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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.
The first thing we are going to set up in the laboratory today is a disc diffusion for antibiotic susceptibility testing. First thing you need to do is to take a Mueller-Hinton agar plate and a tube of PBS. Label both with the name of your isolate– mine is just isolate number one– your initials, and the date. So the first step is to make a uniform suspension of the bacteria that you are to test. You’ll need an overnight growth of your bacterial culture, preferably with a single colony so that you can be sure that there is no contamination present.
Take a sterile swab and pick one to two colonies, depending on the size of the bacteria you are testing, and place these into the sterile saline, mixing thoroughly.
Shake the suspension well. And then what we want is to obtain a suspension equivalent to 0.5 McFarland standard. One way to check for this is visually, using a known 0.5 McFarland standard, and then using something with, for example, black stripes behind and visually compare to check if you have the same turbidity or cloudiness in your suspension and in the known suspension. If you are happy the suspension is the same, then this is what we will use. The next step is to take a fresh sterile swab, mix well in your bacterial suspension. Wipe around the edge of the tube before you bring it out of the tube to knock off any excess liquid.
Take your Mueller-Hinton agar plate, and then very gently, whilst turning the swab continuously, spread the suspension evenly in three directions. This should give you confluent growth of the bacteria, which is what you need. Now allow your plate to dry for at least a minute or so. The next step is to choose the antibiotics you wish to test. In this instance, I’m going to test ciprofloxacin, vancomycin, and cefoxitin discs. Take either a sterile disposable pair of tweezers or some sterilised metal tweezers. Remove one disc from the tube and place gently onto the surface of the agar.
Do this for each antibiotic you wish to test, keeping them spaced evenly apart, putting a maximum of six discs on one plate so that when the antibiotic is released into the agar, the antibiotics do not mix with one another. Once complete, turn the plate over and place in an incubator overnight. The temperature and the conditions of the incubator will depend on the bacterial species you are testing. And these can be found on the latest EUCAST clinical break guidelines. So here are two plates that I incubated overnight after setting up yesterday. And we’re now going to measure the zones around the disc to determine whether this particular bacterial strain is susceptible or resistant to the antibiotics.
So how you do this is to take a ruler and measure the diameter of the area with no growth. It’s called the zone of inhibition, which is around each disc. Take the measurements in millimetres. For my bacterial isolate one, the zone around the cefoxitin disc is approximately 8 millimetres. For my bacterial isolate two, the zone around the cefoxitin disc is 32 millimetres. We then look at an interpretation table, the EUCAST Clinical Breakpoints table– I’ve printed off an excerpt here– and look to see where this falls for cefoxitin. For bacterial isolate one, which was 8 millimetres, this is less than 22 millimetres, which is the clinical cutoff, which means bacterial isolate one is resistant to cefoxitin.
Bacterial isolate two was 32 millimetres, which is larger than the clinical breakpoint, which means that bacterial isolate two is susceptible to cefoxitin. As bacterial isolate one is resistant to cefoxitin, this means that it is an MRSA, what is known as methicillin-resistant Staphylococcus aureus. Isolate number two is an MSSA, or a methicillin-susceptible Staphylococcus aureus. But in the case of vancomycin, EUCAST does not support disc diffusion testing for vancomycin with Staph. aureus. And in this case, you would want to do a broth microdilution test, which is what we will move on to in the next session.

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.

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Bacterial Genomes: Antimicrobial Resistance in Bacterial Pathogens

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