Feedback from previous write-up

Key strengths:

Some great discussion points

Good background

Professionally written – e.g., use of passive tense and scientific language.

Areas for improvement:

Ensure you include units in table of results

The independent variable must be on the x-axis of the graph

Try to calculate your % uncertainties next time (where applicable).

RP 6- Aseptic Techniques

Introduction:

aseptic techniques equip you with the skills necessary to carry out experiments involving
microorganisms safely. These techniques are not only essential in laboratory research but are
also applicable in industries such as medicine, microbiology, and biotechnology, where the
control of microbial contamination is of utmost importance. provides a hands-on experience
that allows you to practice key skills such as sterilizing equipment, preparing agar plates, and
using inoculation techniques to transfer microbial cultures. This practical will help develop a
deeper understanding of the significance of aseptic techniques in scientific research and the
critical role they play in maintaining accuracy and reliability.
Aim:
The aim of the AQA A-Level Aseptic Techniques Required Practical is to develop a thorough
understanding and proficiency in employing aseptic techniques within a laboratory setting.
Hypothesis:
"By employing proper aseptic techniques, including sterilization of equipment, preparation of
sterile agar plates, and careful inoculation of microbial cultures, it is hypothesized that the
growth of contaminating microorganisms will be minimized, leading to the successful
cultivation of pure microbial colonies on the agar plates."
Background:
Aseptic techniques are fundamental practices used in various scientific and medical disciplines
to maintain a sterile environment and prevent the introduction of unwanted microorganisms.
These techniques are crucial in fields such as microbiology, medicine, biotechnology, and
laboratory research, where the control of microbial contamination is essential for accurate and
reliable results.
The primary goal of aseptic techniques is to prevent the growth and spread of microorganisms,
including bacteria, fungi, and viruses, that can interfere with experimental outcomes or pose
risks to human health. By following strict aseptic procedures, researchers can minimize the
introduction of contaminants and ensure the purity of cultures, samples, and laboratory
equipment.
Key Principles of Aseptic Techniques:

 Sterilization: Sterilization is the process of eliminating all forms of microorganisms from

a surface, material, or environment. This can be achieved through various methods such
as heat (autoclaving), filtration, chemical disinfection, or irradiation. Proper sterilization
of equipment, media, and work surfaces is essential to create and maintain a sterile
environment.
 Flame Sterilization: The use of a Bunsen burner flame is a common method to sterilize
inoculation loops, needles, and other metal instruments. Passing the instruments
through the flame heats them to a temperature that kills any microorganisms present,
ensuring the transfer of sterile cultures.
 Aseptic Transfer: Aseptic transfer techniques involve the controlled movement of
microbial cultures from one location to another without introducing contaminants. This
can include methods such as streaking, where a sterile loop is used to spread bacteria
evenly across an agar plate, or pipetting, where sterile pipettes are employed to transfer
liquids without contamination.
 Sterile Work Environment: Creating a sterile work environment is crucial for maintaining
aseptic conditions. This involves regularly cleaning and disinfecting laboratory surfaces,
minimizing air movement that could introduce contaminants, and using laminar flow
hoods or biosafety cabinets when working with highly sensitive samples.
Importance of Aseptic Techniques:
Aseptic techniques are essential for several reasons:

 Preventing Contamination: By employing aseptic techniques, researchers can prevent

the introduction of unwanted microorganisms into their experiments, ensuring that only
the intended microorganisms or cells are being studied.
 Reliable Results: Aseptic techniques help to minimize variations in experimental
outcomes caused by contamination, allowing for more accurate and reliable results. This
is particularly crucial in microbiology and medical research, where even small amounts
of contaminants can significantly affect data interpretation.
 Safety: Aseptic techniques play a vital role in maintaining the safety of researchers and
the general public. They help prevent the spread of infectious diseases and protect
against exposure to harmful microorganisms.
 Quality Assurance: Aseptic techniques are critical in industries such as pharmaceuticals,
biotechnology, and food production, where maintaining sterile conditions is essential for
product quality and safety.

Materials:
 Laboratory Coat: A lab coat provides a protective barrier between the operator and the
experimental setup, reducing the risk of introducing contaminants.
 Disposable Gloves: Disposable gloves are essential to prevent direct contact between
the operator's hands and the samples or equipment. They help maintain aseptic
conditions and minimize the transfer of microorganisms.
 Sterile Petri Dishes: Petri dishes, typically made of glass or plastic, are used to hold the
agar medium for microbial culture. Sterile petri dishes ensure that no contaminants are
present before inoculation.
 Sterile Agar: Agar is a gel-like substance derived from seaweed that provides a solid
medium for microbial growth. Sterile agar is prepared in petri dishes and serves as the
substrate for cultivating microorganisms.
 Sterile Inoculation Loops or Needles: Inoculation loops or needles, typically made of
metal wire or glass, are used to transfer small amounts of microbial cultures to the agar
medium. These instruments must be sterile to prevent contamination.
 Bunsen Burner: A Bunsen burner is a gas burner used to create a flame for sterilizing
inoculation loops or needles by passing them through the flame. It is an essential tool
for aseptic transfer techniques.
 Sterile Pipettes: Sterile pipettes, both disposable and reusable, are used for transferring
liquids, such as microbial cultures or sterile solutions, without introducing contaminants.
 Ethanol or Disinfectant Solution: Ethanol or a suitable disinfectant solution is used to
disinfect the surfaces of the work area, equipment, and hands to maintain a sterile
environment.
 Incubator: An incubator provides controlled temperature conditions for the growth of
microbial cultures. It is used to promote the growth of microorganisms on the agar
plates after inoculation.
Results: Area of zone of inhibition (mm)
Antibiotics E. coli B. Meg
Chloramphenicol (c) 0 0
Erythromycin (e) 0 5
Fusidic acid (fc) 0 10
Oxacillin (ox) 0 0
Novobiocin (no) 0 0
Penicillin G (PG) 0 1
Streptomycin (S) 0 18
Tetracycline (T) 0 0

Bar chart to show Area of zone of inhibition of E. Coli and B.Meg

 Chloramphenicol: This antibiotic showed no effectiveness against either E. coli or B.

Meg. Lack of inhibition suggests that these bacteria may possess intrinsic resistance
mechanisms or that the concentration of the antibiotic used in the experiment was not
sufficient to inhibit their growth.
 Erythromycin: Erythromycin exhibited no effectiveness against E. coli but showed some
effectiveness against B. Meg with a value of 5. This indicates that B. Meg may be more
susceptible to erythromycin compared to E. coli.
 Fusidic acid: Fusidic acid showed no effectiveness against E. coli but exhibited moderate
effectiveness against B. Meg with a value of 10. These results suggest that B. Meg may
be more susceptible to fusidic acid compared to E. coli.
 Oxacillin and Novobiocin: Both antibiotics demonstrated no effectiveness against both E.
coli and B. Meg. Lack of inhibition suggests that these bacteria may be inherently
resistant to these antibiotics.
  Penicillin G: Penicillin G displayed no effectiveness against E. coli but showed limited
effectiveness against B. Meg with a value of 1. This indicates that B. Meg may have some
susceptibility to penicillin G, albeit less.
 Streptomycin: Streptomycin showed no effectiveness against E. coli but exhibited
significant effectiveness against B. Meg with a value of 18. This suggests that B. Meg
may be highly susceptible to streptomycin.
 Tetracycline: Tetracycline demonstrated no effectiveness against both E. coli and B.
Meg. Lack of inhibition suggests that these bacteria may possess inherent resistance
mechanisms or that the concentration of the antibiotic used was insufficient.

Discussion:

One crucial aspect addressed in the practical is the distinction between gram-positive and
gram-negative bacteria. Gram staining is a widely used technique to classify bacteria based on
their cell wall composition. Gram-positive bacteria possess a thick layer of peptidoglycan in
their cell walls, while gram-negative bacteria have a thinner peptidoglycan layer surrounded by
an outer membrane. Understanding these differences is crucial as it influences the choice of
antibiotics and the effectiveness of aseptic techniques. Nagesh et al. (2019) provided a
comprehensive overview of aseptic techniques in their article "Aseptic Technique: Concepts
and Applications in Microbiology". This article emphasizes the importance of maintaining
sterility, proper disinfection methods, and aseptic transfer procedures, highlighting the
significance of aseptic techniques in preventing contamination and ensuring accurate
experimental results. Allen et al. (2020) discussed the structural differences between gram-
positive and gram-negative bacteria in their article "Cell Wall Architecture and Integrity in
Gram-Positive and Gram-Negative Bacteria". This study dived into the variations in cell wall
composition, including the role of peptidoglycan and the significance of the outer membrane in
gram-negative bacteria. Understanding these structural variances is crucial in determining the
effectiveness of antibiotics and the susceptibility of bacteria to aseptic techniques. Boonma et
al. (2017) conducted a study titled "Antibiotic Susceptibility Testing Methods: From Disk
Diffusion to Machine Learning" which explored various methods for determining antibiotic
susceptibility, including the disc diffusion method.
Things that could be done to improve validity:

 Control groups: Including appropriate control groups can enhance the validity of
experimental results. Control groups should be designed to account for factors that
could influence the outcome of the experiment but are not related to the independent
variable being tested. For example, in the context of antibiotic effectiveness, including
control groups with no antibiotic treatment or using a known susceptible strain of
bacteria can provide a baseline for comparison.
  Replication: Conducting multiple repetitions of the experiment can increase the validity
of the results. Replication helps to assess the consistency and reliability of the findings.
By replicating the experimental procedure and obtaining comparable results across
multiple trials, confidence in the validity of the outcomes can be strengthened.

Conclusion:

In conclusion, the AQA A-Level Aseptic Techniques Required Practical has provided me with a
comprehensive understanding of the significance and application of aseptic techniques in
laboratory settings. By emphasizing the importance of maintaining sterility and employing
proper disinfection methods, the practical has equipped me with the necessary skills to prevent
contamination and ensure accurate experimental results. The differentiation between gram-
positive and gram-negative bacteria further enhanced my knowledge of bacterial cell wall
composition and its implications for antibiotic effectiveness. Through the practical, I have
developed essential laboratory skills and knowledge, setting a solid foundation for future
scientific endeavors that require aseptic techniques.