Testing antimicrobial susceptibility

How can we test if an organism is inactivated by an antimicrobial? As well as methods using several dilutions of a candidate compound across a microplate, there are some agar plate-based methods that we can use to get an idea of how susceptible a microorganism is to a given agent.

Disc Diffusion Assay

In this assay, filter paper discs are treated with a test antibacterial compound and then placed onto a bacteria inoculated agar plate. Discs treated with a positive control compound (usually a highly effective disinfectant) and an untreated disc (negative control) are usually tested alongside the candidate compound. In this kind of test, the size of the area around the disc where bacteria can't grow (termed the 'zone of inhibition') gives an indication of the compound's antimicrobial efficacy.

Control experiments

When investigating antimicrobial agents using growth plates, it's important that appropriate controls are used so that you can have confidence that your experiments have worked (or failed!). Either way, controls allow you to make the best interpretation of the effects you observe

Positive controls are those that are used to demonstrate that an experiment is capable of working. For example in a diffusion disc assay, a positive control would let you know that the bacteria have grown on an agar plate and what inhibition of growth on that plate looks like. If your positive control doesn't turn out as expected, you might need to repeat the experiment!

Negative controls check those things that shouldn't affect an experiment don't produce an effect. For example, if you dilute a reagent with a buffer, you might test that the buffer doesn't have an effect on your experiment.

AMT ring

Another form of a disc diffusion assay uses a commercially available item an antibiotic multi-susceptibility test ring (AMT ring) to compare many antibiotics simultaneously. The ring comprises many discs, each of which is treated with a different test compound. The size of any zones of inhibition that arise gives an indication for an effective candidate antimicrobial.

Epsilometer test

Related to the disc diffusion assay is the commercially available epsilometer test. each stip of paper is impregnated with an increasing concentration of a candidate antimicrobial from the bottom to the top. The assay is processed by adding a test strip to a freshly bacteria-inoculated agar plate. After an incubation period, the minimum inhibitory concentration (MIC) of a drug required to prevent microbial growth is determined by reading the concentration on the strip where antimicrobial growth is first inhibited.

Visualization of three susceptibility tests. For diffusion disk assay, the surface of the agar plate is shown as a big brown sphere. Three small, white circles are placed on it equally distances from each other. From the upper one, black arrow comes out towards the value of 0 millimeters. From the right one, another black arrow comes out towards the value of 0 millimeters. The left sphere is surrounded by a big red ring, and its diameter has the value of 38 millimeters. For ATM ring test, the surface of the agar plate is shown as a big brown sphere. In the middle of the plate, white ring is placed. From the ring, short, white stripes are coming out towards small circles, representing samples of antibiotics Pink circle with no zone around it refers to novobiocin, dark red circle with biggest red zone of inhibition refers to penicillin, black circle with no zone of inhibition refers to fusidic acid, orange circle with second biggest zone of inhibition refers to clindamycin, light pink circle with third biggest zone of inhibition refers to metronidazole, yellow circle with very small zone of inhibition refers to tetracycline, and last light yellow circle with no zone of inhibition refers to chloramphenicol. For E-test, on the agar plate presented as a brown circle, three long, thin rectangles are coming from the middle of the plate towards the top, lower right, and lower left side of the plate. Around each rectangle, the visible zone of inhibition can be seen as red ovals. The biggest zone is for the upper rectangle that refers to penicillin, the middle size zone is for the lower right rectangle that refers to clindamycin, and the smallest zone is for the lower right one that refers to metronidazole.

Figure 1: A diffusion disc assay. In the image above bacterial growth appears as a confluent brown lawn, with red blood agar showing through areas with no growth. Only a single zone of inhibition has developed around one of the discs. The other two discs, though there appears to be a little growth prevention around the other rings, this is too undefined to be recorded as a true zone of inhibition. An AMT ring assay and an epsilometer test. In both plates, bacterial growth appears as a brown lawn. On the left, we see the AMT ring placed on an agar plate. Three compounds show potential antimicrobial activity due to the presence of clear zones of inhibition around the discs. On the right, we see three epsilometer test strips set on an agar plate. Each shows a clear, teardrop-shaped zone of inhibition. The MIC is determined by reading the concentration from the strip where bacterial growth is first inhibited (the point of the tear-shaped zone of inhibition).