DEFINITION:

The process of separating lighter portion of colloidal solution, mixture, or suspension from the
heavier portion by centrifugal force of several thousands of gravities at low temperature in a vacuum, by
a special apparatus called Centrifuge Machine In a suspension of particles, particle sedimentation rate
depends on the nature of the particles.

CENTRIFUGE MACHINE:

In laboratory table top centrifuge machine is used. It has rotating unit called rotor, has fixed
holes drilled at an angle (to the vertical). Samples tubes are placed in these holes or slots and the motor
in spun. As the centrifugal force is in the horizontal planes and the tubes are fixed at an angle, the
particles have to travel only a little distance before they hit the wall of tube and then slide down at the
bottom. This angle rotor are very popular in the laboratory for routine use. This radial acceleration
causes the dense particles to settle at the bottom of the tube while the low-density substances rise to
the top.
Types of centrifugation:

1. Differential Centrifugation:
Centrifugation based on sedimentation coefficient of the substance under investigation;
applied to homogenate to derive various subcellular fractions.

2. Isopycnic Centrifugation:
In which the solvents in of same density as that substance to be isolated.

3. Density Gradient Centrifugation:

Ultracentrifugation in a liquid such cesium chloride solution. The density of which
increases along the line of centrifugal force, the substance under test or preparation
seeking their level of density.

4. Microscopic Centrifugation:
A high speed centrifuge with built in microscope, permitting the specimen to be viewed
(seen) under centrifugal force.

Aging: if the colloidal solution is allowed to stand undisturbed for a long time, the particles
settled down. This takes a long time and the precipitation will not be complete.
 In natural medium in which the particles are suspended and the force applied to the particles. One
important factor affecting the sedimentation of particles is viscosity. The rate at which a macromolecule
sediment is characterized by its sedimentation coefficient. This has been defined by Svedberg and
Pederson as the sedimentation velocity per unit of centrifugal field, commonly referred to as its
sedimentation coefficient, S.

The rate of sedimentation is dependent on the applied centrifugal field (G) being directed radially
outwards, determined by the square of the angular velocity of the rotor (ω, in radians s–1) and the
radial distance (r, in cms) of the particle from the axis of rotation. According to the equation, G = ω2r

Since one revolution of the rotor is equal to 2p radians, its angular velocity, in radians s–1, can be
expressed in terms of revolutions per minute (revmin–1).

TYPES OF CENTRIFUGE
Low-speed Centrifuge
These machines are used routinely for the initial processing of biological samples, have maximum speed
4000 to 6000 revmin–1 and have maximum relative centrifugal fields of 3000 to 7000 g. Applications
include rapid sedimentation of blood samples, and of synaptosomes.

High-speed Refrigerated Centrifuge

Maximum speed 25,000 revmin-1 and relative centrifugal field of about 60,000 g. The rotor can be of
fixed angle or swinging bucket type. This type of centrifuge is used to collect microorganisms, cellular
debris, large cellular organelles and proteins precipitated by ammonium sulphate.

Ultracentrifuge
They are subdivided into:

analytical ultracentrifuge

preparative ultracentrifuge.

Preparative ultracentrifuge can spin rotors to a maximum speed of 80,000 rpm and can produce
a relative centrifugal field of about 6,00,000 g. The rotor chamber is refrigerated, sealed and evacuated
to minimize excessive rotor temperature. Centrifuge tubes must be accurately balanced within 0.1 g of
each other. Applications include study of macromolecule/ligand binding kinetics, steroid hormone
receptor assays, separation of the major lipoprotein fractions from plasma, and deproteinization of
physiological fluids for amino acid analysis.

Analytical ultracentrifuge can spin rotors to a maximum speed of 70,000 rpm and can produce a
relative centrifugal field of about 5,00,000 g, and consist of motor. The rotor contained in a protective
armored
chamber that is refrigerated and evacuated. An optical system enable observation of sedimenting
material during centrifugation to determine concentration distribution in the sample at any time during
centrifugation.The optical system measures the difference in refractive index between the reference
solvent and the solution by the displacement of interference fringes caused by slits placed behind the
two liquid columns.

TYPES OF ROTOR
Preparative centrifuge rotors are four main types: swing-out (swing bucket), fixed angle, vertical and
zonal.

Swing-Out Rotors
In these rotors, the sample solutions in tubes are in individual buckets, which move out perpendicular to
the axis of rotation, as the rotor rotates. The centrifugal force is exerted along the axis of the tube in
these rotors. Since the centrifugal force is axial, some particles are sedimented against the wall of the
tubes.

Fixed Angle Rotors

In these rotors, the tubes are at fixed angle, varying from 14° to 20°. Rotors with shallow angles are
more efficient in pelleting because the sedimentation path length is shorter. Moreover, reorientation of
the solution in the tubes enhances the loading capacity of isopycnic gradients.

Vertical Rotors
The tubes are held in a vertical position. The diameter of the tube and their design enables them to
generate very high centrifugal forces. Vertical rotors are not suitable for pelleting, but can be used for
isopycnic centrifugation.

Zonal Rotors
Zonal rotors are of two types: batch type and continuous flow. Continuous flow type rotors are less
frequently

Zonal rotors can be used for separation of human blood cells, fractionation of membranes from a
rat liver, nuclear pellet, fractionation of tissue cultures cell, post-nuclear supernatant, and harvesting of
virus from tissue culture fluid.

Various types of rotor, their characteristics and applications

TYPES OF CENTRIFUGAL SEPARATION
Differential Pelleting

Differential centrifugation separates particles according to size and density. However, the centrifugal
force to pellet the larger particles from the top of the solution is also often sufficient to pellet the
smaller particles near the bottom of the tube. Hence, a pure preparation of the smallest particles cannot
be obtained in a single step.

Rate-zonal Centrifugation
The sample is layered as a narrow zone on the top of a density gradient and thereby, minimizes the
convection currents in the liquid column during centrifugation. This is ideal for the particles of defined
size (e.g. proteins, ribosomes and RNA). However, particles of the same type are often heterogeneous.
In such cases, particles can be separated by some other means, such as density.

Isopycnic Centrifugation
Particles are separated on the basis of density. Prolonged centrifugation does not affect the separation
as long

as the density gradient remains stable

PRINCIPLE OF CENTRIFUGATION:

The basic principle is that the more dense components of mixture migrate away from the axis of
the centrifuge while less dense components of the mixture migrate towards the axis.

Centrifugation is measured in revolution per minute (RPM). The greater the number of RPM, the
greater the force of gravity (g) (each gravitational force).

PROCEDURE:

1. Place the centrifuge machine on same leveled stable table.

2. Clean the rotor and container before operation.
3. Take the sample in small centrifuge tube.
4. Take equal volume of sample in test tube and arrange it symmetrically and balanced by placing
the test tube opposite to another so that the load is distributed if only one T.T. sample need
 centrifugation then balance it with equal volume of another T.T. containing distal water and
place
it on opposite side.

5. Switch on the machine and set the rotation per minute (RPM) and duration of centrifugation by
respective knob e.g. separation of serum from clotted blood needs 3000 rpm for 3 minutes.
While the preparation of urinary sediments for microscopic examination needs 1000 – 1500
rpm

for 3 minutes.

6. Cover the lid and machine automatically started, if lid is opened during working, machine
automatically stopped.

7. After finishing work, adjust speed knob to the lowest position and turn timer knob to zero and
switch off the machine.

8. Do’not open the lid until rotor becomes stationary.

After centrifugation the protein, cell and other large particles settle down and clear
supernatants is

produced. Which can be taken out with the help of adjustable / manual pipette.

In urine sample, discard the supernatant takes the sediment, place on glass slide and cover with
cover slip and seen under microscope for casts, cells and crystals.

DISADVANTAGES:

If centrifugation is done at high speed, then required, the shape of cells are destroyed so that
microscopic examination cannot be done properly.

DEFINITION:

Plasma:

The fluid portion of the blood in which the particulate components are suspended.
Fibrined is present.

Serum:

The clear portion of any body fluid.

 Blood Serum:

The clear liquid that separates from the blood when it is allowed to clot completely.

APPLICATION OF CENTRIFUGATION:

It has various applications like in food industry, water treatment, to isolate and determination
the biological properties and function of sub-cellular organelles and large molecules, to find the effect of
centrifugal force on cell, developing embryos and protozoa.

CLINICAL APPLICATION:

In medical or clinical laboratory, it is used to separate plasma from heavier blood cells.

 To separate serum from clotted blood for determination of serum creatinine. S. uric acid and
serum electrolytes etc.
 To prepare the urinary sediments for microscopic examination.
 OSPE OF CENTRIFUGATION

2ND Year MBBS

Q.No.1. Define centrifugation.

Ans.1. The process of separating lighter portion of colloidal solution, mixture or suspension from the

heavier portion by a special apparatus is called centrifuge machine.

Q.No.2. Enlist four types of centrifugation?

Ans.2. I. Differential centrifugation ii. Isopycnic centrifugation

iii. Density gradient centrifugation iv. Microscopic centrifugation

Q.No.3. Write down the principle of centrifugation.

Ans.3. That the denser components of mixture migrate away from the axis of the centrifuge while

the less dense components of the mixture migrate towards the axis because centrifugal force is
in the horizontal plane. The radial acceleration causes the dense particles to settle at the bottom while

low density substances rise to the top.

Q.No.4. Write RPM and time duration required for the preparation of serum from clotted blood?

Ans.4. RPM – 3000 / mint for 3 minutes

Q.No.5. Write RPM and time duration required for preparation of sediments of urine for

microscopic examination?

Ans.5. RPM 1000 – 1500 / minute for 3 minutes

Q.No.6. Write down two disadvantages of centrifugation.

Ans.6. i) If centrifugation is done at high speed than required the shape of cells are destroyed.

ii) If centrifugation is done at low speed than required the sediments / serum are not formed
completely.

Q.No.7. Define blood plasma from blood serum?

Ans.7.Blood Plasma: The fluid portion of the blood in which particulate components as suspended but

fibrinogen is present.
 Blood Serum: The clear liquid that separate from the blood when it is allowed to clot
completely. Fibrinogen is absent.

Q.No.8. Write two clinical applications of centrifugation?

Ans.8. i. It is used to separate plasma from heavier blood cells.

ii. To prepare the serum from clotted blood.

iii. To prepare the urinary sediments for microscopic examination.

Q.No.9. Write first two steps of procedure of centrifugation.

Ans.9. i) First place the centrifuge machine on leveled and stable table.

ii) Clean the rotor and centrifuge test tubes before operation.

Q.No.10. Write last two steps of procedure of centrifugation.

Ans.10. i) Open the lid of centrifuge machine when rotor stop and take out the sample test tube.

ii) Adjust the RPM knob at the lowest position, turn the timer knob at zero and switch off the machine.

Q.No.11. Write down four precautions of centrifugation.

Ans.11.

1. Hazards risks are reduced by using disposable gloves. Discard gloves in a container for
incineration after completion of work.
2. Keep the caps on the test tube in case of infected specimen during centrifugation.
3. The sample should be transferred carefully by using plastic (bulb sucker) pipette. Mouth
pipetting is contraindicated.
4. High risk specimen must be centrifuged separately from another specimen.

Q.No.12. Enumerate four chemical tests for which serum is required.

Ans.12. i) serum creatinine ii) serum uric acid

iii) serum electrolytes iv) serum cholesterol

Q.No.13. After completion of centrifugation how will you prepare the slide for microscopic examination.

Ans.13. In urine sample, discard the supernatant takes the sediment, place on glass slide and cover with

cover slip and seen under microscope for casts, cells and crystals.

Q.No.14. In the absence of centrifuge machine, how will you form serum from the clotted blood?

Ans.14. By aging process.

 Aging: if the colloidal solution / blood / urine is allowed to stand undisturbed for a long time, the
particles settled down. This takes a long time and the precipitation will not be complete.

Q.No.15. Write four components of centrifuge machine.

Ans.15. i) rotor ii) lid iii) RPM knob

iv) timer knob v) electric motor