[MUSIC] Hello everyone, and my name is Lina Cavaco. And I'm going to present to you on additional lecture on the microbial resistance and susceptibility testing. And this case would be about broth Dilution methods which are widely used in the laboratories. So this is one of the mainly used methods that are available because it can be automated and so on. So we are going to give some insights into these methods. We are going to introduce what are these dilution methods and focusing on particularities and overview of the dilution methods and the broth dilution methods as such. And we are going to introduce the general procedures about these methods, how they are going on, and how you are controlling them. And also a bit about the reading and interpretation and control of these methods in your laboratory when you're performing this. So getting back to this general slide that I have shown before, we are still in the quantitative measurement. We want to check this with the dilution method so that it gives us a MIC as result. And, we have just talked about the Agar dilution and now we are going to move into the liquid media. Basically, the principle is exactly the same, just that the media is liquid in this case. And we can do it at a larger scale with tubes but we can also do it in a very small scale with the microtiter plates where the volumes are up to 100 microliters for example in a test. So the test is going on in a large amount of antimicrobials at the same time and this can be very easily done for many strains. The result will be a quantitative result as a minimum inhibitory concentration. That gives us a definition of the fact of the drug on that particular strain, and can have a prediction of the outcome of the infection, for example, and the treatment of the infection and some research perspectives from this as well. So as I mentioned, MIC is a large and a quantitative way of testing. And when you obtain a MIC result, I also shown this slide for the agar dilution. It's the same principle, that we are not testing all the concentrations. We are testing two full dilution, as the standard is telling us to do. And then we are testing, in not on a continuous but in separate concentrations so that when we obtain this MIC result, this minimal concentration, the true MIC would be falling between these values. So that for example when we obtain a 16 it might be between 18 and 16 where the bacteria are not able to grow anymore but, this is what we see. Again, similarly when we test by dilution, and in this case in broth, it's a similar picture. We test the same strain many times. If we tried this you don't obtain the same result all the time. Even if you do it very very the same way and with the same media even, you would obtain with the same drug a number of results that would be falling on the same spot many times but also one below and one above. And that's generally accepted because it's part of the accepted error of this method. So the true MIC would range somewhere in-between here and that the variation is accepted one above and one below. So again, when we test dilution ranges that's the same as agar dilution as I mentioned. We want to choose the right concentrations to test, and this is really depending on what we need, and which bacteria we have, and which antimicrobial. So we want to test the concentrations that fit in the growth and the not growth for that antimicrobial and for that bacteria species. And we also want to check that the interpretative criteria, the break points, are somewhere in the middle of these concentrations that we are testing. Again, we are testing many concentrations. We might have limitations regarding the money or the plates that we want to use. So we cannot always test too many concentrations. And we also want our control strains to be somewhere in this scale so that we can see growth and no growth in the range. So that we can check that the results are alright. As we are speaking about broth dilution, and that's not always the case as dilution we make our own plates. For broth dilution many times we buy the plates from a provider and then we are limited to the choices that we have. Some of the providers, if we have, we will spend enough money and enough number of plates, they might do what you want. But that's not always the case. We might need to choose one design that is available. So again for the procedure itself we need the culture, we need the bacteria that are recently grown. So young culture, not too old. And we need it to be growing on non selective media so that there's no antibiotics beforehand that will damage the result. We will adjust an inoculum using a densitometer or a nephelometer so that we have the right amount of this bacteria in the test. And, We will then further dilute it in the broth already. So here we are dealing with liquid media. We're not just diluting in the suspensions, then we are diluting in the broth. And we will use this broth for the test. Again if we have a bacteria that doesn't grow well with this kind of broth we might need to have supplements. That is really the written in the standards which one you are going to use. For example as campylobacter is defined here, we need to have some lysed blood in the media otherwise they wouldn't grow. But, for the main bacteria that we test in our labs, the normal Mueller Hinton Broth, which is cation-adjusted so that the ions are not affecting the results, they are at the right level then we are using it directly without the supplement. So in regards to the procedures, while it is relatively easy, we are preparing the suspension, as I mentioned. We dilute in the broth and then we have the broth ready. If we are lucky enough, we might have a device automated machine that takes care of the rest. Maybe we are not so lucky, we can it by hand. But anyway, we have these panels. Maybe here you can see a larger view of a panel. And these panels, if you buy them commercially, they have, at the bottom, they have the antimicrobial drug all ready as a dried substance. So when we into, include the inoculum the broth with the bacteria in this wells, normally we fill them with 50 or 100 microliter, depending on the design of the plate. Then we will have the plates ready. And these machines, which is an autoinoculator, just one example of one. They actually fit the tube where you prepare the dilution with the broth. They fit the two upside down and they will dispense the right volume into each well. So you don't need to do anything but pressing one button. And to incubate these plants, you will just need to cover them. For bacteria like, you might need to have these covers that have little holes on them, but for other bacteria, it's fine to have just the plastic seals without the holes. And once you put them in the incubator you also need to check that you're not putting too many on the stack because of the oxygen flow, but you put them in the incubator, you will incubate them for the time that's necessary for that species and that panel. And you also have a purity control that you do on the side on another plate just to check if the culture is fine. After this incubation you will want to determine the end points, similarly as you would do for the agar dilution. But here you would look at liquid culture. So you would look at the wells for a little dot with culture that you can see. I will show it in the next slides. Where you would observe first the control wells because you have always in the plate one or two control wells that don't have any antimicrobial where the bacteria should grow so you can see how the growth looks like and you will also check if the growth is well distributed and where there's growth, there should be growth there is. Skips is like if a there's growth and then there's not growth in one well and growth again so it's kind of a jump over concentration that should not happen too much. You need to check that at first and make sure that you don't have them around. I will also explain a little more about that. And you also might need to make some considerations into which antimicrobials you're looking at. Because the reading is not always black and white for all antimicrobials. If you have one like sulphonamides, you might have slight growth that is not real. Because it's a bacteria static drug and other wise you would these plates. Another thing that I didn't mentioned besides these machines for in inoculating the plates actually there's also machines for reading this so if you are lucky enough you can also read this not only with the well because then you also have a measurement with a fluorescent agent and you can do this automatically. If it's fine to read by eye, that's also a great way to read. And many times you actually need to both because you want to be sure that what you see or what the machine sees, you see, as well but here, we have some very, well, Kind of semi- or manual methods of reading. So imagine we have a microbroth plate. We would put it here. This is a very manual way of reading where you just have a mirror here and we look at the mirror and see because when we are looking from below and we will look something like this. This is growth, where the bacteria did grow, and there's a spot there, and the places where you see nothing, then there's no growth. Then you can also have something where you put the panel here, and you get an image there, but you still read it by eye. So, you will press here, the buttons where you see the growth or no growth. So it's a semi automatic. There's also the fully automatic. I just don't have an image here. But they would decide themselves where there's growth or no growth. And when you get such an image, the normal thing that you would see. Let's imagine, this is one drug. All these are one drug and then another. So there would be on drug for each column. If you look here, this is the lowest concentration for this drug, this is the highest. So if we look here, growth up to here and here no growth. This would be our MIC for this drug. And it's quite clear. If it looks like this, sometimes it looks like this, you have good growth and it gets smaller and smaller. If this is a bacteriacidal drug you will go all up to here and think that the MIC is here. Sometimes you have something like this. There's growth and then there's a jump and then there's growth again. This should not happen. This is something that you would think that you should repeat the test, especially if there's more than one jump. Because here this could be a contamination with another bacteria or it could be something that is not really right with the plate. Here is almost the same situation, just one time. If it's just one time that it happens in the whole plate, just one jump. It could be acceptable in some citations, but then you would, in principle, read the MIC here, and pay attention if this happens more often. Here again, there's a good growth, but then there's this tiny spot, but also here this is covering. This tiny spot here. If this is an antibiotic like sulfonamides or trimethoprim, the MIC would be here because this tiny spot is this bacteria's static growth that leaves the bacteria to grow a little bit. And in this case, it's just a case where the range of antimicrobials used is all the way above what the bacteria can tolerate. So we don't know if MIC is here. We would say that it's equal or lower than this. And here, if there's growth all the way up we don't know if the MIC is here but, actually it's not here because otherwise there wouldn't be no growth here. It's certainly above this. So it's above the highest concentration tested. And that's how we read it. If we just illustrate this in a tube, it would be similar. This is growth, this is our MIC, and this is our growth control that we can compare. So again, an overview about the broth microdilution and here we're just calling it microdilution, or macrodilution, to distinguish. It's the same process, it's broth dilution. It's just that the size is low. And if I just go be here we say well more than one milliliter is macro, and milo hundred or around that volume. That's what you use in this panels. Is a micro. But we start with the colonies, we adjust this inoculum. We diluted our broth. We put on the panels. We incubate, and the next day we wait. And again, a similar. A say, where we have now a more real life panel looks like. The girls controls, that's the first thing we should look at. We also should look like if something looks fuzzy, because it could be a contamination, or not. We don't know. And then we look, well MIC readings, where are they? If we took this, well it's probably the two, sorry. If we take this, there is this one jump that we have to pay attention. If there's more in the plate, here because there's two jumps we actually consider really repeating. In this one we're still in doubt if we should repeat. In this one we are sure that we definitely it should repeat, and this one in principle it's, the MIC is, actually it should be a four, and not sure why we have written in eight? There's no dot and in here, dot not really growth visible that means that it's below or equal this one. Here, where there's all the way up growth, that's above the 64 that is here. And this one, because it's fuzzy and strange. It's not the typical as the growth here. We would think that might be a contamination there. So we would maybe try to repeat this test. So again, when we do this it's not 100% that we do it always right. There could be some errors. And here we have eliminated the errors in the medium or the areas in the panels if we have bought them. If we buy them from a commercial source, then that quality control is not in our hands. That's something we can do in our laboratories. To avoid having all these validations and all these things in our hands is to acquire something that is already quality controlled. But we still have the cultures that could be contaminated. The medium composition as I mentioned could be avoided by having the medium from another source. If we insist in doing it ourselves, then we have to check all these things. And the volumes and the inoculums and so on that we need to check. One thing that is really good to do for all the process, not only for the agard dilution or the broth dilution is to check, well when we do this adjustment with the nephelometer when we make this we are expecting a certain number of bacteria in per milliliter. We can check this once in a while to make a real count to see if we are in the right range. But as I mentioned before, we can standardize some of it by buying it from commercial source. And the rest we should do it by ourselves by quality control procedures and checking the results. So when we actually get the results, we get the MIC or this below, or equal or above a certain MIC and we can use the tables to check the interpretation either in this resistance intermediate susceptible, or the wild-type non wild-type, and we can check that this data is useful [COUGH] to be used in the future. Also to recheck the quality, the interpretative criteria again if they have changed or to follow the resistance levels or to use it for the patients. Of course, also for research because we are going to look at these patterns of resistance and think, well, this thing could have this mechanism. That is very important. And that's how we follow some of the superbugs that are around that we know they have certain patterns. So, thank you very much for this presentation. I hope to see you for the next lectures. [MUSIC]
