[MUSIC] Hello everyone. My name is Lina Cavaco, and I'm going to present you another lecture of this course on antimicrobials, and antimicrobial resistance and methods for susceptibility testing. And this lecture is about one of the main methods, one of the standard methods for testing antimicrobial susceptibility in bacteria. And as mentioned on the title slide this is about agar dilution. As an outline to this lecture we are going to talk about susceptibility testing by dilution methods as part of agar dilution is of course the dilution methods. We are going to go into the particularities and the process and overview of the process of the testing. We are going to look into the procedures and the quality assurance of those procedures, taking in regard all the factors that are in play when we do the tests with agar dilution methods, including all the preparation steps and actual procedure with inocullum, incubation, and reading. And when we get the results how we overview and interpret them and if there's any issues with the quality control, how we control that the quality is good and how do we act if something goes not so well. Just getting a little back to some of the slides that I have shown before in another lecture of the overview of the quantitative methods. We are here in the quantitative methods for testing antimicrobial susceptibility. In the dilution methods, so based on the dilution of antimicrobial drugs, in media, and we are going to talk about dilution in agar mostly, but the dilution methods can be included in liquid media or in solid media in agar and in this presentation we are to focus on the solid media on the agar. The results with that we are going to obtain with this kind of dilution test is a quantitative result which is stated as a minimum inhibitory concentration, and we are going to talk about this of course in the lecture ahead. We are going to focus mostly on this dilution method that is the agar dilution. In the agar dilution we have a solid media and we want to have separate concentrations to test. And this happens that we are preparing first the media, the agar with the separate concentrations. It is not a continuous gradient and that's something we have to make sure. We cannot do all the concentration that could exist in the world. It has been standardized that what we use in such a dilution testing method is two full dilutions. And these two full dilutions are made in a range that is makes sense for the drug, and that it makes sense for the bacteria that we are testing, so that we can see where they are susceptible or resistance. It doesn't make sense to use too low, or to use too high which would be not growing or growing too much. What we're going to do is to have two fold dilutions which has standardized and we are going to check the MIC. As the range is not the continuous, we will have a result that is for example, 16 but the real result, the true result would fall somewhere between 8 and 16 because what we know from the results of the test is that the bacteria don't grow on 16 which and they do grow on the 8 concentration. Somewhere in between is where the bacteria are not able to resist anymore, so it is. But of course we take this result as the one and we know that that is the limitation of our method. We cannot go behind that issue unless we do a nonstandard method where we do more dilutions than the two fold ones. Knowing that, we know that also that we can have these standard dilutions one, two, four, the two-fold dilutions that we are testing, but that also even though we test exactly the same strain, and this graph we show Antimicrobial X. We don't know which one now and we have a certain strain, just one strain and we tested a number of times. Let's say we tested a really large number of times, the same strain, we will not obtain 16 all the time. We might obtain 16 many times, but we will obtain 8 sometimes and 32 sometimes, which means that there is some insecurity about uncertainty about which value we get. And that's part of the method. It's generally accepted that in this method plus one dilution or minus one dilution is within the acceptable, so that we are in the range of the true MIC which would be somewhere in this range. This of course is a limitation of the method but we have to live with it. The issue is sometimes that this lies on the breakpoint. But then we have to classify the strain as good as we can. Also we test of course the concentrations that we can test or that we want to test and we have to choose which concentration we want to test. We normally choose for a particular antimicrobial agent. The range that is expected to be used in a clinical situation for example, and the range that includes the breakpoints for the interpretation of the results, and also the range that are expected for this bacterial population. We will expect when we test all these concentrations that our bacteria some will grow and some will not grow, sorry, that the bacteria will grow in some wells and not in others for example, or in some plates or not in others. And but that the range covers both. The non growth and the growth, so that it's wide enough to get so that you can see the difference. Also, we want to interpret the results, so we want to have the breakpoints in here. If we are following CLSI or UCAST it doesn't matter if we are following clinical, susceptible intermediate resistant or if we are following wild tap and non wild tap. We will want it to be somewhere in the middle of the range if possible. In some very reduced testing ranges, we only test one above, one below. But that's really a reduction of the process. That's called the cutoff or the breakpoint method. And that's really to save space and money and reagents that you only do two. Normally you need to do a larger range of concentrations depending on the decision that you want to make. If you are doing agar dilution you are preparing the concentrations yourself. You are limited also by the amount of work and the amount of material that, that takes. If you are doing another process that is commercially, you are a bit stuck to what the commercially available plates have. Or you might want to design them yourself. So again, not only the break points and your strains should be in this range that you test, but also the quality control strains. Because if you want to test the quality control of one test, you need to have that the quality control strain does grow in some concentrations, and does not grow in the higher ones. So that you can see where the MIC for the quality control strain is, so that you also can read if that MIC is in the acceptable results, or if there's any mistake in it. So if we go ahead, you actually want to do this process and you want to make your plates and prepare the test. So the first thing to arrange is to get the antimicrobial drug. And you need to get it as pure as possible. Sometimes it's not available from the chemical companies and you have to get it from the provider of the drug. Sometimes there's even only one provider for the whole world for that. You need to get the drug as pure as possible. And in the right, in a powder, for example, to make your concentrations. Then you prepare solutions and you will incorporate this into the agar medium. Which is, well, when it's melted, then you will incorporate it your agar, your antimicrobial agent. The medium also needs to be at the right temperature so that you do not degrade the drug, but you mix it well and that you get the plates with different concentrations where you have a number of plates for each concentration. And in the end you will have a set of plates, where the whole range is representative. You also need plates without any antimicrobial, because you need growth controls. You need plates where all the bacteria are able to grow, because there's no antimicrobial, and when you can see that nothing went wrong with the growth. It only, with the actual antimicrobial effect. And then you want to apply your inoculum to apply your bacteria on it. For this, you will want to make the suspension as it should be, and I will show this a little bit ahead. But you also want to apply the replication of this inoculums so that you have the same bacteria on each plate in the same way. So you sometimes need to have a multichannel pipette or actually an equipment that is designed for this, this kind of replicators that put your bacteria on every plate in the same way. And this is just an example. Some replicators allow you to have a number of bacterial strains on each plate. Which is good, because then you can use the same agar plate for many bacteria. And if you do this in this process, you're actually doing a golden standard method that should give you good results. As I mentioned before, this preparation of the plates, this range preparation and organization of just getting the antimicrobial and preparing everything is quite a large work. It pays off, if you have many strains to test and if you want to test them at the same time almost. So but when you are actually doing the test, you want to prepare these antimicrobials to make them, put them into the agar. You want to prepare the agar in a melted fashion. So you will prepare the media and you melt it, and then you'll leave it at a temperature just a little bit above the melting point of the agar so that it doesn't degrade the antimicrobial drug, but is melted. And then, you will mix in the amounts that you need from the dilution of antimicrobials to get the final concentration right in the agar. Also, you need to make your petri dishes with the agar, with the right depth, so that you don't have too much agar, too little. That is very standardized as well. And you make sure that you're cooling them in the right way so that they are not having any problems like bubbles or something that fell into the petri dishes. Many things can happen here. So you want to have smooth, nice plates with the different concentrations. And you'll leave then to solidify and and you'll keep them for some time. You will validate them with the quality control strains in the first test, before you use them with your strains. And you will also run the quality control strains together with your test. There's also some particulars in relation to the drying of the plates that you need to make sure. That you need to make these tests, together with your control strains and the test strains that you want to test. So that you make sure that the results are valid when you read them. Here, it is a large table, I know, and it is just a demonstration from the CLSI that is an example of how you make these plates. And it is quite confusing sometimes because first you have your antimicrobial. It's in powder and it has a certain purity. You need to calculate and you go down here? You need to calculate how much of the powder that you want to use for making a certain concentration of the drug as a stock solution. And you need to divide this by the volume, to multiply by the volume you need, and divide this by the purity that is something that is given to you by the provider of the antimicrobial drug. That will tell you how pure the compound is. And once you have a stock solution and you have a range that you have decided. You have decided to test from 0.03 to 32. That's what you want to have in the final in the plates. Then you need to do some calculations about how much volume do you need to prepare? How much agar and how much of the solutions? And you'll prepare for that. Normally, when you prepare the final plates, you use about 10% of the solution with the antimicrobial. And 90% of the actual agar as a maximum of 10% of the solution, so that agar is still okay, in the ingredients and the nutrients in the agar are still okay. So but this might be a little confusing. We can just check that this is, if we start with this concentration. We might need to make some calculations about how much we want to have in your agar. And here, we will first dilute it at least to ten times, now we want to have 32 in the end plate. So if we want to use 10%, we want to have it ten times concentrated. We would do first a solution from the stock solution that is, at this 320. And then we can take it as, actually a new stock, kind of, and then we will use. Well if we use, sorry. Yeah, you make it a certain volume and you have the 32 that you want. So you use 90 of the media and 10 of your solution. So from the 320 you get to 32. And then you go down, because you start with the highest concentration. And here you want to use half of it, so you actually mix this solution half of your solution with water. And go on and prepare your plate, so they are half concentrated. And here you just mix it with more water, so you only have a quarter of the solution. So that you get down to here. But you don't follow this way all the time, because it might cause many errors. And CLSI had described that you actually should do it step by step. So if you would do this, always just adding more and more water, you could run into some mistakes. So actually, they have decided that you do it three by three. So for the next three, you take this one as a stock for the next ones. So it is a kind of a three step approach. It is not very logical here, but it will be when you try to do it in your lab, you try to calculate this. Sometimes we just get several people to do the calculations to make sure that we are right in the end, and that we get towards the right concentrations in the end. But the basis is that we want to have twofold dilutions in the end of all the concentrations at ten times. So that we dilute them one to ten in the ends to get them on the plates right. So here is just another figurative where you start with the saline or water and the antimicrobial stock solution, and you start doing these dilutions. So that we get with this three step approach, that we get the ten times concentrated, and then the plates with the ten times less, with the 10% dilution. Here, this slide is actually one has a particularity that here we are not using just a normal media. But because it is for testing Campylobacter this Agar in the end is actually added with some blood. So it is even a more complex situation where the blood is a supplement for this Agar that we are using here. So when we want to do this process in the laboratory, because there is a large number of concentrations always involved. We want to prepare all the material with in advance, and we want to prepare the bases Agar in a large amount. And in this case for Campylobacter would be this Agar supplemented with the blood. And then we have glassware for all the concentrations separately. We might need magnets for the agitation of the Agar separately sterilized. So that we can prepare readily from the stock solutions and the steps, each portion of the Agar in one of these. Actually a good idea for this process is that this glassware, where the melted Agar is going to be mixed with the stock solutions. And the solutions of the antimicrobial drug is that this is actually warmed before. So we keep it in a water bath so that the temperature is more or less uniform, so that everything doesn't get stiff so easily. And that the Agar is still melted when you are pouring down the plates. So there is a big apparatus to this. Many times it's actually needed to have extra hands and to have somebody to help with all these process. Because you have to pipette, prepare solutions, mix to Agar. And Agar is melted and will get solidified quite quickly. So there is a number of procedures that are necessary to have with help. But once you do this, you have the plates. So you can keep them for some time. And this here depends on which Agar, sorry, which antimicrobial you are diluting on the plates. It could be that some antimicrobials are very sensitive to light and there some are very sensitive to oxygen. So you might not be able to keep these plates for a very long time. But some others are fine, and you can keep them for a longer time. That you need to validate for your own lab how it works. But once you have them, then you have the whole range of the concentrations, and you can start your tests. And a good advice would be to start it as soon as possible and coordinate it, so that you have fresh plates. Then you will start the test with your bacteria, and that's the first step where the bacteria come in. And where you have the bacteria on the plates and you make some suspension with the 0.5 McFarland density. Where you use the same equipment that I show you before in another lecture. Measuring the density and checking with the measurement with the standard. You will dilute further for the desired concentration that you want to put on the spot. And It might depend on which replicator, which inoculum system that you have for these spots. And then you will use the Agar plates to spot this solution on the Agar in a certain way. You need to recognize where your spots are. So sometimes you need to draw on the plates to make sure that you are following the right isolates in the right way. There would be some replicators where they have a certain design where you can follow which is your number one to three etc strain. You will need to organize also the plates according to the concentrations. You should inoculate always the lowest. So first a control plate without antimicrobial and then the lowest concentrations and then up to the highest. When you are putting them on the replicator so when you are inoculating them to make sure that the antibiotic is not taken from the replicator to the other plates. And then as I mentioned you might need to draw a bit or mark the plates so that you're sure which isolate is which. So when we have done this you apply the bacteria on the spots and you use these devices. You might use a pipette if you don't have the device, but then you also need to know where is what. And you also make a purity control by taking some of this culture and spreading it on another plate where you can check, if the culture is fine and there's nothing contaminating it. Then you will incubate these plates. And these plates when you spot them on they have little drops sometimes you need to let them dry a little bit before you get into the incubator. So that these drops are not spreading around. And then you incubate them and check them the next day after the incubation time has gone. When you read them you will want to look at growth or no growth. You will determine these endpoints. And these endpoints are basically, well, you have a concentration that is lower where you see the growth but then you have the next concentration where it's higher and you don't see growth. Let's say that there's growth on the plates which two micrograms and that there's no growth on the plate with four micrograms per millilitre. This is your endpoint and four micrograms, in this case, would be your MIC. In this case, there are also some things to look at, what is growth, what is not growth. Sometimes there is a little bit of haze or and you need to know which antimicrobial you're dealing with because with some, like this one, you might have to disregard this little bit, with others that's not the case. Here just to make it a little bit more visible what I was telling about the designs and where to follow your inoculum, we have this is in our lab we have a replicator that makes this spot with this design. Where we know that we have like this part this is very horizontal, and then we have these circles, but by following this circle we know that this number one up to 18 here, and then it goes around so that we have. And we always have the control strains in the middle to check for the growth control and for the quality control of the ranges. That we know this strain has a certain ranges, where the result should be and the same for this and this which of course, these are four different strains, so they have different expected results. But we should follow that in the assay. The way that this works on the replicator is that we have a block in kind of a plastic material that looks just like that and has wells just like that. And then we have this part with the pins that fit just into the same holes and it touches the holes with the suspension, with the liquid and then it goes and touches the plate that is put here and you move it with the peddle on the floor so that you're preparing this on older plates exactly in the same way. After you do this you will incubate your plates. There's also differences in relation to how much time that should be, but and also in the atmosphere if we are dealing with campylobacter or with other organisms that don't require oxygen but require another mixture of gases. That's also very important to check. And we will read the plates. And now we are into reading. We have our scheme, we also have a sheet where we have the exact same scheme where we know in which well, we have put which strain with the identification of the strains, and now we try to read them. This is our control, and here we imagine that we only have test these strains and for test for quality control strains, but we are mostly looking at these red, green, and blue strains for the result. This is the controls, so there's no antimicrobial here. Here we have 0.25 milligrams per liter. And if we look between here and there, we can see the blue spot is gone. What we know for the blue spot is that this bacteria, this strain that we put here, cannot tolerate the 0.25. The MIC is either at 0.25 or lower. We don't know more because we have not tested lower concentrations, let's imagine that. The MIC here, it's equal or less than 0.25. For the others, there's still growth so we have to look further. Now the green one is gone and here we have 0.5 milligrams per liter. For the green one, we will say that the MIC is 0.5, because we have tested the lower one, and that there was growth. And here, we don't have growth. But for the red one, let's imagine we didn't test more, so we have no concentration of one which would be the next one. The thing we can say is, well it's higher than 0.5 because there's growth here but we don't know how much higher. That's how we do the reading, it's by looking at all these dots and where do the dots disappear or not disappear or are there's growth or no growth. Here just an overview again. We start with a inoculum and picks some colonies, adjust the suspension, check with the densitometer and nephelometer, measure it, all these replicator. Well in this case it's a slightly different figure but we have the circular one that I just show you. And the different concentrations of agar and then we try to look where the dots are or not. And here it's a real life situation with real plates and here, if I was in the classroom I would ask my students where did the dots disappear? Here we're in the video, we cannot do that but you see the dots still here, most of them. Most of the strains that were tested here, they're still growing here. They're growing is less than here on the 2, but they are still there. Most of them disappeared when we went up to 8. When we look at the end points, we would say that for most strains, the MIC is this 8. In regards to make determination, as I've showed you you have these values. Then you have for example, obtained this result, that was an 8 milligrams per liter. Of course that doesn't tell you much yet, in terms of is the antimicrobial working or not? Because for that species of bacteria and that drug, eight is an abstract number. It needs to be compared with some tables, to make sure that you can interpret it. And in the tables, most of the cases, there would be a resistant intermediate or susceptible if you're using the clinical grade points. Sometimes the intermediate doesn't exist. And there would also, if you're using this epidemiological cut offs, then you would be separating the wild type the susceptible ones from the non wild type to resistance one. And this you look up in the tables that are given by the standards, so that you know from your quantitative result what it would be qualitatively if you would use the antimicrobial. But you still have quantitative result and that would give you good information. And if these break points changed you always have the quantitative result to reevaluate your results. But sometimes things can also go wrong and I have referred this for the [INAUDIBLE] fusion in the dilution method is similar, sometimes cultures can be contaminated or the media could be a little bit off because the media has some nutrient that are not okay, the pH is wrong or there's some inhibitors or the salt content. You can also make mistakes when you're preparing it with the volume or with the preparation of the inoculum putting too much or too little. And also when we prepare the plates that they are too much or too little volume or too dry or too wet, which can also happens. And you need to standardize this with using the quality control procedures and strains where you verify that the results you have are good. When you actually are interpreting and qualifying on these categories, you expect that the results that you would get in VIVO in the human are in the patient will be good if it is a susceptible or bad if it is a resistant strength and you still have the quantitative results. Here the data is very useful not only for the clinical part if it's a patient that you're taking care of but it's also for following the trends and that's very used in monitoring of a country or of a certain type of product where you're following the bacteria on those sub strengths. Where you want to check is the resistance getting worse, is the resistance in the same level and so on. And also for research where you want to look for example in for certain resistance patterns that show you, well, probably there is a specific resistance here. If you have find a type in these tests that show you that maybe there is a certain resistant chain then you can look for it and check also which plasmas are transporting it, how has it disseminated. How did it travel from one country to another, there's a lot to do there. And of course, that is done always with the basis of the quality control, so that the results are okay and it's not just because a mistake that everything was followed up. Okay. This was for the agar dilution. I still have one presentation on the methods for broth dilution and then we have some other lectures on another module. [MUSIC]
