LABORATORY CENTRIFUGE

Principle

A centrifuge is a device that accelerates gravitational separation of substances that differ significantly in their masses.

Components

Centrifuges contain following components:

1. A rotor or centrifuge head
2. A drive shaft
3. Motor
4. Hanging buckets
5. Power switch
6. Timer
7. Speed/gravity control
8. Tachometer
9. Brake
10. Protective shield to minimise aerosol
11. Safety lock

Uses

It separates particulate materials from a solution in which they are suspended. For example:

• Removing cellular elements from blood to provide cell free plasma or serum for analysis.
• Concentration of cellular elements and other components of biological fluids for 21 microscopic examination or chemical analysis.
• Elimination of chemically precipitated proteins from an analytical specimen.
• Separating protein bound or antibody bound legend from free legend in immunochemical or other assays.

Separate two liquid phases of different densities.

• Extracting solutes in biological fluids from aqueous to organic solvents.
• Separating lipid components, e.g., chylomicrons from other components of plasma or serum and lipoproteins.

Types of centrifuge

Centrifuges generally may be classified into following types:

1. Horizontal head or swinging buckets type:

It allows the tubes, placed in the cups of the rotor, to assume a horizontal plane when the rotor is in motion and a vertical position when it is at rest. During centrifugation, particles travel in a constant manner along the tube while the tube is at right angles to the shaft of the centrifuge. Thus, the sediment is distributed uniformly against the bottom of the tube. The surface of the sediment is flat. Supernatant liquid is simply removed by a pipette with negligible disturbance of the packed sediment. It is ideal for separation of erythrocytes from plasma or of a protein precipitate from a solution.

2. Fixed angle or angle head:

Tubes are held in a fixed position at angles from 25-40° to the vertical axis of rotation. Particles are driven outward horizontally but strike the side of the tube so that the sediment packs against the side and bottom of the tube with the surface of the sediment paralleled to the shaft of the centrifuge. As the rotor slows down and stops, gravity causes the sediment to slide down the tube and usually a poorly packed pellet is formed. It allows more rapid sedimentation of small particles as the fixed angle rotors can be run at a higher speed.

3. Axial type:

It is a centrifugal concept that allows tubes of blood to be spun in a vertical orientation.

4. Ultracentrifuge:

They are very high-speed centrifuges that usually use fixed head rotors. Mostly used in the separation of lipoproteins and ultra-microscopic particles. As considerable heat is generated during their operation, as a result of friction, so they are always provided with a refrigerated chamber.

5. Special types:

There are some special types of centrifuges for specific purposes. Mechanically they fall under one of the above-mentioned types. The three most important types are:

• Immunofuge or Serofuge: This type of centrifuge is used in immunohaematology. It is a horizontal head centrifuge with a fixed tube size head and fixed speed. It is commonly used in blood bank for spinning down the red blood cells.
• Cytospin: This is a horizontal head centrifuge having fixed speed and time. It is provided with special devices in the swinging head, which allow the cells in fluid phase to settle down on a glass slide. Because of the slow speed morphology of the cells is not disturbed. It is used for cytology.
• Blood bag centrifuge: This is also a horizontal head centrifuge but is provided with large buckets to hold blood bags. This is used in preparation of blood components i.e. packed red cells, platelets and plasma etc.
• Gerber centrifuge: This is a special centrifuge. It can hold and spin the Gerber tube, a special glass tube for milk analysis.

Operation

1. Only those tubes that are recommended by the manufacturer of the centrifuge should be used. The tubes should have a tapered bottom, particularly if the supernatant is to be removed.
2. The rotor must be properly balanced. Specimen tubes should be placed on opposite pans of a balance and equalised in weight. The placement of the tubes should also be symmetrical. Tubes filled with water may also be used to equalise the weight. The total weight of each rack should not exceed the limit stated by the centrifuge manufacturer. Imbalance of the rotor causes vibration that may increase wear and tear in the centrifuge and more frequent breakage of the tubes.
3. The lid should then be closed and locked.
4. Required time for centrifugation should be adjusted with the timer knob.
5. The centrifuge should then be switched on and allowed to attain speed for required centrifugation force, which should be adjusted with speed/gravity knob.
6. Lid should not be opened until rotor has completely stopped.

Maintenance

1. Cleanliness of a centrifuge is important in minimizing the possible spread of infectious agents such as hepatitis viruses or mycobacteria. In case of breakage the racks and the chamber of the centrifuge must be carefully cleaned. Any spillage should be considered a biohazard and dealt accordingly.
2. Broken glass embedded in cushions of the tube holders may be a continuing cause of breakage if cushions are not inspected and replaced in the cleanup procedure.
3. The speed of a centrifuge should be checked at least once every 3 months, by stroboscopic light or a vibrating read external tachometer of known accuracy.
4. The centrifuge timer should be checked weekly against a reference timer such as stopwatch and the difference should not be more than 10%.
5. The temperature of a refrigerated centrifuge should be measured monthly under reproducible conditions and should be within 2°C of the expected temperature.
6. Commutators and brushes should be checked at least every 3 months. They should be replaced when they show considerable wear.