Aseptic Technique and Serial Dilutions

Aseptic technique is a set of procedures used to prevent contamination of cultures by unwanted microorganisms from the environment. Serial dilutions are used to produce manageable concentrations for counting bacteria or testing antimicrobial agents. Both are required practical skills for the Edexcel A-Level Biology (9BI0) specification.

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Why is Aseptic Technique Important?

In microbiology, contamination can:

• Invalidate results by introducing unintended organisms that confound the experiment.
• Create a health hazard if environmental organisms include pathogens.
• Compromise culture purity — mixed cultures are difficult to analyse.

Aseptic technique aims to:

1. Prevent contamination of the culture by environmental microorganisms.
2. Prevent the release of cultured microorganisms into the environment.
3. Protect the experimenter from infection.

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Principles of Aseptic Technique

General Rules

Procedure	Rationale
Work near a Bunsen burner (or in a laminar flow cabinet)	Rising convection currents carry airborne microorganisms upward and away from the work area
Sterilise all equipment before and after use	Kills existing microorganisms
Flame the necks of bottles and test tubes before and after opening	Kills microorganisms around the rim and creates convection currents that prevent entry of airborne contaminants
Do not leave lids off for longer than necessary	Minimises exposure to airborne microorganisms
Keep Petri dishes closed as much as possible	Prevents entry of airborne contaminants
Tape Petri dishes with two strips of tape (not sealed completely)	Allows aerobic conditions inside the plate; prevents anaerobic pathogens from growing
Incubate at 25°C (in school labs)	Reduces the risk of culturing human pathogens (which grow optimally at 37°C)
Wash hands before and after handling cultures	Removes transient microorganisms and prevents self-contamination
Disinfect the work surface before and after the experiment	Kills microorganisms on the bench surface

Sterilisation Methods

Method	Application	How it works
Autoclaving (121°C, 15 psi, 15 min)	Sterilising media, glassware, contaminated waste	Pressurised steam kills all microorganisms including endospores
Flaming (passing through Bunsen flame)	Sterilising inoculating loops, bottle necks	Direct heat kills microorganisms on the surface
UV light	Sterilising surfaces in laminar flow cabinets	Damages DNA, killing microorganisms
Chemical disinfection (e.g. 70% ethanol, Virkon)	Cleaning work surfaces and spills	Chemicals denature proteins and disrupt membranes
Dry heat (oven, 160°C, 1–2 hours)	Sterilising glassware (e.g. pipettes)	Heat kills microorganisms by oxidation
Filtration	Sterilising heat-sensitive liquids (e.g. antibiotic solutions)	Membrane filters (0.22 µm pore size) physically remove microorganisms

Exam Tip: Autoclaving is the gold standard for sterilisation because it kills endospores that can survive boiling (100°C). Always state the conditions: 121°C, 15 psi pressure, for at least 15 minutes.

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Inoculation Techniques

Streak Plate Technique

Used to obtain isolated colonies from a mixed culture.

1. Sterilise an inoculating loop by heating to red heat in a Bunsen flame. Allow to cool briefly.
2. Pick up a small amount of inoculum (from a broth or colony).
3. Streak across one sector of the agar plate.
4. Flame the loop, cool, and streak from the end of the first set of streaks into a new sector.
5. Repeat for 3–4 sectors, flaming between each.
6. The final sector should contain individual, well-separated colonies.

Spread Plate Technique

Used for quantitative work (e.g. viable cell counts).

1. Pipette a known volume of diluted sample onto the centre of an agar plate.
2. Use a sterile glass spreader (L-shaped rod) to spread the liquid evenly across the entire surface.
3. Incubate and count colonies after an appropriate time.

Pour Plate Technique

1. Mix a known volume of diluted sample with molten agar (cooled to ~45°C).
2. Pour into a sterile Petri dish and allow to set.
3. Colonies grow within and on top of the agar.

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Serial Dilutions

Serial dilution is the stepwise dilution of a substance by a constant factor. It is used to reduce the concentration of a bacterial sample to a countable level.

Method

1. Prepare a series of sterile test tubes, each containing a known volume of sterile diluent (e.g. 9 cm³ of sterile distilled water or saline).
2. Transfer a known volume (e.g. 1 cm³) of the original sample to the first tube. Mix thoroughly.
3. Transfer 1 cm³ from the first tube to the second tube. Mix.
4. Continue through the series.

Dilution Factor

For a 1-in-10 (×10) serial dilution:

Tube	Dilution factor
1	10⁻¹ (1/10)
2	10⁻² (1/100)
3	10⁻³ (1/1,000)
4	10⁻⁴ (1/10,000)
5	10⁻⁵ (1/100,000)
6	10⁻⁶ (1/1,000,000)

Calculating the Original Cell Count

After plating and incubation, count the colonies on the plate where between 30 and 300 colonies are visible (this range gives the most reliable count).

Original cell count (CFU/cm³) = Number of colonies × Dilution factor⁻¹ × (1/Volume plated)

Worked Example

A student plates 0.1 cm³ from the 10⁻⁵ dilution and counts 42 colonies.

Original count = 42 ÷ 0.1 ÷ 10⁻⁵ = 42 × 10 × 10⁵ = 4.2 × 10⁷ CFU/cm³

Exam Tip: Always show your working in serial dilution calculations. State the dilution factor, the volume plated, and the colony count clearly. The formula can also be written as: CFU/cm³ = colony count / (dilution factor × volume plated).

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Practical Skills: Investigating Antimicrobial Agents

The Disc Diffusion Method

This practical tests the effectiveness of different antimicrobial agents (e.g. antibiotics, disinfectants, plant extracts) against bacteria.

1. Prepare a lawn of bacteria on an agar plate using a sterile swab or spread plate technique.
2. Place paper discs soaked in different antimicrobial agents onto the agar surface (using sterile forceps).
3. Include a negative control disc (soaked in sterile water) to confirm that the disc alone does not inhibit growth.
4. Incubate at 25°C for 24–48 hours.
5. Measure the diameter of each zone of inhibition (clear area around the disc where bacteria have not grown).

Interpreting Results

Observation	Interpretation
Large zone of inhibition	The antimicrobial agent is effective at killing or inhibiting bacterial growth
Small zone of inhibition	The agent is less effective
No zone of inhibition	The bacteria are resistant to the agent

Calculating the Area of the Zone of Inhibition

Area = π × r²

Where r = radius of the clear zone (measured from the edge of the disc to the edge of the clear area).

Using the area rather than the diameter gives a more accurate representation of effectiveness, because the area accounts for the two-dimensional spread of the agent through the agar.

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Health and Safety in Microbiology

Precaution	Rationale
Do not eat, drink, or apply cosmetics in the laboratory	Prevents ingestion of microorganisms
Wear a lab coat and tie back long hair	Protects clothing and prevents contamination
Report any cuts or broken skin	Open wounds provide entry points for microorganisms
Dispose of contaminated materials in autoclave bags	Ensures safe decontamination before disposal
Wash hands with antibacterial soap after handling cultures	Removes transient microorganisms

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Key Terms

Term	Definition
Aseptic technique	Procedures used to prevent contamination of cultures and protect the experimenter
Sterilisation	The complete destruction of all microorganisms, including endospores
Disinfection	The reduction of microorganisms to a safe level (not complete destruction)
Serial dilution	The stepwise dilution of a sample by a constant factor to reduce its concentration
Colony-forming unit (CFU)	A unit of measurement for viable bacteria; one CFU corresponds to one colony on a plate
Zone of inhibition	The clear area around an antimicrobial disc where bacterial growth has been prevented

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Summary

• Aseptic technique prevents contamination and protects the experimenter.
• Sterilisation methods include autoclaving, flaming, UV light, chemical disinfection, and filtration.
• Serial dilutions reduce sample concentration for countable plating.
• The disc diffusion method tests the effectiveness of antimicrobial agents by measuring zones of inhibition.
• Practical skills include correct dilution calculations and safe microbiological practice.

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A-Level Deep Dive: Aseptic Technique and Serial Dilutions

Spec mapping

The Edexcel 9BI0 specification places aseptic technique and serial dilutions within Topic 6: Immunity, Infection and Forensics, where they function as the foundational laboratory craft on which every other microbiological investigation depends. Without aseptic workflow there is no axenic (single-species) culture, and without quantitative serial dilution there is no reliable measurement of bacterial population size. The lesson is the procedural backbone of Core Practical 4 and is synoptically connected to lesson 1 (pathogen identification depends on uncontaminated cultures), lesson 2 (the lag–log–stationary–death curve is sampled and quantified using these techniques), and Topic 7 (clinical microbiology — antimicrobial susceptibility testing, anti-thrombosis-unit cultures and CSF sampling all use the same aseptic protocols) and Topic 8: Genetics, Populations, Evolution and Ecosystems (recombinant-DNA workflow — bacterial transformation, plasmid preparation and selectable-marker plating all assume sterile media and contamination-free spreading). Relevant statements concern: describing aseptic technique in the context of culturing microorganisms; explaining the purpose of each step (flaming, working near a Bunsen, taping plates incompletely, incubating at $25°\text{C}$25°C in school settings); calculating viable cell concentrations from serial-dilution and CFU-count data; and applying these techniques to Edexcel 9BI0 Core Practical 4 (refer to the official Pearson Edexcel 9BI0 specification document for exact wording).

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Worked example with full mark scheme

Question (8 marks):

A microbiologist withdraws a $1.0\,\text{cm}^3$1.0cm3 sample of Escherichia coli broth and performs a tenfold serial dilution through six tubes, each containing $9.0\,\text{cm}^3$9.0cm3 of sterile saline. From the bottom-most tube she pipettes $0.1\,\text{cm}^3$0.1cm3 onto a sterile nutrient-agar plate and spreads it with a flamed glass spreader. After 48 hours of incubation at $25°\text{C}$25°C, a control plate (un-inoculated medium from the same batch) shows zero colonies and the experimental plate yields 47 distinct colonies.

(a) Calculate the original viable-cell concentration in the broth, in CFU per cm³, showing each step of your working. (4)

(b) Explain why the un-inoculated control plate must show zero colonies for the result in (a) to be valid, and state two further controls or precautions a careful experimenter would apply. (4)

Solution with mark scheme:

(a) Step 1 — identify the dilution chain. Six tubes of tenfold dilution give a final dilution factor of $10^{-6}$10−6.

M1 (AO2.1) — final dilution factor $10^{-6}$10−6. Common error: counting five or seven tubes, or treating the original $1.0\,\text{cm}^3$1.0cm3 as a dilution step (it is not — the first tube is $10^{-1}$10−1).

Step 2 — plated-volume correction. $0.1\,\text{cm}^3$0.1cm3 of the $10^{-6}$10−6 tube represents cells from $10^{-6} \times 0.1 = 10^{-7}\,\text{cm}^3$10−6×0.1=10−7cm3 of original broth.

M1 (AO2.1) — plated-volume correction: $10^{-7}\,\text{cm}^3$10−7cm3 of original on the plate.

Step 3 — back-calculate. $47 \div 10^{-7} = 4.7 \times 10^{8}\,\text{CFU}\,\text{cm}^{-3}$47÷10−7=4.7×108CFUcm−3.

A1 (AO1.2) — answer in standard form with units: $4.7 \times 10^{8}\,\text{CFU}\,\text{cm}^{-3}$4.7×108CFUcm−3. Marks lost for omitting units or quoting spurious precision (47 has two sig figs).

A1 (AO3.1a) — comment that 47 sits within the reliable $30$30–$300$300 countable range; below 30 Poisson noise dominates, above 300 colonies merge.

(b) M1 (AO1.1) — the un-inoculated control proves the medium itself was sterile before inoculation; without it, experimental colonies cannot be unambiguously attributed to the inoculum.

M1 (AO2.1) — control 1: a diluent blank (sterile saline plated without inoculum) demonstrates the dilution series was contamination-free.

M1 (AO1.1) — control 2: triplicate plates at the countable dilution average out plating variability; report mean CFU/cm³ $\pm$± standard error.

A1 (AO3.1a) — precaution: incubate inverted so condensate cannot drip onto agar; combined with $25°\text{C}$25°C (not $37°\text{C}$37°C) this minimises selection for human pathogens in a school setting.

Total: 8 marks.

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Specimen question modelled on the Edexcel 9BI0 paper format

Question (6 marks): Describe the aseptic procedure for inoculating a sterile nutrient-agar plate from a broth culture using an inoculating loop, and explain how each step prevents contamination.

Mark scheme decomposition by AO:

Marking point	AO	Credit-worthy content
1	AO1.1	States that the loop is flamed to red heat in a Bunsen flame (kills all microorganisms and endospores on the wire) and allowed to cool briefly in still air (so as not to kill the inoculum on contact).
2	AO1.2	States that the broth bottle's neck is flamed before and after withdrawing the loop (heat kills surface contaminants and the rising convection current sweeps airborne organisms away from the opening).
3	AO2.1	States that the Petri-dish lid is lifted at the minimum angle needed to admit the loop (limits the area through which airborne contaminants can fall onto the agar) and that streaking is rapid.
4	AO2.1	States that the loop is re-flamed before being set down (prevents transfer of inoculum to bench surfaces and decontaminates the loop before the next operation).
5	AO3.1a	Explains that work is conducted in the updraft of a Bunsen flame, whose convection currents lift airborne particles away from the open plate.
6	AO3.2a	Concludes that the plate is taped (incompletely, with two strips, to maintain aerobic conditions) and incubated inverted at $25°\text{C}$25°C, so condensation forming on the lid cannot drip onto the agar surface and disrupt colonies.

Total: 6 marks split AO1 = 2, AO2 = 2, AO3 = 2. Edexcel rewards candidates who explain mechanism (AO2/AO3 — why the flame works, why the lid angle matters, why inversion matters) rather than merely list the steps (AO1).

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Synoptic links