5.1 Factors affecting disk diffusion tests

As you have learned, the size of the zone of inhibition in a disk diffusion test depends both on how effective the antibiotic is at preventing bacterial growth and how fast it can move through the agar (molecular weight and size of the molecule). However, several other factors can affect the outcome of these tests and lead to incorrect results.

If the inhibition zone is bigger than it should be, the test organism will appear more susceptible to the antibiotic than it really is. Conversely, a too small zone of inhibition suggests the test organism is more resistant to the antibiotic than it really is. You will learn more about how different factors can lead to errors in the test results in Activity 7.

Activity 8: Factors that affect the size of the inhibition zone

Timing: Allow 5 minutes

How might these factors affect the size of the inhibition zone? Consider whether each factor is likely to increase or decrease the size of the inhibition zone.

For each factor, select the appropriate response from the drop-down menu.

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Answer

Factors that lead to the zone size being too big (falsely susceptible)

  • If the turbidity of the starting inoculum is too low, not enough bacteria will grow on the plate. This means that the point where there is enough growth to be visible will be further away from the disk, making the test look falsely susceptible.
  • If there is a delay in incubating the plates after they have been set up, the antibiotic can diffuse too far from the disk before growth starts, leading to an apparent increase in antibiotic susceptibility.

Factors that lead to the zone size being too small (falsely resistant)

  • Disks that are past their use-by date or have not been stored properly are likely to contain antibiotic that has degraded and its concentration fallen.
  • If the inoculum is too heavy (turbidity was too high), the critical mass of organisms to be visible will be reached too close to the disk.
  • If plates are read after too long an incubation period, the bacteria may grow back in towards the disk. Reading plates too early can also lead to errors.
  • If the agar is too thick, the antibiotic will diffuse down as well as out, which reduces the amount available to make the full-sized zone. Note that if the agar is too thin, the zone will be too big.

Factors that lead to the zone size being either too small or too big

  • If the composition or pH of the media is incorrect, the growth of the organism or the action of the antibiotic may be affected.
  • Incubating the plate at the wrong temperature can affect both the rate that the antibiotic diffuses out and the rate the organisms grow.
  • If the disks are placed too close together, the antibiotics can either inhibit or enhance each other. There should be no more than the recommended number of disks for the size of the plate to prevent this effect.

5 Quality procedures in AST

5.2 Standard operating procedures (SOPs)