[Music] welcome back to microbiology lab my name is Kevin toka please make sure to like this video and subscribe to my channel for future videos and notifications in this video we're going to discuss the first biochemical test of the semester and that is the starch hydrolysis test also called the amylase test and every one of these tests that we're going to be looking at for the most part is gonna be detecting some protein or enzyme and the enzyme of interest here is actually amylase now some bacteria can perform what's called amylose hydrolysis or starch hydrolysis one form of starch is amylose but we'll just call it start i dra lysis and the way that they're actually able to do this is using an enzyme called amylase you may have seen this enzyme before if you've taken anatomy and physiology this is an enzyme that's present both in the small intestine and then also in a different form in the mouth it's actually an enzyme that's gonna break apart starch into individual monosaccharides and disaccharides and so here's the general reaction right here do you have starch which is a large complex carbohydrate it's a polysaccharide starch can be broken down by the enzyme amylase into monosaccharides specifically glucose and then disaccharides which would be just two glucose monomers linked together this is also a good opportunity to discuss how we write biochemical reactions so in a biochemical reaction we have a reaction arrow that typically goes from left to right and on the left side of that arrow we have what's called the reactants or the substrates on the right side of the arrow we have the products in this case our reactant or substrate is starch and our products are glucose which is the monosaccharide as indicated right here and the disaccharides and so the way we read this is some enzyme converts starch into these products glucose and disaccharides and the way that the reactants are converted to products is through an enzyme and typically enzymes are written either above or below this reaction air the convention I use is to always write it above the arrow and I'll normally color code the enzyme blue so if you're looking at a reaction arrow and there's something blue written above the arrow chances are it's an enzyme and I'll always indicate that also note that enzymes pretty much always with few exceptions end in ACE ASE all right and in this test amylase is going to be the enzyme that we're detecting the way that we actually run this test these starch hydrolysis tests is we have a plate here that's actually going to be coated in starch initially initially it looked very similar to a TSA plate but it's going to be coated in starch which happens to be the substrate for our enzyme amylase okay now if bacteria do not have amylase then they're not going to be able to convert starch into the products in other words they're not going to be able to break down starch okay so if we think about this logically if bacteria do not have the enzyme amylase then all the starch is going to be left on the plate because they're not going to be able to break that down however if bacteria do possess the enzyme amylase they're going to be able to break down the starch and so anywhere where the bacterial art that have the enzyme amylase you're not gonna have any starch okay and so the way we test to see if there's any starch is we use iodide and specifically we're going to be using grams iodine this is actually the iodine that we used in the Gram stain okay so we're gonna add grams iodine to the plate right now on this plate this is not how we do it specifically in the lab we'll look at that in a minute but here we have one species on the Left written as a and one species on the right written as B and when you pour iodine on the plate after you've incubated it and grown the bacteria anywhere where there's starch it turns purple okay so we see on the right side here pretty much all of this is purple for this species but over on the left side for a there's areas particularly you can see around the smear where there's no purple in fact you can see directly through the plate let's talk about what that means first we're gonna look on the right side and look at the these bacteria are actually amylase negative and what this means when we had the negative sign here that means that these bacteria whatever species it is does not have amylase so why is this all purple well remember if the bacteria do not have amylase they're not going to be able to break down starch so all the starch remains on the right side of the plate the starch is still there they were unable to break it down due to the lack of amylase and so when we pour the iodine on it iodine stains starch purple and so everywhere there's purple that means they're still starch so pretty much if you have a plate and there's no clearing or it's all purple that means you have an amylase negative organism so if you see all a purple left on the plate or it's a very dark purple or no clearing you have an amylase negative organism now in contrast on the left side we can clearly see that not all of this is purple in fact everywhere around the smear directly we actually see that there's no purple and we can see what's a clearing around the smear in fact if you hold this up to a light you can actually see directly through the plate these organisms on the left are amylase positive let's talk about the rationale for why that is if you have an amylase positive organism that means these organisms have the enzyme amylase they possess this enzyme therefore they are able to break down starch into the products but if they're able to break down starch that means that there's gonna be areas on this side of the plate where there's no starch and so when you pour the iodine on there it doesn't stain purple where there's no starch and so that creates what's called a zone of clearing that will manifest around the smear itself and so if you have an amylase positive organism the major thing you look for is whether or not there's clearing and if there is clearing it's Emily's positive now yes you can worry about the color the purple or what not but the easiest way to tell the difference between an amylase positive organisms such as on the left and an amylase negative organism on the right is in the positive case there's clearing around the smear in the negative case there's no clearing at all around the smear now when we do this test we're not gonna do one species on one half of the plate and a different one on the other side what way we're actually gonna do it is one species per plate and we did what's called a single streak the reason we do a single streak is because it's a lot easier to read whether or not those clearing around that smear because it's just a line and so we're only expecting if there's clearing that to be directly around this line okay so we already know we're gonna inoculate by single streak we're then gonna incubate the organism and when we take it out we're gonna pour grams iodine on it and remember if there's a zone of clearing you have an amylase positive organism so let's consider the right case over here first this is an amylase negative organism where this gray line represents the smear well when you have an amylase negative organism you'll be able to see the smear most likely when you pull it out and and put iodine on it but you'll be able to see no clearing zone around that streak okay around that smear the opes see nothing around it however if you have an amylase positive organism once you pour the iodine on there anywhere where there's no starch you're gonna see clearing and so more or less what you're gonna see is you'll see this streak where you actually put the bacteria that's the gray line right here but around that and it's not gonna be perfect like this but you'll actually see a zone of clearing and if you actually hold this plate up to the light you'll be able to see directly through this clearing zone and to be able to see what's on the other side okay and so that's pretty much all there is to the start hydrolysis test when you learn this you're gonna be focusing on the results mainly and the result is if you have clearing that's the key clearing you have an amylase positive organism if you do not have clearing you have an amylase negative organism and remember amylase is just the enzyme that breaks down starch also called amylose into monosaccharides and disaccharides all right hopefully this video gave you a good intuition of the starch hydrolysis test please make sure to LIKE this video and subscribe to my channel for future videos and notifications thank you very much