Centrifuge: Introduction, Principle, Types, Handling Procedure, Uses and

Centrifuge: Introduction, Principle, Types, Handling Procedure, Uses and Keynotes

Introduction of Centrifuge

Benjamin Robins (1707–1751), an English military engineer invented a whirling arm apparatus to determine drag. Later in 1864, Antonin Prandtl proposed the idea of a dairy centrifuge to separate cream from milk and his brother Alexander Prandtl became able to exhibit a working butter fat extraction machine in 1875. A centrifuge is a piece of equipment used to separate heavier particles from lighter ones by the action of centrifugal force. The first analytical ultra-centrifuge was developed by Svedberg in 1920. Biological centrifugation is a technique that uses centrifugal forces to separate and purify a mixture of biological particles in a liquid medium. It is a key method for isolating and analyzing the cells, sub-cellular fractions, supra-molecule complexes, and isolated macro-molecules such as proteins and nucleic acids.

Parts of Centrifuge

Motor: Electric motor is a part of the centrifuge which helps to drive.
Control Panel: The control panel placed on the front casing serves the purpose of controlling centrifuge operation.
Chamber: The entire system is housed within a chamber. The centrifuge head contains the cups or shields that cover the rotor and turns on a spindle. A safety shield in the chamber surrounds the rotors.
Rotor: Rotors in centrifuges are the motor devices that house the tubes with the samples. Centrifuge rotors are designed to generate rotation speed that can bring about the separation of components in a sample. There are three main types of rotors used in a centrifuge, which are: Fixed angle rotors,
Vertical rotors and Swinging bucket rotors/ Horizontal rotors. Fixed angle rotors-Tubes are held at an angle of 14 to 40°to the vertical. Particles move radially outwards and travel a short distance. Useful for differential centrifugation
Reorientation of the tube during acceleration and deceleration of the rotor. Vertical rotors-Held is vertically parallel to the rotor axis. Particles move short distances. The time of separation is shorter. The demerit of this, the pellet may fall back into the solution at end of centrifugation. Swinging bucket rotors/ Horizontal rotors- Sing out to horizontal position when rotor accelerates. A longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing the pellet. Normally used for density-gradient centrifugation.
Sample: That is to be separated.
Latch: Discontinue the use of any centrifuge that does not have a latch; the latch keeps the centrifuge lid closed in the event of tube breakage or other problems while the centrifuge is operating. All such units should be replaced or modified to include a latch; contact the manufacturer for information.
Interlocks: If available, purchase centrifuges that have lids with interlocks to prevent the user from opening the lid while the rotor is spinning.
Lid: Discontinue the use of any centrifuge that does not have a lid and does not retrofit the unit with a lid that does not have a latch.
Note Inner safety lids: When possible, purchase centrifuges that have inner safety lids for the buckets or rotor. If the units being purchased allow the outer lid to be opened while the rotor is spinning at low speeds, be sure they have protective inner lids.

Basic Principle of Centrifuge

The centrifuge works on the principle of gravity and the generation of the centrifugal force to sediment different fractions. The rate of sedimentation depends on the applied centrifugal field (G) being directed radially outwards G depends on-

Angular velocity (ω in radians/sec)
Radial distance (r in cm) of the particle from the axis of rotation
G = ω²r

Rate of sedimentation

It depends on factors other than centrifugal force like the mass of the particle, density and volume, the density of the medium, shape of the particle, and friction.

Sedimentation time

It depends on the following factors and they are-

Size of particle
The density difference between particle and medium
Radial distance from the axis of rotation to the liquid meniscus (rt)
Radial distance from the axis of rotation to the bottom of the tube(rb)
Revolutions Per Minute (RPM)

Relative Centrifugal Force (RCF) calculation

Using revolutions per minute (RPM) and the radius of the rotor (r), the RCF of a centrifuge can be calculated using the below formula. RCF is the measure of the strength of rotors of different types and sizes. This is the force exerted on the contents of the rotor as a result of the rotation. Relative centrifugal force is the perpendicular force acting on the sample that is always relative to the gravity of the earth. The RCF of the different centrifuges can be used for the comparison of rotors, allowing the selection of the best centrifuge for a particular function. The formula to calculate the RCF can be written as: RCF (g Force)= 1.118 × 10¯⁵ × r × (RPM)²
where r is the radius of the rotor (in centimeters), and RPM is the speed of the rotor in revolutions per minute.

Types of Centrifuges

Depending on the particular use, centrifuges differ in their overall design and size. A common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated. Types of centrifuge depend on the maximum speed of sedimentation, presence or absence of vacuum, temperature control refrigeration, the volume of sample and capacity of centrifugation tubes, etc.

Small Benchtop Centrifuge

It is very simple and small having a maximum speed of 3000 rpm. It does not have any temperature regulatory system and is used normally to collect rapidly sedimenting substances such as blood cells, yeast cells, or bulky precipitates of chemical reactions, common in the clinical laboratory for blood for plasma or serum, urine, and body fluids separation. It can take approx (up to) 100 tubes, depending on the diameter.

#Various types of Centrifuges Demonstration as shown below-

Microcentrifuge

Microcentrifuge is also called microfuge or Eppendorf centrifuge and it takes tubes of small volume (up to 2 ml i.e. microcentrifuge tubes). It is very common in medical laboratory sections like biochemistry, molecular biology, microbiology, Immunology/serology, blood banking, laboratory medicines, etc. It can generate forces up to ~15,000 x g with or without refrigeration.

# Micro Centrifuge demo as shown below-

https://www.youtube.com/watch?v=NuFqs9zOky8

High-Speed centrifuges

The maximum speed of 25000 rpm, provides 90000 g centrifugal forces. Equipped with refrigeration to remove heat generated. The temperature was maintained at 0-4 °C by means of the thermocouple. It is used to collect microorganisms, cell debris, cells, large cellular organelles, and precipitates of chemical reactions, and is also useful in isolating the sub-cellular organelles like nuclei, mitochondria, and lysosomes.

Ultra-centrifuge

It has the following properties-

65,000 RPM (100,000’s x g)
limited lifetime
Expensive
require special rotors
care in use – balance critical
research applications
The high speeds used in such devices generate considerable amounts of heat and therefore cooling arrangements and vacuum are required in ultracentrifuges Operate at speed of 75,000 rpm, providing a centrifugal force of 500,000 g. The rotor chamber is sealed and evacuated by the pump to attain a vacuum.
Refrigeration system (temperature 0-4°C).
The rotor chamber is always enclosed in a heavy armor plate.

Miscellaneous Types of Centrifuge

Hematocrit centrifuges: Hematocrit centrifuges are specialized centrifuges used for the determination of packed cell volume (PCV) or hematocrit (HCT) of erythrocytes in a given blood sample.

#PCV( packed cell volume) / HCT(hematocrit) determination by using Microhaematocrit Centrifuge as shown below-

Refrigerated centrifuges: They are centrifuges that are provided with temperature control ranging from -20°C to -30°C and an RCF of up to 60,000 x g that is ideal for the separation of various biological molecules.
Vacuum centrifuges: These are also called concentrators and utilize centrifugal force, vacuum, and heat to speed up the laboratory evaporation of samples. These centrifuges are capable of processing a large number of samples (up to 148 samples at a time).

Continuous flow centrifuges: They are is a rapid centrifuge that allows the centrifugation of large volumes of samples without affecting the sedimentation rates and they also have larger capacities which saves time as the sample doesn’t have to be loaded and unloaded over and over again like in traditional centrifuges. The high volume of samples i.e. up to 1 liter of samples can be centrifuged by this centrifuge at a time period of 4 hours or less.

Gas Centrifuges: They are used for the separation of gases based on their isotopes and based on the same principle of centrifugal force as all other centrifuges where the molecules are separated on the basis of their masses.
They are used mainly for the extraction and separation of uranium -235 and uranium 238.

Centrifugation

Centrifugation for isolation and purification of components is known as preparatory centrifugation, while that carried out with a desire for characterization is known as analytical centrifugation.

Preparative centrifugation

It is concerned with the actual isolation of biological material for subsequent biochemical investigations and is divided into two main techniques depending on the suspension medium in which separation occurs-

Homogenous medium – differential centrifugation
Density gradient medium–density gradient centrifugation

Differential centrifugation: Separation is achieved based on the size of particles in differential centrifugation. It is commonly used in simple pelleting and obtaining the partially pure separation of sub-cellular organelles and macromolecules. It is used for the study of the sub-cellular organelle, tissues, or cells (first disrupted to study internal content). During centrifugation, larger particles sediment faster than smaller ones. A series of progressive higher g-force generate partially purified organelles. In spite of its reduced yield differential centrifugation remains probably the most commonly used method for the isolation of intracellular organelle from tissue homogenates because of its; relative ease. It is convenient, time economy, and the drawback is its poor yield and the fact that the preparation obtained is never pure.

Density gradient centrifugation: It is the preferred method to purify subcellular organelles and macromolecules. A density gradient can be generated by placing layer after layer of gradient media such as sucrose in a tube, with the heaviest layer at the bottom and the lightest at the top. Classified into two categories:

Rate-zonal (size) separation and
Isopycnic (density) separation

Gradient materials used are Sucrose (66%, 50 C), Silica solution- Glycerol, CsCl, Cs Acetate, ficol (high molecular weight sucrose polymer, and epichlorohydrin), sorbitol, polyvinylpyrrolidone.

Rate zonal centrifugation: It is gradient centrifugation and takes advantage of particle size and mass instead of particle density for sedimentation. e.g. common application includes the separation of cellular organelle such as endosomes or proteins ( such as antibodies).

Isopycnic centrifugation: A particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of particles. e.g. separation of Nucleic acid in CsCl (Cesium chloride) gradient.

Analytical centrifugation

It has a speed of 70000 rpm and RCF up to 500000 g. Motor, rotor, chamber that is refrigerated and evacuated optical system. The optical system has a light absorption system, a schlieren system, and Rayleigh interferometric system with 2 cells – an analytical cell and a counterpoise cell. Optics used – schlieren optics or Rayleigh interference optics. In the beginning, the peak of the refractive index will be at the meniscus. With the progress of sedimentation, macro-molecules move down and peak shifts giving direct information about the sedimentation characteristics. It uses for checking the purity of macromolecules, the relative molecular mass of solute (within 5% SD), the change in relative molecular mass of supermolecular complexes, the conformational change of protein structure, and ligand-binding study.

Principles of Common Centrifuge operation

Centrifuge applies centrifugal force to separate suspended particles from a liquid or to separate liquids of different densities. These liquids can include body fluids (e.g. blood, serum, urine), commercial reagents, or combinations of the two with other additives. By creating forces many times greater than gravity, centrifuges can greatly accelerate separations that occur naturally as a result of density differences. In its simplest form, a centrifuge is a metal rotor with holes to accommodate vessels of liquid, spun at selected speeds by a motor. Low-speed centrifuges generally operate at up to 10,000 revolutions per minute (rpm) and may be non-refrigerated or refrigerated. High-speed centrifuges generally operate at 10,000 to 30,000 rpm and some are refrigerated to cool the rotor chamber.

Proper Handling Procedure

Ensure that the centrifuge tubes are properly balanced and that the speed and tube length is in accordance with the tube and the centrifuge manufacturer’s recommendations.
Do not use tubes that are not properly sized for the rotor. If using a swinging-bucket rotor, ensure that the tubes are placed in accordance with the manufacturer’s instructions; long tubes (e.g., greater than 100 mm) placed in the corner tube holders closest to the rotor shaft may break when the rotor buckets swing out.
For centrifuges with swinging-bucket rotors, fasten a protective inner safety lid (if available for your model centrifuge) onto the bucket; for those with fixed-angle rotors, fasten an inner safety lid to the rotor before centrifugation.
Ensure that the safety lid is properly sealed and positively locked into place.
Ensure that the rotor has completely stopped spinning before opening the lid, even if an “open lid” indicator lights and the safety interlock disengages. In some cases, the rotor may not be visible; therefore, the user should allow a reasonable amount of time for the rotor to stop and should feel the top of the lid for the cessation of vibration before opening it.
Never attempt to stop a moving rotor with your hands or with a tool or object (e.g., a paper towel).
All personnel should follow universal precautions when performing centrifugation and other functions that may expose workers to splashed blood or body fluids. These precautions include personal protective equipment like wearing gloves, facial protection (e.g., face shields), gowns or laboratory coats, and plastic aprons; these are described in detail in the Clinical and Laboratory Standards Institute (CLSI) tentative guideline and the Occupational Safety and Health Administration’s (OSHA) bloodborne pathogens standard.
Spillage and break of tubes should be considered the blood-borne pathogen hazard.
Cleanliness minimizes the possible spread of infections.
The speed of centrifuge should be checked once in 3 months while the timer to be checked per week

Uses of Centrifuge

In the clinical laboratory, a centrifuge is applicable for the following purposes and they are-

Remove cellular elements from blood to provide cell-free plasma or serum for analysis.
Remove chemically precipitated protein from an analytical specimen.
Separate protein-bound from free ligand in immunochemical and another assay.
Separation of the subcellular organelle, DNA, RNA.
Extract solutes in biological fluids from aqueous to organic solvents.
Separate lipid components.

Key Notes

After centrifuging, the liquid is called “supernatant“ and the solids at the bottom of the tube are called “pellet”.
The denser a biological structure, the faster it sediments in centrifugal force.
The more massive the biological Periclean, the faster it moves in a centrifugal field.
Dense the buffer system, the slower a particle moves.
Greater the friction coefficient, the slower a particle moves.
The greater the centrifugal force, the faster particle sediments.
The sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal.
Centrifugation is a technique that helps to separate mixtures by applying centrifugal force. A centrifuge is a device, generally driven by an electric motor, that puts an object, e.g., a rotor, in a rotational movement around a fixed axis.
Prior to switching the centrifuge on, one shall read carefully all sections of this instruction in order to ensure smooth operation and avoid damage to this device or its accessories, and in order to protect the centrifuge against unbalance, fill in the test tubes up to the same weight.
The heat buildup and convection problems caused within a centrifuge by air resistance can be avoided by spinning the rotor within an evacuated chamber.
Prolonged contact with some disinfectants (e.g., 10% sodium hypochlorite) may damage the rotor and other centrifuge components; be sure that such solutions are removed by rinsing the well with water.

Safety precautions during handling Centrifuge

The following points should take in mind during the operation of the centrifuge ( some points may vary according to the type of centrifuge)-

For safety reasons, inspections of the centrifuge are carried out by the authorized service at least once a year after the period of warranty. The reason for more frequent inspections could be a corrosion-inducing environment. Examinations should end with issuing a report of validation that checks on the technical state of the laboratory centrifuge. It is recommended to establish a document where every repair and review is registered. Both these documents should be stored in the place of use of the centrifuge.

Inspection procedure carried out by the operator

The user has to pay special attention to the fact that the centrifuge parts of key importance due to safety reasons are not damaged. This remark is specifically important for:

Centrifuge accessories and especially structural changes, corrosion, preliminary cracks, and abrasion of metal parts.
Inspection of the rotor assembly.
Inspection of bio seals of the buckets if such are used.
Control of execution of the guaranteed yearly technical inspection of the centrifuge
It is not allowed to lift or shift the centrifuge during operation and rest on it.
It is not allowed to stay in the safety zone within a 30 cm distance around the centrifuge nor leave within this zone some things, e.g. glass vessels.
It is not allowed to put any objects on the centrifuge.

Cover opening

The cover opening isn’t allowed to open the cover manually in the emergency procedure when the rotor is still turning

Rotor

The rotor is not allowed to use the rotors and round carriers with signs of corrosion or other mechanical defects.
It is not allowed to centrifuge highly corrosive substances which may cause material impairment and lower mechanical properties of the rotor and round carriers.
It isn’t allowed to use rotors and accessories not admitted by the manufacturer. Let to use commercial glass and plastic test tubes, which are destined for centrifuging in this laboratory centrifuge. One should absolutely not use poor-quality elements. Cracking of glass vessels and test tubes could result in dangerous vibration of the centrifuge.
It is not allowed to carry out centrifugation with the rotor caps taken off or not driven tight.

Use and maintenance

User(s): Laboratory technicians/ researchers
Maintenance: Biomedical engineering staff and/or service contract with the manufacturer or third-party organization
Training: Training by manufacturer and manuals

# Equipment and instruments most commonly used in Microbiology as shown below-

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