[Music] how do we know if a particular kind of antibiotic stops bacteria on our petri dish from growing we can use an assay called dis diffusion or the Kirby Bower antibiotic sensitivity assay we use little paper discs that are soaked in antibiotic generally scientists buy these discs already soaked in the antibiotic we take the discs and put them on a petri dish that is thick with bacteria the antibiotic diffuses into the augur while the plate is incubated and stops growth let's explore how this works we soaked the antibiotic discs in food coloring so we can see how the antibiotic diffuses into the augur notice that the concentration decreases as you go farther from the disc now let's put them on an inoculated petri dish for this petri dish we'll grow a lawn of bacteria last time we streaked out the bacteria to get individual colonies this time let's cover a whole plate with a thick layer of bacteria the bacteria grows up into a lawn now let's put them on an inoculated petri dish this way we can see where the antibiotics stopped bacteria from growing notice how the bacteria still grew where there was a low concentration of [Music] antibiotic now that you have the basics let's see how scientists do the Kirby Bower antibiotic sensitivity assay to perform this experiment you need gloves if divided tweezers sterile cotton swabs a marker six discs each pre- soaked with a different antibiotic a plate with single colonies a blank plate and a container for Waste here we have two scientists demonstrating the Kirby Bower antibiotic sensitivity assay scientist number one who's wearing purple gloves will perform the assay flawlessly scientist number two who's wearing yellow gloves we'll make nine mistakes let's see if you can spot them first put on the gloves if they are provided next label the bottom of the plat you can write your initials the date and anything else your teacher would like [Music] take out a sterile swab it's important not to contaminate the cotton tip draw a black dot next to the tip this will help you locate which side of the cotton swab is inoculated with bacteria find a single colony to pick make sure you can see the black dot when picking the colony now inoculate a lawn on the blank plate by spreading the bacteria evenly it's important to cover the whole plate you can spread the bacteria again in the perpendicular Direction discard the bacteria covered swab in a waste container divide the plate in sixs this will help you evenly place the antibiotic [Music] discs use use the tweezers to get one antibiotic disc and place it in its own section of the plate gently tap the disc to stick it to the augur the letters te are an abbreviation of the antibiotics full name in this case te stands for tetracycline and the numbers tell us the concentration of the antibiotic on the disc 30 for 30 microG when placing the discs use the lid to Shield the plate from contamination if the disc falls out of the tweezers leave it where it falls do this five more times for each of the antibiotic diss check the labels on the discs to make sure they are all different we want to test how susceptible the bacteria is to each kind of antibiotic place the plates upside down in a 37° c incubator for 24 to 48 hours or incubate as instructed by your teacher after the bacteria have grown you can see clearings around the antibi iotic discs we call these the zones of inhibition the bacteria are only susceptible to some kinds of antibiotics the purpose of the Kirby bow assay is to determine how susceptible the bacteria is to each type of antibiotic can you think of a way to measure the susceptibility remember how the antibiotic is most concentrated near the [Music] dis well what concentration do we need to to kill the bacteria you can measure the diameter of the clearing to determine how susceptible the bacteria is you can draw a line with a marker to help you measure be sure you measure in [Music] millimet each antibiotic disc is soaked with a different concentration of antibiotic the numbers on the disk refer to the concentration also some antibiotics are just more effective than others even at the same concentration therefore we can't use the diameter alone to determine the susceptibility of the bacteria use the reference table to determine whether the bacteria are susceptible resistant or if they have an intermediate phenotype the reference table provided by your teacher takes into consideration these these factors as well as how each antibiotic is metabolized by the human body always pay attention to unexpected results after all penicillin was a surprised Discovery let's take a look at these students plates do you see anything interesting what can you tell me about these colonies they are resistant to the antibiotic and how did they get there look at this disc it's too close to the edge to measure the diameter but can we still get accurate data for this antibiotic well we can measure the radius take the radius and double it to get the diameter let's review the Kirby bow antibiotic sensitivity essay plate a Lawn Place antibiotic discs grow the back Bia measure the diameter of the zone of inhibition and refer to the reference table and that's it