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# Made for Spinning: The Centrifuge

##### Centrifuges – What Are They?

Technically, any device that applies centrifugal force to objects by rotating them around a central axis might be called a centrifuge. Centrifuges are devices that apply high-speed spinning to objects, liquid or gas samples, to separate their individual components. Centrifuges contain a rotor where tubes with the samples can be placed to spin, i.e., to be centrifuged. However, there is a much more common type of centrifuge in almost every house: washing machines with perforated drums are centrifuges that rotate clothes at high speed to remove the excess water. The spinning speed is expressed in rotations per minute (rpm), or in terms of relative centrifugal force (rcf). RCF is expressed as a multiple of the Earth’s acceleration due to gravity (g) so that a centrifuge running at 5 rcf applies a force of 5 g to the spinning samples (Sorenson BioScience Inc, 2012).

Centrifugal and centripetal forces are applied to objects in a circular motion (see Figure 1) (Lucas, 2019). Centripetal force is necessary to keep the object moving in a curved line and is directed towards the inside of the rotational axis, i.e., it’s a pulling force. The centrifugal force arises as a consequence of the first and is directed outwards the rotational axis, i.e., pushes the object away from the point-of-origin of the initial centripetal force (Lucas, 2019). If the first stops being applied to an object in rotational motion, the object will move in a straight line, as a result of the imbalance of forces (Figure 1 A).

Figure 1

Relationship between centrifugal and centripetal forces. (A) When a centripetal force is applied to the grey ball, it pulls it toward the center of the rotational axis. The centrifugal force arises from the inertia of the ball, i.e., the tendency to stay at rest or keep at uniform linear motion unless some external force is applied. (B) Another example of the application of centripetal and centrifugal forces. Here, the centripetal force corresponds to the tension between the children that keeps them together, rotating around the stick. When the tension is too high, the child in the outer side is projected in a straight line that is determined by the direction of the centrifugal force (image credit: PowerMasters, Motorsports Academy)

##### How to Choose The Best Centrifuge?
###### Laboratory centrifuges

Science laboratories, from academia to industry, usually have a myriad of different centrifuge types. The choice to use one type of centrifuge over another considers the volume of the liquid sample, the need (or not) for sample refrigeration and the degree of component separation that is expected. The last condition refers to the differences between the densities of the components of the mixture that are to be separated. The greater the density difference between the solvent and the solute, the faster the separation occurs. Finally, the separation of the components is influenced by the type of centrifuge rotor. Figure 2 summarizes the types of rotors commonly found in laboratory centrifuges, and how they influence the sedimentation of the sample in the tubes.

Figure 2

Centrifugal forces in different rotor types, and correspondent sedimentation pathlength (Mikkelsen & Cortón, 2004)

###### Microcentrifuges

Microcentrifuges (available for purchase on Amazon) are designed to accommodate samples with little volume – a few microliters to 1-2 ml (Figure 3). Routinely, they may spin at a maximum of 16000-18000g. At low speed, microcentrifuges can be used to process living organisms, with minimum damage. For example, to wash living cells and remove the old culture medium to replace with fresh medium. In this case, upon centrifugation, the pool of cells is displaced to the bottom of the tube and forms a pellet. The supernatant (i.e., the liquid) is then removed by aspiration, and the new medium is added to the tube to resuspend the cells, which are then transferred to a cell culture flask and allowed to grow in an appropriate incubator.

Figure 3

Types of microcentrifuges. (A) Refrigerated microcentrifuge for 1.5 – 2 ml tubes; (B) Mini centrifuge for 200 – 250 µl tubes (image credit: amazon.com)

Microcentrifuges come with adapters for different microtubes, like PCR tubes (of up to 250 µl volume) and tubes of 0.5 ml, 1.5 ml or 2 ml. It is also common for laboratories to have room temperature and refrigerated microcentrifuges, which are used when samples require cold temperatures. Living cells and protein extracts are examples of samples that must be almost always centrifuged at cold temperatures.

###### Benchtop centrifuges

Benchtop centrifuges (available for purchase on Amazon) are bigger than microcentrifuges and can also be refrigerated. They are suitable for the processing of samples with larger volumes, up to a few liters (Biocompare, 2019). Their spinning range can go from a few hundred g’s up to 5 000 g (Biocompare, 2019), and the applications of this type of centrifuge are similar to the ones of microcentrifuges, but with larger samples.

###### Vacuum centrifuges

Vacuum centrifuges (available for purchase on Amazon) are used to remove the solvent, i.e., to concentrate or dry samples, and usually have a side container to collect the liquid (Figure 4). Samples are spun under vacuum and the temperature can be increased to help to remove the solvent. In molecular biology, vacuum centrifuges are used to concentrate nucleic acids or proteins, in which case the centrifugation should be carried out at a low temperatures to avoid damage, such as denaturation, of the components.

Figure 4

A vacuum centrifuge is routinely used to concentrate/dry liquid samples (image credit: Fischer Scientific).

###### Refrigerated centrifuges

Refrigerated centrifuges (available for purchase on Amazon) are the best option for the centrifugation of temperature-sensitive samples, such as living cells, nucleic acids or proteins (Mikkelsen & Cortón, 2004). Classical microcentrifuges and benchtop centrifuges may have refrigeration capacity as low as -10ºC. However, specially designed refrigerated centrifuges can go as low as -40ºC to -50ºC. They are sold with a variety of adaptors so that they can accommodate a wide range of volumes, and their spinning speeds can go as high as 65000 g (high-speed centrifuges – Figure 5) (Biocompare, 2019; Labcompare, 2019).

Figure 5 High-speed refrigerated centrifuge

###### Multipurpose high-speed centrifuges

Multipurpose high-speed centrifuges (available for purchase on Amazon) offer a wide range of rotors and supports that accommodate distinct centrifuge tubes, from microtubes to bottles, and even microtiter plates. This type of centrifuge is ideal for large-volume, daily-working laboratories, as they allow to concentrate several applications into a single device.

Figure 6

Benchtop multipurpose centrifuge on the left, with the correspondent rotor, accommodating test tubes and microtiter plates, making it possible to centrifuge different sets of samples in one run (image credit: analytica-world.com)

###### Ultracentrifuges

Ultracentrifuges are designed to spin at exceptionally high speeds, being capable of generating an acceleration as high as 1 000 000 g. There are two types of ultracentrifuges: analytical and preparative, with important applications in the fields of biochemistry, molecular biology, and polymer science (Mikkelsen & Cortón, 2004).

###### Analytical ultracentrifuges

Analytical ultracentrifuges – accommodate high pure samples of up to 1 ml. They are equipped with an optical detection system to follow the sedimentation process in real-time. These centrifuges are used to determine the molecular weight, size, and shape of particles, to determine sedimentation coefficients, and to study chemical equilibrium between macromolecular species (Mikkelsen & Cortón, 2004; Wikipedia Contributors, 2019).

Figure 7 Analytical ultracentrifuge (image credit: Beckman Coulter)

###### Preparative ultracentrifuges

Preparative ultracentrifuges – are suitable for the separation of fine particulate fractions, through gradient separation centrifugation. To perform a gradient separation the sample tube is filled from top to bottom with an increasing concentration of a dense substance in solution. Each component will sediment in a part of the tube immediately above the gradient density that is superior to its own (Figure 8). Sucrose gradients are used to separate cellular organelles, such as mitochondria, nuclei, ribosomes, etc., and cesium salt gradients are used to separate nucleic acids (Dumetre & Darde, 2004; Perper, Zee, & Mickelson, 1968).

Figure 8

##### Centrifuge Tubes

There is currently on the market a wide range of centrifuge tubes, each designed for specific applications. Table 1 lists some of the routinely used centrifuge tubes and their respective applications.

Table 1

Types of centrifuge tubes and respective applications

 Microplate Clinical and research immunologyDNA isolation and precipitation; cell centrifugation applications. 250 µl microtubes and strips Applications requiring the use of a PCR machine (PCR, cloning ligation, DNA denaturation, cDNA synthesis, etc.) 1,5 – 2 ml microtubes All routine lab applications, for which reactions must be up to 1,5 – 2 ml. 15 ml test-tubespolypropylene Up to 12 000 rcfDNA isolation, purification, and precipitation of nucleic acids. Cell centrifugation applications 15 ml test-tubespolystyrene Up to 1800 rcfDNA isolation, purification, and precipitation of nucleic acids. Cell centrifugation applications Centrifuge bottles Ideal for cell harvesting or any application that requires large volumes Plasma Preparation Tubes (PPT™) Closed tubes containing EDTA (anticoagulant), to collect blood and inhibit clotting. Blood collection tubes with anticoagulants-sodium heparin or lithium heparin-acid citrate dextrose-sodium citrate(…) Blood collection for general blood analysis in the clinical. Tubes are closed, sterile systems, and the collection is performed with the help of an appropriate sterile needle, Blood collection tubes with anticoagulant and preservative agents Blood collection and prolonged storage of the sample before centrifugation. Sodium fluoride is commonly used as a preservative agent

##### Centrifuges: Be Careful With The Spinning!

In academia, industry, or hospitals, centrifuges are routinely used devices to spin samples and separate their contents. Despite being almost always operated by qualified technicians, centrifuges may be dangerous devices if not used properly. One of the key aspects to always pay attention to when using a centrifuge is to balance the distribution of the samples in the rotor. The combined weight of all samples must be symmetrically distributed. Figure 8 shows how to load samples in different rotors. Sample imbalance can lead to rotor and sample damage, due to increased vibration of the device, and in extreme cases, i.e., it can lead to a rotor crash.

Figure 8

Appropriate sample distribution in different different types of rotors. The weight of all samples is assumed to be the same (image credit: Eppendorf Handling Solutions).

Another important issue is to ensure that sample containers are not filled up to more than 80% of its volume, to avoid spilling the sample to the rotor. This is especially important when samples contain corrosive agents or pathogenic agents like viruses or bacteria. Following this idea, all dangerous samples that pose some form of biological hazard or health threat must be spun in air-proof, leak-proof containers, to avoid the formation of aerosols, that can contaminate the operator.

##### Centrifuges: Basic Devices to Help Research and Development

Thinking of centrifuges may not pop up all the amazing things they’ve brought us. If you work in a chemistry/biology laboratory, or in a hospital, centrifuges are probably not the stars of the place! However, their use helped to bring tremendous advances to scientific research and patient care. Centrifugation made it possible to separate individual cellular components, such as proteins, DNA, or individual organelles, allowing for their study and better comprehension of cellular biology. It is also used in water treatment centers, helping to improve water quality by removing contaminants. In hospitals, there are specially designed centrifuges for distinct applications, essential for sample preparation in diagnosis and clinical testing. There is no doubt today, that the invention of centrifuges and their development over the last decades, has helped to improve substantially our quality of life.

The main limitation of the use of centrifuges is, in general, their cost, as some devices may be very expensive, especially if high-speed, or ultra-speed centrifuges are required. Centrifuges also consume a lot of energy, due to the elevated g forces necessary for high-speed spinning. Finally, centrifuges can be very noisy, especially the older devices, and can cause bench vibration, possibly affecting other running experiments.

##### Final Words

Centrifuges are spinning devices routinely used in labs all over the world to separate the components of liquid samples. The capacity to isolate individual components of samples, such as the chemicals in solution, or cellular constituents like nucleic acids or proteins, is fundamental for advances in all fields of biology, chemistry, and many more. In hospitals, specialized centrifuges are key to processing samples, such as urine and blood, helping in patient diagnosis and treatment. Only qualified technicians must operate centrifuges, given the associated risks, mainly related to sample handling and loading of the centrifuge rotor. A myriad of centrifuges and accessories are available on the market, which are suitable for specific applications. Despite still being relatively expensive, there is no question of the fundamental value of centrifuges in research and clinical contexts.

##### References
1. (2019). Laboratory Centrifuges. Retrieved November, 2019, from https://www.biocompare.com/Lab-Equipment/Laboratory-Centrifuges/
2. Dumetre, A., & Darde, M. L. (2004). Purification of Toxoplasma gondii oocysts by cesium chloride gradient. J Microbiol Methods, 56(3), 427-430. doi: 10.1016/j.mimet.2003.11.020
3. (2019). Refrigerated Laboratory Centrifuge. from https://www.labcompare.com/General-Laboratory-Equipment/220-Refrigerated-Laboratory-Centrifuge/
4. Lucas, J. (2019, May 2019). What are centrifugal & Centripetal Forces. Live Science. from https://livescience.com/
5. Mikkelsen, S. R., & Cortón, E. (2004). Centrifugation Methods Bioanalytical Chemistry (pp. 247-267): John Wiley & Sons.
6. Perper, R. J., Zee, T. W., & Mickelson, M. M. (1968). Purification of lymphocytes and platelets by gradient centrifugation. J Lab Clin Med, 72(5), 842-848.
7. (2012). RMP vs RCF. from http://www.sorbio.com/index.php/rpm-vs-rcf
8. Wikipedia, & Contributors. (2019, 15 November 2019). Ultracentrifuge. Retrieved December, 2019, from https://en.wikipedia.org/w/index.php?title=Ultracentrifuge&oldid=92623441
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