PRACTICAL SCAFFOLD BOOKLET_2023

Introduction
Microorganisms known as pathogenic bacteria are capable of infecting humans, animals, and plants with diseases.
The diversity of these single-celled organisms is immense; thousands of distinct species have so far been discovered.
While many bacteria are harmless or even helpful, pathogenic bacteria have unique properties that allow them to
penetrate and multiply within the tissues of their hosts, causing infections. The ability to produce toxins, the
capacity to cling to host cells, and the evasion of the host's immune defences are just a few of the techniques they
use to do this. Numerous ailments, from mild skin infections to fatal systemic diseases, can be brought on by
pathogenic bacteria. Escherichia coli, also known as E. coli, Staphylococcus aureus, and Mycobacterium tuberculosis
are a few examples of dangerous bacteria.

Antibiotics are drugs specifically designed to combat bacterial infections. They function by killing the bacteria
straight up or preventing their development and reproduction, leaving the remaining germs for the immune system
to destroy. Antibiotics' discovery and development revolutionised medicine and vastly increased the survival rates
for a variety of infectious diseases. These drugs can target a number of bacterial activities, including DNA replication,
protein synthesis, and cell wall formation. However, because different antibiotics are made to focus on particular
bacterial species, their effectiveness is not universal. Additionally, the rise of antibiotic-resistant bacteria as a result
of improper and excessive use of antibiotics poses a serious threat to global health.

Aim
To determine which dose possesses the minimum concentration of ampicillin to create a zone of inhibition amidst E.
coli growth.

Hypothesis
It is hypothesised that as the dose of ampicillin increases, the zone of inhibition in the petri dishes growing E Coli will
progressively grow as well.

This is because, generally, more of a certain drug is going to have a stronger affect than less of it which would result
in a larger zone of inhibition as the dose increases rather than smaller.

Variables
The independent variable is…. The dependent variable is….
The concentration of ampicillin. The presence and size of a zone of inhibition (ZOI)
created in the nutrient agar plates growing E. coli.
It will be changed by using a different dose each time
containing different concentrations (µg/ml): 2000, 1000, This will be measured with a 15cm ruler in millimetres.
500, 250, 125, 62.5, 31.3, 0.
Controlled Variables
Controlled variables How you kept this constant Why is this being kept constant?

Bunsen Burner. Keep the duration and If the intensity or duration of the
intensity of the flame constant flame were to vary, it could lead
throughout the experiment. to inconsistent levels of
This helps maintain a sterilization, potentially
controlled environment and introducing unwanted microbes
minimizes the risk of that could interfere with the
contamination. experiment. Keeping the Bunsen
burner constant ensures a
consistent level of sterility.

Species and Age of E. Coli. Ensure that the E. coli Ensuring all the E. coli is of the
samples are obtained from the same species and age helps
same source and prepared isolate the effect of ampicillin
and handled in the same way concentration on bacterial growth
to minimize any potential because different strains and
variability. stages of growth of the bacteria
may react different to the drug.

Amount of nutrient agar gel in each plate. Measure and pour the same Keeping the volume of agar
volume of nutrient agar into consistent ensures that the
each petri dish. This ensures a environment provided to the
consistent substrate for bacteria remains uniform across
bacterial growth and a all plates.
standard environment for the
experiment. Also allow the
agar to solidify for the same
amount of time in each plate.
This ensures uniform thickness
and consistency of the agar
layer
Equipment –

 Broth culture of E. coli

 3x nutrient agar plates
 1x sterile swab
 Blank sterile filter paper discs
 Forceps
 Ethanol in container
 Bunsen burner
 Fire lighter
 Heatproof mat
 Spotting tile
 30‐39 °C Incubator
 Tape
 15cm clear ruler

Method
1. Lap coats, eyeglasses, and sterilised gloves were put on by students performing the practical.
2. All equipment was gathered.
3. A Bunsen burner was set up on a ceramic plate where everything was to be done within a 30cm radius of to
ensure the bacteria did not stray and cause safety concerns and also acted to prevent stray contaminants
 from falling onto the agar in the plates as the group worked.
4. The plates were labelled with the group's initials. Two of the plates were then marked into thirds on the
bottom, and the last one, in halves. These sections were then numbered 1-8 as per the number of ampicillin
concentrations with 1 and 8 on the dish marked in halves.
5. A sterile cotton swab was removed from the cover, the plates were opened, and then the jar containing the
E. coli broth culture was then opened.
6. The cotton swab was dipped into the E. coli and lawns were spread on all nutrient agar plates.
7. The cotton swab was then sterilised by being tossed into an ethanol bath.
8. Forceps were then dipped into the ethanol and then held over the flame to sterilise.
9. A single sterile paper disc was then picked up by the forceps and dipped into one of the numbered ampicillin
concentrations and placed gently into the corresponding numbered section of a plate. This step was
repeated with all doses, the forceps being sterilised with heat from the flame in between each time to
prevent cross contamination and alteration of concentration of ampicillin in the wells.
10. The forceps were sterilised once more in the flame.
11. The plates were closed and taped shut and later placed in an incubator at 30C° for 24 hours.
12. After 24 hours, lab coats, eyeglasses, and gloves were put on once again and the zones of inhibitions around
each paper disc were measured with a 15cm ruler and recorded in millimetres.

Safety and Ethical Considerations

Hazard What is the risk? How will the risk be reduced?

E. coli broth culture Students could contract sicknesses caused by E. Heavy health and safety measures were
coli such as urinary tract infections, abdominal taken to prevent direct contact with the
and pelvic infections, pneumonia, bacteraemia, bacteria. These included lab coats, safety
and meningitis. These conditions can be serious glasses, gloves, and regular sanitisation of
and depending on the individual and on whether equipment.
it remains untreated, can cause death.

Bunsen Burner Due to working in close quarters with an open To minimise risk, hair was fully tied back,
flame, there was a relatively high risk of burns and movement was to be careful and
and things catching fire. considered around the flame.

Forceps Since they are sharp/pointed, small, and metal, Students were to be careful and
they may cause injuries to skin or eyes if used considerate of their movements when
inappropriately. Additionally, injuries that break using the forceps and they were to be
the skin may get infected due to the forceps used only for the purposes of the
being dirty. practical.

Laptop Chords Long chords across the floor are a tripping hazard Students were to unplug their laptop
and with other dangerous equipment and chords from power points in the room
supplies in the workspace, this could act as a during the practical.
precursor for other injuries.

Liquids There were many liquids used in this practical: The ethanol and E. coli containers were
the ampicillin doses, the ethanol bath, and the E. not handled besides from opening and
coli broth culture. As well as slips, these liquids closing the lids to minimise the risk of
pose other health risks; E. coli causing sicknesses spillage and vapours from escaping. If
listed above, and ethanol being harmful by there were any spills, students were to
ingestion, inhalation and by skin absorption. section off the area immediately for it to
Direct ingestion of the liquid can cause coma and be cleaned.
death, inhalation of vapours can cause irritation
in the respitory tract, lack of coordination, and
 narcosis and contact with skin can be extremely
irritating.

Results

Dose Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group7 Group 8 AVERAGE

1 28 20 25 30 26 28 28 30 27.9

2 25 25 15 23 24 26 27.5 30 25.8

3 22 20 23 21 23 22 26 25 22.8

4 20 35 19 18 19 16 22.5 21 19.4

5 17 15 23 15 11 13 19.5 15 15.1

6 12 7 12 11.5 0 10 15 0 11.3

7 8 0 0 7 0 0 0 0 0

8 0 0 0 0 0 0 0 0 0
Raw Data Table:
Orange = Outlier (not included in averages.)

Processed Data Table:

Dose Ampicillin Concentration (µg/mL) Average ZOI

1 2000 27.9

2 1000 25.8

3 5000 22.8

4 250 19.4

5 125 15.1

6 62.5 11.3

7 31.25 0

8 0 0

Graph: