[0:01]So now we are going to learn about the antimicrobial susceptibility test or the AST. After doing all the culture and the culture identification, we want to get to know that which antibiotic is most effective against a particular bacteria so that we can treat the a particular infection with the best antibiotic out there. And there comes the role of the antimicrobial susceptibility testing. This testing helps us to identify the most effective and the appropriate antibiotic for a particular infection against a particular bacterium or the microorganism. So this is test which is done to check the effectiveness of a antimicrobial agent against the bacterium and also to select the best agent with the right dosages. Okay. And one most important, uh, uh, you know, the concept is that the AST is performed only for pathogenic bacteria and not for commensal bacteria. This is not performed against the commensal bacteria, this is not performed against the commensal bacteria. Like, for example, the E coli bacteria is present both in our gut as well as in our oral cavity. But there it is present as the, you know, as it is present as the commensals. So we are not going to do the antimicrobial susceptibility testing against a E coli which is found in the stool of a person. Because that is coming from a area where E coli are normally present as commensals. But on the other hand, if E coli is present in a urine culture, that means it is urine, that means the E coli is, uh, is dwelling in an area which is not made for it, that means the urinary tract. So in that case, we have to do the antimicrobial susceptibility testing and we have to give the antibiotics to the particular, uh, uh, person whose urine has been found culture positive for the E coli and treat that person with that particular antibiotics. Now, coming to the purpose, what is the purpose of this antimicrobial susceptibility testing? So the first purpose is to select the best antibiotic out there for a particular bacteria. Then it is also to control the irrational use of the antimicrobials. Okay. So when we do the antimicrobial susceptibility testing, we find the best antibiotic against a particular bacterium, that means we will not be using the substandard dose or the, you know, the less effective antimicrobial agents against that particular organism. So this will reduce the irrational use of those less effective antibiotics. If we use those less effective antibiotics, that will, uh, uselessly create some more hazards for us, that means that will create the antimicrobial resistance in that particular organism. So we want to avoid that by avoiding the irrational use and that avoidance of the irrational use can be only done if we do the antimicrobial susceptibility testing before giving the antibiotics to a particular person. And then we also this also helps us in the empirical treatment against the drug resistant bacteria. Now comes the types of the anti micro antimicrobial susceptibility testing methods. So the types includes the disk diffusion method, also called as the Kirby Bowler's disk diffusion test, then the dilution method and the episilometer test. Other than these three tests or the methods, we also have some other tests like the genetic tests like PCR and RT PCR, those types of different tests are there, they also help in detecting the antimicrobial susceptibility. And other than that, we have some automated systems also. They help us in detecting the best antibiotic for a particular microorganism. But in our Indian exams, there is a trend to ask the questions which are, you know, outdated. So these, these tests are also outdated. We are not using this test for antimicrobial susceptibility testing. And in these days, we are using only the automated systems for identification of the antimicrobial susceptibility testing, but they ask you the questions which are outdated. So, we will also be learning only those things which are outdated because we want to learn according to the exams here. Okay. So let's talk about the different methods here. Those include first of all, is the Kirby Bowler's disk diffusion method. The Kirby Bowler's disk diffusion method, that is the quanti qualitative test, okay, that is the qualitative test. And, uh, in this method, first we have to prepare the inoculum. That inoculum is prepared by suspending the colony in a normal saline or by inoculating into a broth. So suppose if this is a test tube out there and this is the normal saline there, okay, in this this is the normal saline in this test tube. So we have to suspend a colony in the, in this normal saline. So a colony of bacteria is suspended in this normal saline or the broth. And we will wait till, uh, till this, this, uh, you know, till, till this bacterial broth becomes a little turbid. So as to, uh, so as this opacity of that turbidity reaches to 0.5 McFarland. So once the opacity of this, opacity of the turbidity which is present in this bacterial broth reaches to this 0.5 McFarland, then it will be inoculated over the Mueller Hinton agar, Mueller Hinton agar. This is the agar over which we do the disk diffusion test, disk diffusion method of the antimicrobial susceptibility testing. So once it reaches to this opacity level, that is 0.5 McFarland, then we will be doing the lawn culture. I hope you remember the lawn culture method in our culture methods. I have discussed about the lawn culture method there. So by that lawn culture method, we will be doing or inoculating this bacterial broth, this bacterial broth over a Mueller Hinton agar, over a Mueller Hinton agar, we will be, uh, inoculating this bacterial broth.
[7:09]After that, we will be impregnating the antibiotic disks. So see here, there are different antibiotic discs. These are all the antibiotic disks that are impregnated over the Mueller Hinton agar after the inoculation of this, of of that bacterial broth. Then this is incubated at 37° C for 16 to 18 hours. Okay. So after the incubation for 16 to 18 hours, then we have to interpret the result. So how do we interpret it? So susceptibility to the drug is determined by the zone of inhibition of bacterial growth. So see here, in this case, in this case, the zone of inhibition is large. Okay, zone of inhibition is large. But see the this case, here the zone of inhibition is very small. Okay, that means the antibiotic which is present in this disk, is more effective against the bacteria. While the antibiotic which is present in this disk is less effective against the bacteria. That means for the treatment of the treatment in this person, we will be using this antibiotic, we will be using this antibiotic. Okay. So this is how we do the antimicrobial susceptibility testing and we interpret the results of these antimicrobial susceptibility testing in the disk diffusion method. Next comes the dilution method. So in the dilution method, we we will be discussing the tube dilution method, that is the quantitative, that is the quantitative method. Okay. So what do we do in this tube dilution method? So here the antimicrobial agent is in the Mueller Hinton broth, taken in the tubes and are serially diluted. So see here, I have taken the, uh, the antimicrobial agents in these all test tubes. In all these test tubes, there is antimicrobial agents and the Mueller Hinton broth. So this is Mueller Hinton broth with antimicrobial agent, this is also Mueller Hinton broth, this is also Mueller Hinton broth with antimicrobial agent. These all are the Mueller Hinton broth with antimicrobial agents. The difference is that the dilution. So this is the maximum dilution, 64%, okay. Sorry, so maximum dilution is in the, in tube one. The maximum dilution is in the tube one and the maximum concentration of the antimicrobial agent is in this test tube. So in this test tube, the concentration of the antimicrobial agent is maximum, while we are diluting serially in this direction and the minimum concentration of the antimicrobial agent is in this test tube. Okay, is in this test tube. So this test tube has the minimum amount of antimicrobial agent. Okay. So after this serial dilution, then each tube incubated with fixed amount of suspension of test organism. So then we add the test organisms in each of the test tube. But remember, the amount of the specimen will be same for all of these test tubes. So we will be adding here also, here also, here, here, here, here. So everywhere we will be adding the antimicrobial, every in every test tube we will be adding the, sorry, in every test tube we will be adding the suspension of the test organism and the quantity of that suspension should be equal in all of them. Then the tubes are incubated at 37° C for 18 hours. And then we detect the minimum inhibitory minimum inhibitory concentration of the antimicrobial agent is noted and the and the tube where no visible growth of the microorganisms occurs. That is the minimum inhibitory concentration. So see here, the two things should be noted. One is the test tube eight. So here in, in these three test tubes, we are seeing turbidity, that means obviously there is growth of the bacteria. But in the tube eight, we are not showing the, we are not seeing any turbidity, that means it is the minimum inhibitory concentration. So see here the minimum inhibitory concentration is 8 mg per ml, 8 mg per ml. But when we are doing the culture from this, uh, but when we are doing the culture from this test tube, then we are getting the colonies, we are getting the colonies, that means it is not the, I mean bacteria less uh broth. It, it, it has some bacteria there, but they are not able to produce the bacterial, you know, the turbidity there. So it is the minimum inhibitory concentration because no visible growth of microorganism occurs in this test tube number, test tube number eight. Suppose it's eight, but that is the concentration. But you call it eight, okay, for easy understanding. Next comes the tube number 16. Here also when we are doing the culture, then we are seeing the colonies, that means it also contains some of the bacterias. So next come we come to the 32. So when we culture from this test tube 32, then we are not getting any colonies out there in the sub culture, that means this is the the minimum bacteria concentration is present in the, present in that test tube, which is having the concentration of the antimicrobial agent at 32 mg per ml. Okay, at the 32 mg per ml. So that's the way of determining the minimum inhibitory concentration and the minimum bacterial concentration, okay. Next, so this is all about the tube dilution method. Now we come to the epsilometer test. So in the epsilometer test, we are using the principle of both the dilution as well as the diffusion of antibiotics are used. Okay. And this is done by using a strip in which different antibiotic concentration is present along its length. So see here, we have this strip of impregnated with the antibiotics and there are, uh, I mean there is the concentration of written over that strip. Concentration of the antimicrobial agent is written over that strip and this is the in the increasing order. Here the concentration is one, here the concentration is two microgram per ml, here the concentration is 32, here the concentration is 128 microgram per ml in that strip. Okay. So after impregnation of that strip in that agar, then we do the lawn culture of the bacteria. Okay. Lawn culture of the bacteria is done over this agar and after that incubation, we see that there is a elliptical zone of no growth. So in this region, there is no growth of the bacteria, that means the antibiotics are diffusing from the strip in this direction and they are preventing the growth of the microorganism. They are preventing the growth of the microorganism. So after the diffusion of the antibiotic from this maximum concentration, the growth is inhibited in this area only. But the antibiotic which is present, the antibiotic concentration or the antimicrobial concentration, which is present in this portion, these are not able to inhibit the growth of the bacteria. That's why the growth is seen even here, along with the, I mean, along the strips also. But since this amount, this eight microgram per ml of the concentration of the antimicrobial agent is preventing the growth of the bacteria. So above this, no growth is seen along the sides of the strip. But below the 8 microgram per ml, we are seeing growth even along the strips also. Because that concentration is not able to inhibit the growth of the bacteria. I hope you are understanding this concept. So here, how do we know which, I mean, what is the concentration at which the microorganisms are not growing. So that is determined by the age of the ellipse, which is intersecting the strip. So see here the edge is intersecting the, uh, the age of the ellipse is intersecting the strip at the level of the eight microgram per ml concentration. So this is our the, this is our minimum inhibitory concentration value. This is our minimum inhibitory concentration value. Okay. So this is the three methods of doing the antimicrobial susceptibility testing. Now I have told already also that there are different other methods as well like the, uh, the genetic methods, PCR, RT PCR and some automated machines are always there to identify or to do this antimicrobial susceptibility testing. Those were or those are far much better than these conventional tests. Those are far much better than this conventional, okay, but still we are discussing these conventional things because in exam, in exams, they ask us about these conventional things only.
[20:31]So that's all for the antimicrobial susceptibility testing.
