To determine the sensitivity of a bacteria to different antibiotics a sample of the pure culture is inoculated in a culture medium in which the diffusing capacity of each of the substances to be tested is known. This is the Mueller Hinton culture medium.

Material

• Pure Culture of a microorganism
• Plate with Mueller-Hinton growth medium
• Metal tweezers
• Antibiotic discs

First to carry out the test inoculate the culture medium with a large bacterial load so that a bacterial lawn grows. A sterile swab that is soaked in the liquid culture or impregnated in a solid culture is used for inoculation. 

The bacterial load on the swab is transferred to the middle of the plate in a star shape and then it is spread on the whole surface so that it is all covered, Once the plate has been inoculated the antibiotics to be tested are chosen.

The absorbent paper discs are soaked in the antibiotics and they are taken out using metal tweezers that have been sterilized in a flame after being immersed in alcohol. The discs are put on the surface of the inoculated culture medium and pressed down gently so that they stick to it. There should be enough space between them to be able to read the results clearly and so that there is not any interference between the effect of one substance and another.

Photo: Enrique Rodríguez.

The inoculated plate and the antibiotics are turned upside down and they are put in the incubator for 24 hours at 37ºC. After this time, the results are read by measuring the diameter of the growth inhibition halo that appear around the paper discs. Their effectiveness is assessed by checking the diffusion capacity, according to the antibiotic, in the culture medium and therefore, the size of the halo for a sensitive, moderately sensitive/intermediate sensitivity, or resistant bacteria.   

	Photo: Ania Tribaldos
Photo: Fernando Durán Gran

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