Analytical Microbiology

MIC402
Centrifugation techniques
K.M. Mazharul Alam
Lecturer,
MNS Department,
BRAC University
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Study Outcome

◼ Introduction of centrifugation technique

◼ Sedimentation coefficient and rate
◼ Basic principle of centrifugation
◼ Different types of centrifugation techniques

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References

◼ Principles and Techniques of Biochemistry and Molecular Biology (Sixth

edition)
KEITH WILSON & JOHN WALKER

3
Introduction

◼ The biochemical analysis of subcellular structure, supra-molecular

complexes and isolated macromolecules is of central importance for
understanding of molecular biology of the cell.

◼ An important prerequisite for studying the biochemical and physiological

properties of organelles and biomolecules is the preservation of their
biological function and properties during separation of cellular
components.

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Introduction: Sedimentation

◼ Sedimentation is the process by which particles in suspension

in a fluid are deposited as a sediment on the bottom of the
fluid container
◼ The sedimentation process is determined by the size, density,
and shape of the particles in a suspension.
◼ Larger and denser particles settle faster than smaller and less
dense particles. Spherical particles settle faster than non-
spherical particles
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Blood Sedimentation Rate
◼ The sedimentation rate (sed rate) blood test measures how
quickly red blood cells (erythrocytes) settle in a test tube in
one hour.
◼ The more red cells that fall to the bottom of the test tube in
one hour, the higher the sed rate.

◼ Another name for this test is erythrocyte sedimentation rate

(ESR).

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Erythrocyte Sedimentation Rate (ESR)

◼ The erythrocyte sedimentation rate (ESR) is a non-specific

measurement used to detect and monitor an inflammatory
response to tissue injury.
◼ ESR is affected by three factors: erythrocytes, plasma
concentration, and mechanical/technical factors.

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Erythrocyte Sedimentation Rate (ESR)
◼ Erythrocyte sedimentation rate (ESR) is the measure of ability of erythrocytes (
red blood cell) to fall through the blood plasma and accumulate together at the
base of container in one hour.
◼ The erythrocyte sedimentation rate (ESR or sed rate) is the rate at which
red blood cells in anticoagulated whole blood descend (fall down) in a standardized
tube over a period of one hour.
◼ It is a common hematology test, and is a non-specific measure of inflammation.
◼ When your blood is placed in a tall, thin tube, red blood cells (erythrocytes)
gradually settle to the bottom. Inflammation can cause the cells to clump.
Because these clumps are denser than individual cells, they settle to the
bottom more quickly. 8
Erythrocyte Sedimentation Rate (ESR)
▪ There are 3 stages in erythrocyte sedimentation:
▪ Rouleaux formation: ln normal blood, the red blood cells remain more
or less separated by being negatively charged. or an inflammatory
process is present, the high proportion of fibrinogen in the blood
reduces the negative charge and causes red blood cells to stick to
each other. The red cells form stacks called 'rouleaux,' which settle
faster, causing high ESR levels. It takes 10 minutes approximately
▪ Sedimentation or settling stage (40 minutes)
▪ Packing stage - 10 minutes (sedimentation slows and cells start
to pack at the bottom of the tube apart from the plasma)]

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 Sedimentation Test

Westergren tube
Deoxygenated
blood

1
Sedimentation Test

5 mm 10 mm 20 mm

Zero hour 15 mins 30 mins 1hour

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Sedimentation Rate
◼ Sed rate test will be reported in the distance in millimeters that red blood cells have
descended in one hour. The normal range is 0-22 mm/hr for men and 0-29 mm/hr for
women.
◼ The upper threshold for a normal sed rate value may vary somewhat from one medical
practice to another.

◼ There are many possible causes of a high sedimentation rate. For this reason, a sed rate
is done with other tests to confirm a diagnosis.

◼ After a diagnosis has been made, a sed rate can be done to help check on the disease or
see how well treatment is working. 12
 Sedimentation Test
▪ When inflammation is present in the body, certain proteins cause red blood cells to stick
together and fall more quickly than normal to the bottom of the tube.

▪ These proteins are produced by the liver and the immune system under many
abnormal conditions, such as an infection, an autoimmune disease, or cancer. The level of
acute phase reactants such as C-reactive protein (CRP) and fibrinogen increases in the
blood in response to inflammation.

▪ An increased number of red blood cells (polycythemia) causes reduced ESR as blood
viscosity increases.

▪ Hemoglobinopathy such as sickle-cell disease can have low ESR due to an improper 13

shape of red blood cells that impairs stacking

Sedimentation
◼ Particles suspended in a solution are pulled downward by Earths
gravitational force. Greater the density or mass, the faster they sink.
◼ Because, the earths gravitational force is weak, a solution containing
particles of very small masses usually remain suspended due to random
thermal motion
◼ Gravitational force only depends on mass. Forces much larger than
Earths gravitational fields are required to cause appreciable
sedimentation of these particles of very small masses.
◼ Solution?
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Centrifugation
◼ The centrifugation method separates different components or the
suspended particles from the homogenous solution through centrifugal
force. A natural gravitational force can also concentrate particles but in
an indefinite time
◼ To increase particle separation efficiency, a centrifugal force function to
separate particles by sedimentation property. It separates particles
based on their physical properties like size, shape, density etc.
and chemical properties like molecular weight and viscosity.

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Centrifugation

◼ Centrifugation is the separation process that relies on the action of

centrifugal force to separate particles in a solid–liquid mixture into two
distinct phases consisting of the sediment and centrifugate (also called
supernatant liquid).
◼ Centrifugation is used to collect cells, to precipitate DNA, to purify virus
particles, and to distinguish subtle differences in the conformation of
molecules.

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Centrifugation
◼ A centrifuge is an instrument that rotates at variable speeds, generating a
centrifugal force, which causes sedimentation of particles.
◼ The rate of sedimentation, or velocity of a particle (v) is proportional to
the centrifugal force (G).
v∞ G
◼ The rate of sedimentation per unit of centrifugal field is called
sedimentation coefficient, and is generally expressed as Svedberg (S) units.
v∞ G , since G= ω2r • ω = angular velocity (rad/s)
• r= radial distance (cm)
therefore
v ∞ ω2 r
v = s ω 2r 17

s = v/ ω2r
Svedberg Unit
◼ A Svedberg is a non-SI physical unit that is used to
characterize the behavior of a particle in
sedimentation processes, notably centrifugation.

◼ The Svedberg is technically a measure of time, and is

defined as exactly 10-13 seconds, so this quantity has
been designated 1 Svedberg unit.

◼ The unit is named after the Swedish chemist Theodor

Svedberg (1884-1971), winner of the Nobel prize in
chemistry in 1926 for his work in the chemistry of
colloids and his invention of the ultracentrifuge. 18
The Nobel Prize In Chemistry 1926
"For His Work On Disperse Systems"

◼ In 1926, Theodor Svedberg won the Nobel

Prize in Chemistry for his work on the behavior
of molecules in solution.

◼ Svedberg is famous in the biological sciences

for his development of the high speed
ultracentrifuge and the technology for
photographing the behavior of molecules during
centrifugation.
 Sedimentation Coefficients
◼ The rate of sedimentation per unit of centrifugal force is called
sedimentation coefficient, and is generally expressed as Svedberg (S)
units.
v∞ G , since G= ω2r, therefore
v ∞ ω2 r
v = s ω 2r
s = v/ ω2r

◼ The Svedberg is the most important measure used to distinguish

ribosomes, which are important in phylogenetic studies.
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 Sedimentation Coefficients : Problem Solving

◼ A scientist is trying to isolate a ribosome subunit by centrifugation. He

observed that, the sedimentation rate is 26 micrometers per second
(26×10−6 m/s) under the centrifugal acceleration of 107 m/s2. What is
the sedimentation coefficient of the particle?

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 Sedimentation Coefficients
◼ The Svedberg coefficient is a nonlinear function

◼ A particle's mass, density, and shape will determine its S value.

◼ The S value depends on the frictional forces retarding its movement, which, in turn, are related to the average
cross-sectional area of the particle
◼ Sedimentation coefficients are not additive.

◼ Sedimentation rate does not depend only on the mass or volume of a

particle, and when two particles bind together there is inevitably a loss
of surface area.

◼ Thus when measured separately they will have Svedberg values that
may not add up to that of the bound particle. 22
 Sedimentation Coefficients

◼ The Svedberg coefficient is a nonlinear function

◼ A particle's mass, density, and shape will determine its S value.

◼ The S value depends on the frictional forces retarding its movement,

which, in turn, are related to the average cross-sectional area of the
particle

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Ribosomes

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Courtesy: http://micro.magnet.fsu.edu/cells/ribosomes/ribosomes.html Courtesy: https://www.quora.com/What-are-70S-ribosomes

Sedimentation Coefficients
• The two eukaryotic ribosomal subunits have sedimentation
coefficients of 40 x 10-13 and 60 x 10-13. As one Svedberg (S) unit is
10-13, the two ribosomal subunits are referred to as the 40S and the
60S ribosomal subunits.

• The molecular mass of the 40S and 60S particles are 1.5 and 3.0
million g/mol, respectively. Thus, the complete ribosome has a mass
of approximately 4.5 million g/mol.

• The complete ribosome is referred to as the 80S ribosome.

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 Basic Principle of Centrifugation

◼ The rate of sedimentation is dependent upon the applied centrifugal field

(G), being directed radially outwards.
◼ This is determined by the square of the angular velocity (ω, in
radian/sec) and the radial distance (r, in cm) of the particle from the axis
of rotation.
⮚ G= ω2r --- (1)

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Principle Of Centrifugation …
◼ Since 360o equals to 2п radians, one revolution of rotor can be
expressed 2п rad. Accordingly, the angular velocity can be
expressed in terms of rotor speed in revolutions per min or r.
p.m (rev min-1).
⮚ ω = 2п rev min-1 -------- (2) G= ω2r--- (1)
60
⮚ G = 4п2 (rev min-1)2 r -------- (3)
3600
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Mathematical Problem

◼ For the pelleting of the microsomal fraction from a liver homogenate, an

ultracentrifuge is operated at a speed of 40 000 r.p.m. What is the
angular velocity in radians per second?
Calculation of
ω = 2п rev min-1 RPM
rpm= (ω*60)
60 2п
={(2*3.1416*40 000)/60} rad/s
=4188.8 rad/s
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Relative Centrifugal Field (Rcf)

◼ Many particles or cells in a liquid suspension, given time, will eventually settle
at the bottom of a container due to gravity (1xg). However, the length of
time required for such separations is impractical.
◼ Other particles, extremely small in size, will not separate at all in solution,
unless subjected to high centrifugal force. When a suspension is rotated at a
certain speed or revolutions per minute (RPM), centrifugal force causes the
particles to move radially away from the axis of rotation. The force on the
particles (compared to gravity) is called Relative Centrifugal Force (RCF).
For example, an RCF of 500 x g indicates that the centrifugal force applied
is 500 times greater than Earth gravitational force. 29
Relative Centrifugal Field (RCF)

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 Relative Centrifugal Field (RCF)
We know G= ω2r and ω = 2п rev min-1
60
So, G= 4п2 (rev min-1)2 r
3600
◼ Hence, RCF = G = 4п2 (rev min-1)2 r (g = 981 cm/sec2)
g 3600 x 981

Or, RCF = 1.118 x 10-5 (rev min-1)2 r

⮚ When conditions of the centrifugal separation of particles are reported, rotor

speed, radial dimensions and time of separation of the rotor must also be 31

quoted.
Mathematical Problem

◼ A fixed-angle rotor exhibits a radius at the top of the centrifuge tube of

3.5 cm. If the rotor is operated at a speed of 20 000 r.p.m., what is the
relative centrifugal field (RCF) at the top of the centrifuge tube?

RCF = 1.118 x 10-5 (rev min-1)2 r

= 1.118 x 10-5 (20 000)2 x 3.5 g
= 15 680 g

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Molecules And Sub-cellular Structures Can Be Defined By A Sedimentation
Coefficient Which Reflects Their Size, Shape & Density

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Summary

◼ Introduction to centrifugation and centrifuge

◼ Sedimentation coefficient and it’s calculation
◼ Sedimentation rate test (ESR)
◼ Basic principle of Centrifugation
◼ Calculation of RPM and RCF

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Thank You!

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