 In this video, we will perform antimicrobial susceptibility testing. It is used to treat the infections caused by bacteria by using different antimicrobial substances. There are three types of antimicrobial substances. Number one is the natural occurring substances. Number two is the synthetic derivatives of natural occurring substances. And number third is the chemical antimicrobial compounds. There are two techniques basically used for this test. Number one is the dilution technique. And number two is the diffusion technique. So, in generally, in most laboratories, this diffusion test is used. There are different materials required for this practical. Number one is the molar-hinton agar. This is a solidified form of the molar-hinton agar. So, prepared and steelized molar-hinton agar as per the instructions of the manufacturer. Number one is we take 38 grams of the molar-hinton agar in powdered form and dissolve in the one liter of distilled water. Then we will autoclave it at 121 degree centigrade, 15 pounds pressure for 15 minutes. So, after autoclaving, we will pour the molar-hinton agar into the patty plates. This is the disposable patty plates made of plastic. After pouring it, we will keep it straight for some time so that it does not solidify. After solidifying it, we will invert it. We will invert it because you can see here that its humidity has come. So, when the humidity increases, these droplets will fall on the agar. We will do the cultural interference and the results can be positive or negative. And you have to keep in mind that this layer is neither too thick nor too thin. If this layer is too thin, then the zone of inhibition will be too big and falsely large. If it is too thick, then the zone of inhibition will be falsely small. We use this agar because on this agar, all the microorganisms grow. For example, there are eucalysin fluza, astrophilococcus and streptococcus pyrogens. So, Naziria-Gloria, these bacteria will require a chocolate agar for them. But apart from that, all the bacteria grow on the molar-hinton agar. So, we want one because this is a non-differential media and one is a non-selective media. And there is a starch present in it. Some bacteria reduce toxins. What do toxins do? Toxins can be harmful for us. To kill toxins means that toxins do not interfere with our results. So, the starch absorbs those toxins and our results come to be accurate. That is why we use molar-hinton agar. And this is a loose agar. The benefit of this is that the antibiotics are easily diffused. The effect of this is dispersed in the agar and it becomes quite easy for us. And number two material is the antimicrobial discs. These are the antimicrobial discs used for this purpose. These discs are present in different concentrations for different bacteria. There are three types of bacteria. Number one is the gram-positive bacteria. Number two is the gram-negative bacteria. Number three is the acid-fast bacilli. So, these three bacteria are our skin, our layer of skin. So, there is a cell wall in this base. So, the difference between these three bacteria is that the gram-positive bacteria has a layer of more than 60% peptidoglycan in it. And the gram-negative bacteria has less than 10% peptidoglycan in it. And the acid-fast bacilli has a layer of more than 60% mycolic acid in it. So, we use different antibiotics for different bacteria. As per the recommendations of the doctors and clinicians, different bacteria, if the gram-positive bacteria has different antibiotics and antimicrobial substances, now the gram-negative bacteria has different antimicrobial substances. And if the acid-fast bacilli, like the mycobacterium tuberculosis, which causes TB, then it has different antibiotics. So, we use these different concentrations. And the number third material is, we have 0.5 McFarland standard. 0.5 McFarland standard is a turbidity standard. Basically, we use it because the turbidity in it is called gazla pan. So, along with the turbidity, we have to match our inoculum. Inoculum means that we will take sterile water or we will take an anonymous line. In that, we will make bacterial suspension. We will take one or two colonies of that bacterial suspension. Along with the 0.5 McFarland standard, we will match its turbidity. And after matching the turbidity in it, what will happen is that when we will do its learning, we will do its sticking on the molar intranagra of the inoculum, then the growth of it will be a uniform growth or a confluent growth. And that growth will be 1.5 into 10 is to power 8 CFU per amyl. The first step, we will tell you the way to make this 0.5 McFarland standard. Now we will prepare 0.5 McFarland standard. So, whenever we have to deal with any liquid, whether it is acid or any other chemical compound, we have to deal with liquid. We have to wear these goggles on our face to protect our eyes from the plastic. For 0.5 McFarland standard preparation, we will require 1% valium chloride solution. 1% valium chloride solution. And number second thing is the 1% sulfuric acid. So, we will take 99.4 ml of 1% sulfuric acid and add into 0.6 ml of 1% valium chloride solution. So, first of all we take, this is the flask. This is the glass flask. And now we will take mooring slender. This is the mooring slender to measure the liquid. We will take 99.4 ml of 1% sulfuric acid. Now add it in the flask. Now we take 0.6 ml of 1% valium chloride solution. 0.6 ml means we require 600 microlitre. So, for this purpose, we use blue colour micropipette. So, there are three types of micropipettes. Number one is the 0.1 microlitre to 10 microlitre. For this we use white tips. And these are the tips. For 0.1 microlitre to 10 microlitre, we require white tips. They are the small in size. Number second range is the from 10 microlitre to 200 microlitre or 100 microlitre. For this purpose, we require yellow colour pipette. And from 200 microlitre to 1000 microlitre, we require blue colour pipette. So, for this purpose, we require 600 microlitre. So, we use this blue colour pipette and blue colour tip. This blue colour pipette and blue colour tip. Take this tip to micro pipette like that. Press it one to two times. Then we take 1% valium chloride solution. As you see, we set this pipette on 600 microlitre as it is clearly mentioned on meter. First, press the micro pipette like that. And then insert the tip into the bottle. And pick up the 0.6 ml or 600 microlitre. 1% valium chloride solution. Add 1% valium chloride solution into the flask having 1% sulphuric acid. Then for discard this tip, we use discard box. Call it discard box. For discarding the tip, we use over thumb and press this button of the pipette. Now place the micro pipette on its base and discard box on its base. And now we will take 8 ml, cabbed test tube. Open it and add the 0.5 muc file and standard into it. And now cap the test tube. And place on the test tube back. Now we are going to perform main process. First step is that, using a sterile wire loop, touch 3 to 5 ml isolated colonies of similar appearance to the test organism. Emulsify in 3 to 4 ml of sterile physiological saline or Newton growth. Second step is that, in a good light, match the turbidity of the suspension to the turbidity standard which is known as 0.5 muc file and standard. When comparing turbidities, it is easier to view against a printed card or sheet of paper. Third step is that, using our sterile swap, inoculate a plate of Muller-Hinton agar, remove excess fluid by pressing and rotating this web against the side of the tube above the level of the suspension. Strict this web evenly over the surface of the medium in 3 directions, rotating the plate approximately 60 degrees to ensure even distribution. Fourth step is that, using sterile forceps, place the appropriate entry microbial disc evenly distributed on the inoculated plate. Not the precaution of this step. This disc should be almost 15 mm from the edge of the plate and no closer than about 25 mm from disc to disc. No more than 6 discs should be applied in 90 mm disc. Each disc should be lightly pressed down to ensure its contact with the agar. It should not be moved once in place. Now we'll incubate the plate for 16 to 18 hours in incubator at 37 degree centigrade. After 16 to 18 hours of incubation, we will take the plates and examine the zones of inhibition. After 16 to 18 hours of incubation, means after overnight incubation, we will check the results. Results could be of three types, resistant, intermediate and susceptible. Results could be of three types. We have resistant, means that antibiotic, which has no effect on bacteria. Intermediate means that antibiotic, which needs more quantity, means that its dose will have to be given to patients to kill this bacteria. And number third is the susceptible, which can be used in the dose against that bacteria. We have three types of results. This is the plate after 16 to 18 hours of incubation. After that we will take the plate out of the incubator and then we will scale it. We will keep the scale in such a way that the antibiotic disc will keep the scale in the middle and we will measure its zone. As we know, it is 10 mm in 1 cm. So from total 1 to 3 cm, the zone of inhibition is coming. So what we consider is that it is 30 mm. It is the zone of inhibition of 30 mm. After measuring the zone of inhibition, we have made a chart for clinicians with which the dose of that antibiotic and its zone of inhibition is compared and told that the antibiotic is effective or not. We will measure its zone with that standard chart and the patient will recommend that medicine to the doctor. As I told you, the zone of inhibition can come in this way. Number one is the resistant. This type is the resistant. You can see the standard and the size of the zone of inhibition. So this is the standard dose that will be beneficial for the patient.