Hi everyone and welcome to Miss Estrich Biology. In this video I'm going to go through cell fractionation and ultracentrifugation for A-level biology. So this falls under the methods of studying cells topic and this is so that you can study the internal structure or organelles of cells.
And it's through the use of microscopes which I've covered in a previous lesson which I'll link up here. as well as cell fractionation and ultracentrifugation that we've learnt the internal structures and functions of organelles. So cell fractionation, this is used to be able to remove and isolate individual organelles from the inside of a cell.
And that way they can be studied to learn more about their physical structure and also their function. So what has to happen is the cell is... broken open to release all of the organelles and then each organelle has to be separated out.
Now this whole process is done in solution but the solution has three special properties. It has to be cold, isotonic and buffered. So the reason it must be cold is once you break open the cell organelles which would previously be within the cell maybe in lysosomes you and within other structures are released and these enzymes might have the ability to damage the organelles. So we keep it in an ice-cold solution so that the enzyme activity is reduced and that should prevent damage to the organelles. The solution is also isotonic and that means it has the same water potential as all of the organelles of the cell.
And the reason for this is it stops osmosis occurring. So you won't have the organelles either shriveling if water was to move out or bursting if water was to move in. And finally, the solution is buffered.
And what that means is it's kept a constant pH and this will be neutral to prevent any damage to the organelles. Now, for each of these, I've underlined organelles because this is a common error that students make in exam questions. when explaining the reason that the solution must be cold, isotonic or buffered.
Because when you're talking about isotonic, usually in osmosis, you're referring to cells. Sometimes students write to prevent the cell bursting, but we've already burst open the cell. We are working with the organelles.
So that is why I've underlined organelles as a reminder for that. So let's have a look then at the two step process. of cell fractionation.
The first thing is we have to burst open the cell and that is what homogenization means. So this is when you break open or homogenize the cell and you basically just use a blender to do this. You will blend up the cells or whatever tissue is you're working with in that solution that we said would need to be cold, isotonic and buffered.
And in this example I've got spinach leaves blended up to break open all the cells. Next then we need to filter that solution to remove all of the large cell debris. So we can see here just filtering that solution and we're left with this liquid which will contain all of the organelles. So now we've broken open the cell and separated the organelles into this liquid, we now need to do ultra centrifugation and this is how we can separate out all of the individual organelle types.
So a centrifuge spins around at different speeds and as it spins it causes the organelles to separate out according to their densities. And that is how we can isolate each individual organelle type. So we do this through differential centrifugation. The centrifuge is spinning and that will generate centrifugal forces and those forces cause the organelles to form these pellets. and the organelles that are most dense will form the first pellet and move to the bottom.
And that's what we're seeing here in this diagram. We've got a mixture of all of the organelles in the solution, and we've spun it, first of all, at a low speed from one to two, and that will cause the most dense organelles to form a pellet and sink to the bottom. We then remove the liquid, which is called the supernatant.
and take out the pellets of that one type of organelle which can then be examined that supernatant liquid is then put back into the centrifuge and it is spun at a slightly faster speed and then the next most dense organelle forms a pellet at the bottom and we repeat this process over and over until all of the organelles have been separated and this is from going up in increasingly faster speeds each time. So the order that the organelles separate out we've said is due to their density. So the nucleus will be in the first pellet, then depending if you're working with plants it'd be the chloroplasts, if not the next one would be mitochondria, then the lysosomes, the endoplasmic reticulum and lastly the ribosomes.
So you could be asked how to separate out, let's say the chloroplasts from a plant cell. And you would have to go through the whole process that we've said, but you would also need to know that they are in the second pellet. So that would be a marking point, knowing that you'd spin it once and remove the pellet with the nuclei, then re-spin the leftover supernatant and the chloroplast would be in the second pellet.
So that is it. for self-fractionation and ultra-centrifugation. Hope you found it helpful. If you have, give this video a thumbs up.