hello and welcome to miss estrich biology in this video we're going to go through the mitosis root tip squash required practical if you are new here then click subscribe so you don't miss out on any of the latest videos so this practical is the one where you have to prepare a stained squash of cells and then work out the mitotic index and what we're going to go through is the equipment method risk assessment how you then work out the mitotic index and the typical exam questions you get linked to this required practical so let's start with the equipment here we have a range of equipment that you would need to use for this experiment i'm going to go through the pieces of equipment in particular which often come up in exam questions that you have to explain why you use that piece of equipment the rest of it though you can pause and write it down if you need it to write up your experiment so it's these four which typically come up why do you use the hydrochloric acid now this step we add our root tip to hydrochloric acid leave it for 10 minutes and that is so that acid can soften the root tissues so later on when we squash them they are soft enough for that to happen we use a stain and i'm using acetic or scene in my example and the acetic or scene or acetoacetin there and that stain as you can see is a dark reddy purple color and that will stain the chromosomes in the nucleus so they become visible under the microscope when you lower down your cover slip you use a mounted needle the reason for that is as you lower it down gently it prevents any air bubbles coming underneath the cover slip which would affect your ability to view the slide and lastly why do you use the root tip of and it's an onion or garlic but the key thing is the root tip and it's just last five millimeters that is the growing region of the root so that means it'll be in that section that cells will be dividing and therefore will be undergoing mitosis and that's what we need if we want to view cells in mitosis so let's have a look at the method the first thing you need to do and if you're at school the technician would have done this stage for you is grow your garlic or onion on top of a beaker of water for about three days and that can just be a regular onion regular garlic clove and after three days in water these new roots will start to grow and that is what you need for the experiment so once you have these roots then you can do the experiment and you use a scalpel to cut off the last five millimeters and that's what we can see here with this lovely ruler that one of the sixth formers was using when i took this photo and the last five millimeters as we said that's where mitosis is happening and you need to be really careful when you're using the scalpel because it's very sharp so you cut away from you to be safe once we've done that we then add our root tip into a test tube with hydrochloric acid and we also put it into a water bath at 60 degrees c we'll leave that for 10 minutes and this is the stage that we said the acid and with that heat it will start to soften the cells now that they've been left for 10 minutes and they're softened you then pour off the acid and you rinse your root tip in distilled water just to get rid of that acid we can then transfer using forceps the root tip to the microscope slide and any of that distilled water that happens to still be on the slide we need to remove so we put filter paper just next to it and that will soak up any excess water on your slide now we're ready to stain our root tip and you add just two small drops of acetic or seam if you have too much then again you can add some filter paper next to it and it'll absorb the excess stain because you don't want so much that will then spill over the edges you then use your mounted needle to carefully lower down that glass cover slip so that you'll end up with no air bubbles now when you leave it for 10 minutes the liquid will spread across that whole cover slip but if you do have any areas where the stain hasn't transferred to you can just put filter paper at the edges and that will draw the liquid along the main thing is that during those 10 minutes the stain is covering our root tip because we want the chromosomes inside of the nuclei to become stained over that 10 minute period then the very last thing is we need to squash which is what the title of the experiment is root tip squash so you place a bit of filter paper on top and you push down but you need to do this gently if you do it too hard you're going to break the glass cover slip you also need to make sure that you don't accidentally slip and move your cover slip to the side because that defeats the purpose of the squash step this stage is to squash down gently so you get this really thin layer of cells so the light can then pass through them when you're viewing it under the microscope if you slide the cover slip you'll end up putting a layer of cells on top of another layer and the light won't pass through and you won't be able to focus your image you might also actually damage some of the cells and the chromosomes so it ruins your image so that's how we prepare our slide the risk assessment i've picked out two key examples here number one the hazard is the scalpel the risk is you could cut yourself how do you prevent this will we cut onto a white tile so that we're less likely to slip but also cut away from you don't ever cut towards you in case you do slip the hydrochloric acid can be another hazard the risk is that it's an irritant if you have a stronger concentration then it could be corrosive as well the prevention then to making sure you don't get any harm is wear goggles to protect your eyes if you do get any on your hands wash your hands now there are other things you could mention the mounted needle is pretty sharp so that could be another hazard and the risk there could be stabbing yourself um the prevention here is just making sure you're taking care that you are holding it from the wooden ends not the metal end and being very careful when you're moving around with that needle so the last bit then is our observations and i've actually taken this photo down the microscope looking at one of the slides that my students made and what you then look at is for any cells that you can visibly see are in one of the stages of mitosis and i'm going to zoom this in further and we can see here this cell is in anaphase we can see that the chromosomes have been um separated we've got the chromatids i've been pulled to either end of the cell this one here i've highlighted possibly that is in um metaphase with the cells lining up in the middle of the equator the rest of them they don't really look like they are actually in any of the stages of mitosis because we can't see any visible chromosomes so possibly they're all in interphase and unfortunately that happens a lot with this practical if you've done it already then you probably found that you only saw one cell or very very few and unfortunately often is that way which is why you should be given maybe a prepared slide and then you can do the mitotic index from a pre-prepared slide where you did have lots of cells in mitosis so the mitotic index is a bit like a percentage where you're working out what percentage of cells are currently undergoing mitosis and if we have a look it's the number of cells in mitosis divided by the total number of cells times 100. so essentially it's the percentage of cells that are um undergoing mitosis currently so the typical exam questions you get linked to this required practical would be number one two and three are linked to the method so why do you use only the first five millimeters from the root tip of an onion it could be a garlic why do you press down firmly or in our case we said it's actually quite gently but pressed down is the key thing and why do you use a stain now if you want to have a go at these pause the video have a go but i'm going to go straight through now answering those questions so the first three then why do we use only the first five millimeters that is because that is the region where you'll find cells are undergoing mitosis and that's what we want to view under the microscope we press down to make sure you get a thin layer of cells so that light can pass through and the key thing is that light can pass through that would need to be said to get the mark you can't just say so you get a thin layer of cells why do we use a stain that is to stain the chromosomes to make sure they are visible so then if we have a look at an example of the mitotic index um there's 32 cells in this image calculate the mitotic index so they've told us already there's 32 so you don't have to spend lots of time counting every single individual cell and instead you're just looking to identify which cells that are visible are currently in a stage of mitosis and having a look it's just these three which all happen to be in anaphase those chromatids have been pulled to opposite poles of the cell so that's 3 divided by 32 times 100 which is 9.38 so lastly when counting the cells to calculate the mitotic index what should you do to ensure your count is accurate so for this one examine a large number of fields of views fields of view or many cells and that is to make sure you have a representative sample so what we mean by that is this here is just looking at a small section of cells it's one field of view which means you're just looking at one section under the microscope what you should really do is count and calculate it for that field of view move your slide over and do the same thing for another field of view and do that at least 10 times and then you can work out what was the average mitotic index and that means it'll be more representative of the entire root tip which is what your sample is so that's kind of linking to sampling skills but it's sampling in terms of um recording and counting the cells under the microscope so that is it for your mitosis root tip squash practical if you found it helpful please give it a thumbs up and if you've got any questions add them in the comments [Music]