 As-Salaam-Alaikum students, my name is Ahmad As-Salaam Khan and I am going to teach you about chromosomal preparations. This is a very important lecture and this lecture or practical whatever you say is about preparation of chromosomes. This practical is part of our course title essential of genetics. The course code is Bio 301. Students as you know chromosomes are one of the most important genomic structure in ourselves. Why they are important? They are important because they are the unit of inheritance. Not only your gene is unit of inheritance but it has the chromosomes. These are the chromosomes which are transferred from one generation to other generation from one cell to other cell. So what we are? We are because of the genetic material. This is the genetic material which is transferred from our parents to us in the form of chromosomes. Not only this, our whole body is consisted of cells and these cells maintain their shape, their structure, their function because of the genetic material which is contained in the chromosomes because all the information about the cells is written in the chromosomes, in the genes which are in the chromosomes. So you can ask me the question that why we want to prepare chromosomes? The reason is that chromosomes tell us about many diseases. For instance we have a very very dangerous or lethal disease which leads to the abortion of a baby that is the deletion of part of a chromosome. Similarly there are certain problems which leads to the mental retardation in children. For instance trisomy 21 in which we have one extra chromosome the chromosome number 21 which leads to Down syndrome. The Down syndrome babies are mentally retarded, they are uncreatable. So the second aspect of chromosomes is how they help us to understand the basic biology. You know every species have a very specific number of chromosomes. I have 46 chromosomes being a homo sapiens, being a member of the homo sapiens family. Similarly the chimpanzee has 48 chromosomes and so on. So in this way the chromosome, the study of chromosomes help us to understand the evolutionary aspects also. So that's why I say both medically and also for basic science research chromosome study is very important and for that you have to prepare the chromosome. Ok guys let's now move to our practical straight away. So one of the most important thing you should always remember whenever you are going to conduct any experiment is just keep in mind that all the things you are going to use in this experiment are arranged in a really well mannered way so that no mess is created afterwards. So I have all these things which I will be using from now onwards. Ok now let us now add some medium into the falcon tube. This is RPMI 1640, this is the medium. 5 ml will be enough. So just keep in mind that you know the functions of each and every chemical you are using in your experiment. It's very important. So RPMI this medium is actually the growth medium. Without this medium the cells will not grow. At the end of the day what you want, you want the cells to grow. So that's why we are using this medium and now we will add your cells which are in your blood. We will take around 400 to 500 microlitre is enough so we take it and we add it into the medium. Just add 100 microlitre of PHA. PHA is an abbreviation of phytohemaglutinin. PHA is a stimulant. You know your cells, your blood cells they are not dividing at the moment. They are differentiated cells. So to keep them dividing, to make them dividing, we have to add some stimulant and PHA is their stimulant. So now I am going to add 100 microlitre of phytocarp serum or FCS. FCS also contains growth factors which are very helpful for the overall growth of the cells because at the end of the day what we want, we want the cells to divide and to divide in their healthy form. Next I am going to add 100 microlitre of an antibiotic. This antibiotic is Gentromycin. You can also add Steptomycin penicillin. The main thing is that you are adding an antibiotic which prevents cells to get contaminated. This is a very important part of your practical, especially practical which involves culturing of cells that the cells should not get contaminated. This is very very important. Now we have added PHA, we have added penicillin, Steptomycin and we have added CFCS that is the phytocarp serum and now we have the cells in our medium which contains all the important chemicals. I am going to just gently mix it. Okay guys now we will culture these cells in the incubator to let them grow for 72 hours. So they will be incubated into the incubator for next 72 hours. So guys as you can see these are the well grown cells because they have been incubated for 72 hours. Now we will centrifuge these cells so that we get a distinct pallet because this is the pallet which contains your cells, your lymphocytes, your white blood cells which contains the chromosomes. So we will centrifuge it. This is a centrifugation machine. You know about it. It is used to centrifuge whatever you put into it. As a result of the centrifugation you will get two or three layers depending upon whatever materials you are putting into it. One layer, the lower most layer is known as the pallet, the upper layers are known as the suponatin. So we close the lid. So we will centrifuge these cells at 2000 rpm for three minutes. So the centrifugation is complete. I will now open the lid and you can see now the pallet is very well ready, it's very distinct. We will now discard this suponatin and make use of the pallet for further processing. Now you have to be very gentle because your pallet shouldn't be disturbed. Keep around 500 microlitre to 1 milliliter suponatin above the pallet. So now we have around 1 ml 700 microlitre suponatin above the pallet. Now a very important step. We are going to add potassium chloride, 0.56 percent potassium chloride in these cells. This treatment is known as hypotonic treatment because when we treat the cells with potassium chloride what will happen actually the cells will swell. Why we want the swelling of the cells? We want it because in the swollen cells the metaphase will be very distinct, very clear and that is we want bigger cells in which metaphase is very clear because we want to visualize the chromosomes. Now you can see these are the cells after hypotonic treatment. Now we will put these cells in the centrifugation machine for further centrifugation. Again centrifugation will be for 3 minutes, 2000 rpm and for 3 minutes. Now the centrifugation is complete, let's open the lid. Now you can see this very distinct pallet. You will again discard the suponatin and treat these cells for fixation process. Again you have to be very gentle so the pallet is not disturbed. Again keep around 700 microlitre to 1 ml suponatin above the pallet. Now we will treat these cells with fixative for the fixation process. This is the fixative. This is composed of methanol and acetic acid in 3 into 1 ratio. That is 3 parts methanol and 1 part acetic acid. Why do we fix the cells? We fix the cells to keep them in a very stable state so that they don't get destabilized. The processes in them get fixed and they don't get decomposed. Now we will add around 5 ml of fixative. You can add it manually, you don't need pipette for adding. You only need pipette when you are taking out the suponatin. This is 5 ml, we have added the 5 ml fixative, keep in mind the fixative should be chilled so that the cells are completely fixed. Now mix it very gently. Now this is known as the first round of fixation. Just keep it in the refrigerator for 1 hour or so so that the fixation is very well complete and I will now put it in the refrigerator for 1 hour or so, around 1 hour to 2 hours more than enough. So this is the refrigerated medium containing our cells or the fixed cells in short. Now we will put these cells in the centrifugation machine again and we will centrifuge it. We will not only centrifuge the cells 1 time but 3-4 times so that at the end of the day we get a very clear pallet. So let us see that whether or not we have clear enough or transparent enough pallet. Yes students, you can see it's very well in the form we require, it's quite transparent and clear. Let's now move towards our next phase that is the preparation of the slides for pyro typing.