 It is unsafe to operate a centrifuge while the rotor is in motion. Because of this, all modern centrifuges have a safety mechanism to prevent opening the lid while the centrifuge is in use. It also prevents the centrifuge from starting while the lid is open. This makes it impossible to see inside the centrifuge during its operation, so we have created an animation to show exactly what happens inside the centrifuge during a run. In this centrifuge, we have placed a swinging bucket rotor. If we go below the rotor and look into the lower part of the centrifuge, we see an electric drive motor. We can control the speed of this motor with the speed controller. As the motor turns, it turns an axle which we call the drive spindle. Any rotor we choose will mount on and attach to the spindle. The rotor is firmly attached to the drive spindle so that as the motor turns, the rotor will rotate at the same speed. The faster the rotor spins, the higher the g-force placed on the particles in their tubes or bottles. The amount of force generated also depends on how far the particles are from the center of rotation. The center of rotation is the center of the spindle. The farther away we move from the center, the greater the force at a given speed of rotation given in rpms or revolutions per minute. We call this distance the radius of rotation depicted by the small letter R. We consider the maximum radius to be at the bottom of the tube. We'll call this value r sub max. The value at the top of the tube we see is referred to as rmin for minimum force. And in the middle, we see r sub average for average force. It is good to consider all of these values, but unless otherwise stated, we'll consider g-forces to refer to the maximum radius as rmax for the center of our rotation to the bottom of our tube or bottle. This is a value that we use in our centrifugation force equation, where we have the g-force given as rcf or relative centrifugal force. This is equal to a constant 11.6 times r, our rmax, our radius of rotation given in centimeters times rpm divided by 1000 squared. For the purposes of this animation, we have chosen to do a differential centrifugation run with our particle samples contained in 50 milliliters centrifuge tubes. In their 50 mil centrifuge adapter placed in the bucket of a swinging bucket rotor. First, we will mount the rotor and its four buckets onto the spindle of the motor and get it firmly attached. We will then place the adapters for our 50 milliliters centrifuge tubes into the four bucket and we will then place the tubes containing our samples into these four buckets, noting that the samples are balanced by placing the samples symmetrically opposing each other. Before placing them, you would have confirmed that these tubes are carefully balanced. Now that we are ready to close the lid and start the centrifuge, we'll look at the controls. Here on the front, we see three main control knobs. The one on the left is speed control, which we will use to control the speed of rotation or the rotor. In this particular centrifuge, we can adjust it in terms of RPMs or RCF. The centrifuge recognizes the rotor and can do that computation or conversion for us. The middle knob is to control runtime and the third knob on the right adjust temperature. This particular centrifuge is refrigerated, so we can run from four degrees centigrade to ambient temperature. You often want to run samples that are heat sensitive in a refrigerated environment and also as rotors turn, they cause a fair amount of friction which increases temperature, so refrigeration will compensate for that heat so that the samples do not overheat. Beside that control knob, you see a button for start and a quick run, a feature that runs rotors very quickly. In this situation, it would go to max speed for that rotor and spin as long as you hold that button down. As soon as you release the button, it would decelerate. The button below to the right, the stop button would stop the run. You need to press this if for some reason something went wrong with the run or if you were not running in time mode. Now we look at the controls and displays on the left side of this centrifuge. You would still see runtime and speed display. We see the button for switching between RPM display and RCF display. If it's on RPM, it will show the speed. If it is in RCF, it will show G-forces that are being generated. The next button is for bucket selection. You use the RCF display with the swinging bucket rotor. Since there are a number of buckets which you can use, you need to select the bucket from a menu that shows up in a display to tell the centrifuge which bucket is actually mounted onto the rotor so that it may adjust the RCF value for that rotor in that particular bucket. On the extreme left, we see a display called deceleration profiles and a set key. Deceleration is how fast the rotor slows down. If you are running some cells that do not pellet very tightly or if you are running some type of gradient, you do not want the rotor to decelerate too rapidly and disturb those pellets or gradient lines that you created so you would want it set on a slow deceleration. If you have a pellet that packs tightly, you can decelerate more rapidly to save time. So this centrifuge has a number of profiles you can select using the set keys. Now that we have decided the size of tubes and mounted them in the centrifuge and set our run parameters in terms of speed we will spin them at and the time that we will allow the centrifuge to run, we push the start button and allow the rotor to start turning. The beauty of animation allows us to take a look at what is going on inside the centrifuge as the rotor turns. Here we see as the rotor starts turning the buckets go from their vertical position and swing out horizontally so that the tubes are going to be spun horizontally and the particles in the tubes will then also. The force will pull them horizontally to the bottom of that tube. In this run we see that our samples contain particles of different sizes and density. During the first stage of our differential run we will spin these at a slower speed to bring down the more dense particles and we can see that as this run progresses. Here we see the larger reddish brown particles being pulled to the bottom and they'll be forming what we call a pellet and these particles that are less dense are not moving as rapidly so they are sustained in suspension. We call this the supernatons. On this initial run the centrifuge will stop and we will collect the supernaton off of this run into a separate tube and load them back into the centrifuge and spin them at an increased force which will then centrifuge those particles. Here we started our second run at increased force and see that the particles which did not pellet in the first run are now being carried to the bottom of the tube to form a pellet at this point. So next we will see that some particles are still in the supernaton. We again collect those particles put them in a fresh tube and centrifuge them for a third run at a higher g-force yet to pelletize those. Here we see that we'll start a third run and the remaining particles will be pelleted for this run. So using the differential centrifugation in this demonstration we have collected three different densities of particles by increasing the g-forces with each successive centrifuge run. Now as we have indicated the swinging bucket rotor swings horizontally as it spins so that the forces are toward the bottom of the tube and the pellets form in the conical bottom. But there is a limitation on the speed at which we can spin a swinging bucket rotor without generating too much turbulence and friction. But commonly when higher speeds are required we'll use a fixed angle rotor. These can be spun at a much higher g-force because they are machined to a high tolerance of balance and they are solid low friction low resistance rotors. You can see from this animation that they do not pellet in the bottom of the tube but rather spin to the maximum point the r-max which is not the bottom. It's near the bottom on the side of the tube so that is something to watch for when you are spinning samples in a fixed angle rotor.