hello and welcome to learn a level biology for free with miss a stroke in this video we're going to be going through cell fractionation and ultracentrifugation as our second method of how you can study cells and organelles within them so grab cells from paper to make notes and have a go answering the questions as we go through so so far we've already gone through the first video on how to study cells and going through the importance of this that's how the structure and organelles of cells was discovered so the first video is our microscopes magnification calibration of the eyepiece graticule if you haven't had a chance to view that video yet I've linked it at the top here so you can just click to have a look at that video first and then it can come back to this one or alternatively carry on through this video and I'll link at the end as well so you can watch it next so this one is just going to be on cell fractionation and ultra certification so self fractionation this technique is used to break open cells so you can then isolate the different organelles and examine their structure and function so in a previous video we've gone through the different organelles that you'd expect to find in a eukaryotic cell and prokaryotic cell again links at the top and at the end so you can have a look at those videos the reason is possible to discover these structures and details and the function is in part due to the microscope studies but also it's through self fractionation which enabled scientists to isolate large numbers of each organelle to then study them so all of this process involves the cells and then the organelles afterwards being prepared in a solution and the solution has to be cold isotonic and buffered so pause the video at this stage to have a think about why once you break open the cell does the solution have to be those three things okay so cold this is because when you break open the cell you'll be releasing enzymes which aren't typically in contact with the organelles because previously they might be locked up within lysosomes or other parts of the cell and some of those enzymes could damaged organelles and then that would prevent you from being able to study them so by producing the cold solution for the study that will reduce enzyme activity to prevent any enzymes from damaging the organelles isotonic now this term means that the water potential of a solution is the same as the cell but in this case we're not using cells you've already broken them open so it's the water potential is the same as the organelles and the reason for this is that prevents osmosis so that means you won't have any water leaving causing the organelle to shrivel and you won't have any excess water entry and by osmosis causing the organelles to burst and if either of those occurred you wouldn't be able to study the structure and function of the organelle properly now I've underlined organelles because this is a common error that students make in exams link to explaining why it has to be isotonic and cell fractionation the most common mistake is students say to prevent the cell from shriveling or bursting because that's the terminology they used to form osmosis but the cells already been broken open so it can't triple or burst it's already been broken it's specifically the organelles preventing them from shriveling or busting so lastly it has to be buffered and this is to make sure you don't have any sudden changes in pH either too acidic to our climb which again could damage the organelles preventing them from being able to be studied so cell fractionation is a two-step process step number one is homogeneous Asian step to alter centrifugation her modernization is how the cells are broken open or homogenized and this is done using a blender so the cells that you're going to be examining would be placed into the blender along with the solution which is isotonic buffered and cold to prevent damage in the organelles now in this case I've just got spinach so you can isolate the organelles from plant tissue once you've then blended it you need to filter your solution to remove the large cell debris and that will then just leave you with this filtrate which contains the organelles so we've now got all the organelles removed from the inside of the cell the next step is step to ultra centrifugation and this is how you can then separate the organelles in that filtrate so the filtrate will get placed in a tube and put into a centrifuge and this is a machine which spins around at very very high speeds and this creates what we call centrifugal forces which causes the more dense organelle to separate and move to the bottom and in this way you can then separate out the organelles it's actually done as differential centrifugation meaning you do it repeatedly at different speeds and it's increasing speeds so this first but here is just talking about what I was saying on slide before the centrifuge spins at high speed and those centrifugal forces cause pellets which is what we call the solid part that moves the bottom pellets that contain the most dense organelle to me to the bottom what you're then able to do is remove the liquid from the top and we call the liquid the supernatant remove that into a second tube and then you're just left with the pellets which will contain all of the same organelle which you can then go in examine so your second chip with that supernatant you then spin again but a slightly faster speed and this will then cause the second most dense organelle to move to the bottom and then you repeat the process remove the supernatant and you're left with the pellet which contains nextdoor canal you do this over and over increasingly faster speeds until you've isolated all the organelles and just to show you then the order that you'd expect to remove and isolate those organelles the nuclei would be the first to separate because they are the most dense so they require the slowest speed and they would be in the first pellet then go again increasingly increasing the speed and the next most dense with the chloroplasts and mitochondria so they've been your second pellet your third pellet would be your lysosomes and the endoplasmic reticulum and your final pellet would be the ribosomes and you are expected to know that order so you could be asked in an exam question scribe how you'd isolates the chloroplasts from the filtrate and what they'd be after is that you know you increase the speed each time and the chloroplasts are in the second pellet so in summary cell fractionation is a way to isolate organelles to enable their structure and function to be studied it's a two-stage process so the first step is the cell has to be homogenized which means to be broken open and then you have ultra centrifugation and this is when you spin the filtrate at high speed to separate out the organelles according to their density all of this process needs to have the cells which are then broken opens and then it's just in your canals prepared in a cold isotonic and buffered solution and this is to make sure that those organelles don't get damaged lastly differential ultracentrifugation is when the organelles are separated according to their density the less density organelle the faster the speed needed to separate the organelle so that then concludes the two videos on methods to study cells the first one was on microscopes and this one's gone through the cell fractionation and ultracentrifugation so for practice questions and to test yourself just go along to Miss Esther accom for questions on that and it said link to the previous videos which you can have a look at to help you with this topic so click any of the links to see the previous videos and if you haven't already subscribed make sure to click the symbol just here to subscribe to keep up to date with all the latest videos