RP06 - Aseptic technique and antimicrobials

What a zone of inhibition shows

Spread bacteria across agar, add a disc of antibiotic, and a clear ring appears: that ring is your result.

An antimicrobial substance, such as an antibiotic, antiseptic or disinfectant, kills microorganisms or inhibits their growth.

On a lawn plate, bacteria are spread over nutrient agar and the substance diffuses from a paper disc into the agar. Its concentration falls with distance from the disc, so the edge of the ring is where it becomes too low to stop growth.

What a zone cannot tell you

A larger zone can indicate more inhibition, but three limits apply:

• Diffusion affects size. A substance that diffuses slowly can give a small zone even if it is very effective, because it does not spread far from the disc.
• Clear agar does not prove death. Killing and temporary growth inhibition both leave the agar clear.
• The result is specific. It applies to one strain, concentration and incubation period, and does not show the best treatment for an infection.

Asking a specific question

Ask, for example, how the concentration of one disinfectant affects visible growth of one bacterial strain. Concentration is the independent variable and inhibition-zone size the dependent variable. Different substances can also be compared, but differing diffusion rates limit the interpretation.

Use only the approved non-pathogenic culture, such as E. coli K12, and the risk-assessed method under supervision. Do not culture unknown environmental samples.

Aseptic technique and safety

A contaminated plate cannot be trusted, so each step here blocks a route for unwanted microorganisms.

It protects the people handling cultures and the comparison itself: a second organism could change the apparent inhibition.

Keeping contaminants out

1. Disinfect the work surface before and after work, and wash your hands. This reduces the microorganisms that could reach the culture.
2. Use sterile agar, pipettes, spreaders and forceps, and avoid touching surfaces that will contact the culture, so nothing else is carried in.
3. Open vessels briefly, with the lid over the opening as far as possible. This limits exposure to airborne microorganisms.
4. If the approved method uses a burner, flame culture-bottle necks as instructed. The flame kills microorganisms on the rim, and the rising hot air carries airborne microorganisms away from the opening.
5. Cool heated transfer tools before touching a culture, so they do not kill the intended organisms.

Keep flames away from open flammable disinfectants such as ethanol.

Sealing, incubating and disposing safely

• Tape the lid with a few small strips, not an airtight seal. Tape reduces accidental opening and the gaps allow gas exchange; fully sealing a plate can favour anaerobic growth.
• Incubate inverted, so condensation does not drip across the agar and redistribute the lawn or treatments.
• Incubate below body temperature, often about 25 °C (25–30 °C in AQA's example). This reduces the risk of encouraging human pathogens, but does not guarantee safety.
• Keep incubated plates closed. Measure through the closed plate and never reopen it to inspect or smell colonies.
• Decontaminate before disposal. Used cultures and equipment need a validated procedure, such as technician-managed autoclaving.

Running a fair disc test

Change only the treatment: each step below stops something else from altering the size of the zone.

Prepare a range of concentrations of one antimicrobial in the same solvent, using a graduated pipette or syringe, or compare named substances at stated concentrations.

1. Label the base, not the lid, with treatment and replicate, because lids can be swapped. Prepare sterile nutrient-agar plates with equal agar depth.
2. Aseptically transfer the same measured volume of a well-mixed broth culture (bacteria grown in liquid nutrient medium) of the same strain and cell density to each plate. Spread evenly to make comparable lawns.
3. Deliver the same measured volume of each solution, such as 20 microlitres, to identical sterile discs, because disc size and volume both change the dose.
4. Place discs with sterile forceps far enough apart and from the edge that expected zones will not overlap, and do not move them afterwards.
5. Include a solvent-only negative control: the same disc and solvent volume, but no antimicrobial. This tests whether solvent or disc handling accounts for inhibition. Sterile water is suitable only when water is the solvent.
6. Secure the lids, invert and incubate under the same approved temperature and duration. Measure through the closed bases using a stated convention.

Replicates and controlled variables

Use independent replicate plates for each condition, because discs on one plate share that plate's conditions. A positive control, a known effective treatment, shows the assay can detect inhibition but does not replace the negative control.

If concentration is the independent variable, do not also change its volume, or differences could come from the total liquid delivered.

Measuring zones and calculating area

Turn a clear ring into a number you can compare, and state exactly what that number includes.

Define the measurement before collecting results. Here, outer zone diameter is the full clear-zone diameter through the disc centre, including the disc.

If a 6 mm disc has no clearing beyond its edge, record “no additional inhibition” and an outer diameter of 6 mm, not 0 mm. Clear-zone width outside the disc is an alternative, but do not mix the two.

Measure two outer diameters at right angles through the base of the closed plate. Their mean allows for slight irregularity; very irregular zones limit the circular approximation.

From diameter to area

The area of a circle is $\pi r^2$, and $r=d/2$, so the area is $\pi d^2/4$.

The first area includes the paper disc; the second subtracts it to estimate inhibited agar. State which area is reported.