A-level Biology required practical No.

6 Name:__________________ Date:__________

Comparing the effects of different antimicrobial substances on growth of bacteria in a

lawn plate culture.
 Apparatus and techniques AT
o Use laboratory glassware apparatus for a variety of experimental techniques
o Use microbiological aseptic techniques, including the use of agar plates and broth
No Some Mastery
Competency (CPAC) Assessed Evidenc Evidence
e
CPAC 2a - Correctly uses appropriate instrumentation, apparatus and materials
to carry out investigative activities, experimental techniques and procedures
with minimal assistance or prompting. Observation
Student produces a clear plan outlining the key steps required to ensure that
sufficient data is collected (to include accurate equipment details)
Student correctly carries out aseptic technique using appropriate techniques
including
o Sterilising work surface before starting practical
o Working in close proximity to the bunsen burner using a blue flame
o Minimising exposure time by ensuring agar plate lid is off for minimal time
o Using sterilised forceps and sterilising after use
o Storing plates upside down with lid secured with tape

CPAC 2b – Carries out techniques or procedures methodically, in sequence and

in combination, identifying practical issues and making adjustments when
necessary. Observation and questioning
Explain why working near to a Bunsen burner is required for aseptic technique
Explain why plates are incubated upside down with lid secured by two pieces of
tape
Suggest improvements to the method that would improve the validity and
accuracy of the results
CPAC 2c - Identifies and controls significant quantitative variables where
applicable, and plans approaches to take account of variables that cannot
readily be controlled. Observations and questioning as well as write up
 Identify the independent and dependent variables in the investigation
 Identifies the key variables to control and how they are to be controlled /
approaches for variables difficult to control
 Explains why the raw data needs to be processed (calculation of area of zone of
inhibition)
CPAC 2d – Selects appropriate equipment and measurement strategies in order
to ensure suitably accurate results
 Identifies appropriate equipment in their plan to allow for appropriate precision
 Plan outlines requirement for repeats / alternatives if time is a limiting factor
 Appropriate units are identified in the table of results
CPAC 3a – Identifies hazards and assesses risks associated with these hazards,
making safety adjustments as necessary, when carrying out experimental
techniques and procedures in the lab and in the field.
 Complete the risk assessment for this investigation by identifying associated
risks, control measures and calculating risk scores.
 Student takes account of the safety procedures required on the risk assessment
when carrying out the practical and follows the appropriate precautions.
 CPAC 3b – Uses appropriate safety equipment and approaches to minimise risks
with minimal prompting Observation
Student takes account of the safety procedures required on the risk assessment
when carrying out the practical and follows the appropriate precautions.
Treats equipment and materials with respect and demonstrates confidence when
handling them, responding appropriately if things go wrong (e.g. broken glass)
Wears safety goggles as standard and without prompting
CPAC 4a – Makes accurate observations relevant to the experimental or
investigative procedure.
 Student demonstrate accurate aseptic technique and correct measurements of
the zones of inhibition.
 Student records results in a suitable table, following necessary conventions.
CPAC 4b – Obtains sufficient, precise and sufficient data for experimental and
investigative procedures and records this methodically using appropriate units
and conventions
 Student completes the appropriate number of repeats
 Table for results is well constructed with correct headings and units written in
the appropriate format
 Raw data presented clearly and effectively in tables to the highest resolution
 All calculated data follows the rules of significant figures

CPAC 5a – Uses appropriate software and/or tools to process data, carry out
research and report findings.
 Student correctly processes data by calculating area of the zone of inhibition
from their raw data.
 Student follows correct conventions to present their data in the form of a
suitable graph.
 Student writes a conclusion and evaluation to their experiment
CPAC 5b - Cites sources of information demonstrating that research has taken
place, supporting planning and conclusions.
 Student researches the method for how to investigate the effect of antimicrobial
substances of the growth of a named bacteria
 Student makes it clear what the sources were useful for
 There is no plagiarism of sources
 Student cite sources correctly
Plan your Method (2a):

Background
Antibiotics are used to treat bacterial infections. They work in one of two main ways:
 by killing the bacteria by interfering with the building of the bacterial cell wall by inhibiting the synthesis
of peptidoglycan;
 or by preventing the bacteria from multiplying by interfering with DNA replication, so cell division
(asexual reproduction) cannot occur.
A patient is suffering with an E.coli infection and as such, a swab has been taken and grown in culture
(bacterial broth).
Plan out a method to determine which antibiotic (out of a selection of 6) a sample of this E. coli is most
susceptible to so that the patient can be effectively treated.
Wipe down surfaces with antibacterial cleaner, both before and after experiment. Use a Bunsen burner
in the woworkspaceo that convection currents draw microbes away from the culture. Flame the wire
hoop before using it to transfer bacteria. Flame the neck of any bottles before using them to prevent
any bacteria entering the vessel (air moves out so unwanted organisms don’t move in). Keep all
vessels containing bacteria open for the minimum amount of time. Close windows and doors to limit air
currents.

1. before starting carry out any aseptic techniques

2. using either a sterile or wire hoop to transfer bacteria from broth (water, bacterial culture, nutrients)

3. using a sterile plastic spreader spread the bacteria evenly over plate

4.use sterile forceps to place a multi disc antibiotic ring on the plate. Do not hold the ring on the arms only
the centre.

5. lightly tape a lid on do not tape around the entire dish as it prevents oxygen entering and so promotes the
growth of harmful anaerobic bacteria, invert and incubate at 25 degrees for 48 hours

6. disinfect work surfaces and sterilise equipment used to handle bacteria

After incubation:

Work out the area of the inhibition zone(clear circle) and diameter for each antibiotic making sure to not
remove the not to remove the lid from the agar

Formula for inhibition zone:

A=3.14d^2/ 4

N.B – make sure you list your sources correctly at the back of this RP section

Identification of variables (2c):

Independent variable =
(How are you going to change this?)

Dependent variable =
(Make sure you explain exactly how you are going to measure this, which instrument, what units, resolution?)

Control variables =

Variables that I am able to control =

Variable that I am unable to control =

I will try to mitigate these variables by …

Precautions to ensure that results are as accurate as possible:

Risk Assessment – use the HazCards to help you write this (3a):
Hazard Who is at risk? Risk Preventative measures Responsibilities
rating
band
Disinfectant Students and 4 Keep away from naked flame Haseeb and
Flammable
teacher Hassan

Biohazard Students and 6 Use disinfectant; wash hands with Haseeb and
Contamination; soap after dissection; do not
infection
teacher incubate at human body
Hassan
temperature; do not open agar
plate post incubation

Naked flame Students and 4 Keep away from flammable Haseeb and
Fire hazard; burns materials; tie up long hair, wear
teacher goggles
Hassan

Risk rating:
Risk rating Rating action bands
Likelihood X Severity of = Assessed band
injury
1. Most 1. Trivial Minimal risk 1 or 2
unlikely injuries
2. Unlikely 2. Slight Low risk 3 or 4
injuries
3. Likely 3. Serious Medium risk 6 or 8
injuries
4. Most likely 4. Major High risk 9, 12 or 16
 injuries
Results Table (4a/4b) (Ensure you follow the necessary conventions):
Did you, or would you, make any adjustments to the method that you planned? If so, why did you make
the adjustments. (2b)

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Conclusion and Evaluation (5a):
What conclusions can you draw from your data on which antibiotic was most effective? Use scientific terminology in
your answer and refer to your data.

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Questions to consider as part of your conclusion / evaluation:

1. Why was it important to boil the agar before the agar plates were poured?

2. Why was it important to use the same strain of bacterial culture?

3. What does the lack of a clear zone suggest / indicate?

4. How could you extend this investigation to determine the most effective dosage of the antibiotic?
Research Sources (5a/5b):
Which sources did you use to support your planning?

NB: When quoting from secondary sources it is important that you cite the source of the information correctly.
Textbooks - Last name, Initial, (year), article name, Book / Journal, Website - (full URI), and date accessed. You
should say what you used each source for. You should not use tertiary sources (a source which references secondary
sources only. The most famous tertiary source website is Wikipedia and, whilst providing a good starting point, it
should be avoided. Most important of all YOU MUST NOT PLAGIARISE.)
TABLE AND GRAPH SELF/PEER ASSESSMENT:
Assessment of Presentation of Data Self- Peer -
Assessment Assessment
Data presented clearly in a table, drawn and completed during the actual practical!
Independent variable in the left hand column
Dependent variable in the right hand column (s)
Units clearly stated and only in the heading to the column
Units presented with correct convention e.g. /ml
Data presented to the correct resolution (maximum by equipment)
An appropriate number of repeats, based on the equipment used
Assessment of Processing Self- Peer -
Assessment Assessment
Graph has independent variable on x-axis and dependent variable on y-axis
Appropriate scales chosen for X and Y axes allowing for accurate plotting and reading
Both axes correctly labelled with the appropriate units (correct convention)
All points plotted accurately
Data presented as a line graph and including a suitable line of best fit (straight line or
curved as appropriate).
Total Marks

Student/Peer Signature: ___________________________________________ Date: _________________