the paper discs using the same
puncher to ensure all the paper discs are of the same size.

Justification of apparatus and materials

In this experiment, the antibiotics used are Tetracycline, Streptomycin and

Carbenicillin solutions. These antibiotics are more common antibiotics which
are more easily available. The antibiotics have already been prepared in
solutions form. This enables the paper discs to be soaked in the solutions
directly and easier. The bacteria used are S. aureus and E. coli, they are
practically easier to be grown and culture in agar plates. However, these two
bacteria may be harmful to our health, therefore the lids of the agar plates are
not allowed to be opened during observation. This is to keep us from getting
any infections from these bacteria.

Bunsen burner is used in this experiment to minimize the contamination of the

experimental sets while preparing them. For example, the forceps are being
flamed before being used to pick up the paper discs soaked in the antibiotics
solutions. Micropipette is used to transfer the 200 ml bacteria into the agar
medium. The used of micropipette with sterile tips further improved the
accuracy of the result.

Validity and reliability of the results

According to the result from another group, Ampicillin is the most effective
antibiotic towards only E. coli bacteria. Cabernicillin is most effective towards
S. aerues and this is different from the result of my group. This may properly
cause by contamination which affect the accuracy of the result. However, for
the bacteria E. coli, the diameter of clear zone caused by the paper disc soaked
in Cabernicillin is 1.3 cm for my group while the result of another group
showed no clear zone around it. Therefore, it can be concluded that
Carbenicillin is the least effective antibiotics towards E. coli for two groups.
Besides, in order to increase the reliability of the experiment, the variables are
controlled carefully. The constant variables are kept constant while only
manipulating the variables that are being studied.

Sources of errors

One possible source of errors may be the contamination of the agar medium.
This occurred when saliva is accidentally being transferred to the agar during
preparation. Another possible source of error could be the purity of the
antibiotics used. Some of the antibiotics solutions may be contaminated by
some impurities which could decrease their antibacterial properties. Also,
human error can take place especially when measuring the diameter of the
clear zones.