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AST method

In this video, Beth demonstrates the procedure to determine if bacteria are susceptible or resistant to a range of different antibiotics.

Principles of the procedure

The aim of this procedure is to determine if bacteria are susceptible or resistant to a range of different antibiotics. This is done by plating a standardised suspension of the organisms onto an appropriate agar plate, and then applying antibiotic discs to the surface of the plate prior to incubation. The plates are examined the following day for evidence of growth. If the organism is susceptible to the antibiotic there will be a zone of clearing around the antibiotic disc (referred to as the ‘zone of inhibition’). If the organism is resistant to the antibiotic it will grow up to or near to the disc.

Zone diameters are measured and compared with official cut-off values for susceptible, intermediate and resistant isolates. In this practical we will be following the EUCAST ( – European Committee on Antimicrobial Susceptibility Testing) guidelines as all their methods and breakpoints are open access and freely available to everyone. The guidelines are regularly updated (so you should ensure you are using the most updated version).

The selection of discs chosen for susceptibility testing will depend on the organism being tested, the guidelines being used (in this case, EUCAST), and any local antibiotic prescription policy (e.g. which antibiotics would a clinician like to prescribe to treat an infection caused by a certain organism).

Notes on the video

A flame curtain is not normally used in the UK

Safety cabinets are not routinely used for non-respiratory CL2 pathogens so long as aerosols are not being generated

It is not normally advisable to talk while inoculating media, and was only done in this video for the purposes of demonstrating

The demonstration was done using the most accessible equipment, to enable more students to perform the experiment with simple laboratory supplies. There are more convenient methods available such as using a disc dispenser rather than forceps, or using a spectrophotometer instead of visual comparison to a McFarland Standard. In both cases, the cost difference is large, and may not be readily available to all.

This article is from the free online

Bacterial Genomes: Antimicrobial Resistance in Bacterial Pathogens

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