Filtration, centrifugation, cell
disruption
Filtration Sterilization: Types, Mechanism, Uses
Filtration is the preferred method of sterilizing heat-sensitive liquid and gases without exposure to denaturing heat. Rather
than destroying contaminating microorganisms, it simply removes them. It is the method of choice for sterilizing antibiotic
solutions, toxic chemicals, radioisotopes, vaccines, and carbohydrates, which are all heat-sensitive.
The liquid or gas is passed through a filter, a device with pores too small for the passage of microorganisms, but large enough
to allow the passage of the liquid or gas. These filters are made of different materials;
The selection of filters for sterilization must account for the size range
Relative size of human cells, bacteria and virus
of the contaminants to be excluded. The most commonly used filter is
composed of nitrocellulose and has a pore size of 0.22μm. The size of
the bacteria ranges from 0.3 to 0.5 μm whereas the size of the viruses
ranges from 20 nm to 0.36 μm. Thus a filter of 0.22μm retains all
bacteria and spores but not all viruses.
Working Mechanism of Filtration Sterilization
Filters work by physically trapping particles larger than the pore size and by retaining somewhat smaller particles via
electrostatic attraction of the particles to the filters. Besides porosity, other factors also influence the efficiency of filtration,
they are:
• Electric charge of the filter
• Electric charge carried by the organisms
• Nature of the fluid being filtered
Filtration of liquids is accomplished either by pulling the solution through a cellulose acetate or cellulose nitrate membrane
with a vacuum (i.e, by applying negative pressure on the filter paper) or by forcing the solution through the filter paper by
imposing positive pressure above the fluid.
Filtration of air is accomplished using high-efficiency particulate air (HEPA) filters designed to remove organisms larger than 0.3
μm from isolation rooms, operating rooms, and biological safety cabinets.
Depth Filters
A depth filter is a fibrous sheet or mat made from a random array of overlapping paper or borosilicate (glass) fibers. The depth
filter traps particles in the network of fibers in the structure.
Uses
• Filter sterilization of air in industrial processes
• Forced air heating and cooling systems used in houses contains simple depth filter to trap dust, spores, and allergens
• Use in biosafety applications such as biosafety cabinets. Biological safety cabinets contain a filter known as high-efficiency
particulate air (HEPA) filter, which is a type of depth filter.
HEPA FILTER
A typical HEPA filter is a single sheet of borosilicate glass fiber that has been treated with a water-repellent binder. The filter,
pleated to increase the overall surface area, is mounted inside a rigid, supportive frame. HEPA filters come in various shapes
and sizes, from several square centimeters for vacuum cleaners to several square meters for biological containment hoods
and room air systems.
Control of airborne particulate materials with HEPA filters
allows the construction of “clean rooms” and isolation rooms
for quarantine, as well as specialized diagnostic/research
laboratories. HEPA filters typically remove 0.3 μm test particles
with an efficiency of at least 99.97% including most
microorganisms, from the airstream.
Membrane filters
Membrane filters are the most common type of filters used for liquid sterilization in the microbiology laboratory. Membrane
filters are composed of high tensile strength polymers such as cellulose acetate, cellulose nitrate, or polysulfone. Membrane
filters are prepared as circular membranes of about 150μm thickness and contain millions of microscopic pores of uniform
diameters; the size of which is adjusted based on requirements, during the polymerization process.
Porosities of membrane filters range from 0.1μm to 10μm and the most
commonly used membrane filter has the pore size of 0.22μm and 0.45μm.
The membranes are held in special holders and often preceded by depth filters
made of glass fibers to remove larger particles that might clog the membrane
filter. The solution is pulled or forced through the filter and is collected in
previously sterilized containers.
Uses of membrane filter
• Sterilization of fluid materials (pharmaceuticals, ophthalmic solutions,
antibiotics, and other heat-sensitive solutions in laboratories and industries
• Identification and enumeration of microorganisms
Advantages of Filtration Sterilization
• Less capital intensive
• Suitable for heat-sensitive liquids (infusions, vaccines, hormones, etc).
• Large volume of liquids can be filtered reasonably fast
Limitations of Filtration Sterilization
• Only liquids and gases can be sterilized by this process
• Filters are expensive to replace, especially nano-filters
• Inherent limitations of materials used in filters affect the efficacy of this process i.e, breakage of glass filters, rupture of
the membrane filter and absorption of the filtrate by Sietz filter
• Clogging may occur
In microbiology, filtration is used to separate microorganisms or particles from liquids. The main types of filtration used in
microbiology include microfiltration, ultrafiltration, nanofiltration, and reverse osmosis, which are distinguished by the
size of the pores in the filter membranes.
1. Microfiltration (MF):
•Uses membranes with pore sizes ranging from 0.1 to 10 micrometers.
•Effective at removing larger particles, including bacteria and some viruses.
•Commonly used for sterilization of liquids and in water treatment to remove suspended solids.
Microfiltration is the process of physically removing suspended solids from water, through a membrane. Microfiltration is often
used in conjunction with other separation processes such as ultrafiltration and reverse osmosis.
The filters used in microfiltration have a pore size of approximately 0.1 micron (small). Bacteria and suspended solids are the only
element that can be removed through microfiltration.
A typical use for an ultrafiltration system can be:
• A pre-treatment for another water treatment process
• Certain types of effluent treatment
• Certain oil and water separation applications
• Treat wastewater
• Sterilizing beverages and pharmaceuticals without sacrificing flavour
• Processing dairy products while allowing protein through
2. Ultrafiltration (UF):
Uses membranes with pore sizes smaller than MF, typically 0.01 to 0.1 micrometers.
Can remove smaller particles, including some viruses and colloids.
Used in water treatment and for separating molecules based on size.
Ultrafiltration blocks everything microfiltration can with the addition of viruses, requiring a slightly higher pressure to achieve
this.
Although it requires higher pressure than MF, ultrafiltration can be powered by the pressure you get from the tap, making it
popular in the commercial sector for drinking water.
It works the same way as MF by which a contaminated liquid passes through a membrane that is too large to fit through the
membranes pore sizes, yielding a purified liquid stream.
Ultrafiltration filters have a pore size of approximately 0.01 micron (smaller).
UF can be used in the following processes:
• Treating wastewater
• Concentrating proteins
• Chemical process separation
• Separating oil/water emulsions
• Removing pathogens from milk
• Clarifying fruit juices
3. Nanofiltration (NF):
Uses membranes with pore sizes even smaller than UF, typically 0.001 micrometers.
Can remove dissolved salts, ions, and smaller organic molecules.
Used for water treatment and in some industrial processes.
Nanofiltration membranes typically remove 50% – 90% of monovalent ions such as chlorides or sodium.
The design and operation of the filters used in NF are very similar to that of reverse osmosis, with some differences. Those being
the membrane isn’t as ‘tight’ as RO membranes and a lower feedwater pressure is required.
Nanofiltration filters have a pore size of approximately 0.001 micron (smallest).
It has attained the name of the ‘softening membrane’ as it is often used to filter water with low amounts of total dissolved
solids, to remove organic matter and to soften water.
NF can be used in the following processes:
• Water treatment
• Pre-treatment for RO
• Pharmaceuticals
• Textiles
• Bakeries
• Dairy
4. Reverse Osmosis (RO):
Uses membranes with extremely small pore sizes (less than 0.001 micrometers), essentially non-porous.
Can remove dissolved salts, ions, and even some viruses.
Used in water purification and desalination.
Reverse osmosis is the only water filtration procedure to remove all elements completely, leaving just pure water, proving
popular in the pharmaceuticals sector.
Each filtration process has the same procedure, water passes through a semipermeable membrane, depending on the pore size
certain elements are removed whilst others pass through.
The correct filtration process is selected specifically for your individual water issue.
What’s the Difference Between Micro, Nano And Ultra Filtration?
• Ultimately they all do the same job but remove different elements depending on the water issue and the industry using it.
• The pore size of the semipermeable membranes used is the only major difference between microfiltration, nanofiltration and
ultrafiltration.
• The different pore sizes allow for different elements allowed to be passed through, giving ultimate precision to treating your
wastewater issue correctly and safely for the environment.
• To remove all impurities all together, reverse osmosis is used. With tighter regulations and the many benefits it has, RO is
becoming increasingly popular in the industrial sector.
Conclusion
• Dependent on your water issue, microfiltration, ultrafiltration and nanofiltration may be used to purify and help you
manage and treat your wastewater.
• Each water issue is treated separately and a bespoke system is designed and built to reach the quality of water you want
to achieve for your process.
• A combination of more than one system is common to achieve such a precise quality of the water where regulations are
tighter.
A centrifuge is a lab instrument for the density-based
Centrifugation is a term used to describe a method of
separation of fluids, gas, or liquid.
separating mixtures using spinning and centrifugal force.
Several characteristics can separate particles during
centrifugation, including size, shape, density, and viscosity.
Principle of a Centrifuge
The centrifuge utilizes the sedimentation principle due to
gravitational force. The centrifugation technique uses a
centrifugal field to separate particles suspended in a liquid
medium. These are put in the centrifuge’s rotor either in
bottles or tubes. Sedimentation is a process whereby gravity
causes suspended particles to separate from fluids. The
suspended substance may consist of powder or clay-like
particles.
Simple filtration filters particles larger than 5 micrometers
from those less than 5 micrometers, which start Brownian
motion and do not sediment under gravity. These particles
can be separated with the help of the central force.
Some of the common parts of the centrifuge are described
below:
Motor: The motor is the powerful central component of the
centrifuge that creates the spin.
Rotor assembly: A drive shaft and a rotor comprise the rotor
assembly. The drive shaft provides support for the rotor
components. The rotor head is attached to the motor, which
bears the containers to house the tubes containing the sample
to be centrifuged. It converts electrical energy to mechanical
energy. Two rotors with different diameters can have the same b) Swinging bucket/ Horizontal rotors: These rotors, along with
rotational speed. Varying radii and angular momentum results the centrifuge tubes, swing out to a horizontal position during
in a difference in the acceleration of such rotors. Thus, relative the time of acceleration such that particles travel a longer
centrifugal force (rcf) is regarded as the accepted standard unit distance, thereby facilitating easier separation of supernatant
for the rotation speed. There are mainly three types of rotors: from the pellet. These types of motors are employed in density
a) Fixed angle rotors: These rotors hold the tubes at an angle of gradient centrifugation.
14 to 40° to the vertical such that particles travel a short
distance while moving radially outwards and are used in c) Vertical rotors: These hold the tubes vertically, i.e., parallel
differential centrifugation. The sedimentation takes place at the to the motor axis, and the particles move shorter distances
walls of the tubes at an angle since the sedimentation direction with shorter periods for separation. It is used for isopycnic and
is the same as the direction of centrifugal force. The pellets density gradient separation; however, it is not considered
(cluster of sediments) later settle at the corner of the base and useful for pelleting because the pellets are spread out along
the wall surface after colliding with the wall surface. the entire outer wall of the tube by centrifugal force.
3. Containers: Several types of containers, such as test tubes, blood bags, cuvettes, centrifuge tubes, etc., are held in the
rotors such that the sample rotates along as the rotor rotates.
4. Control Panel: It serves the purpose of controlling different parameters such as temperature, rotational speed (rcf or rpm),
etc.
5. Latch: When a tube breaks, or there are other issues with the centrifuge while running, the latch keeps the lid closed.
6. Lid: The centrifuge will only spin if the lid is closed and locked to prevent mishaps.
Types of centrifugation techniques
There are two types of centrifugation techniques, namely, preparatory centrifugation and analytical centrifugation.
Preparatory centrifugation deals with the isolation and purification of components such as tissue, cells, subcellular structure,
membrane vesicles, and other particles of biochemical interest. In contrast, analytical centrifugation is carried out to
characterize purified biomolecules.
Preparative Centrifugation
Based on suspension, preparative centrifugation is divided into two
different types. They are:
i) Differential centrifugation
It separates particles based on shape, size, and density. A suspension
of particles with varying densities or sizes will sediment at varying
speeds, with the larger and denser particles sedimenting more
quickly. Following a series of rising centrifugal force cycles on a
suspension of cells, a series of pellets containing cells with a
decreasing sedimentation rate will result.
ii) Density gradient centrifugation
It separates particles based on their buoyant density or
sedimentation rate. A sample mixture is placed on the top of a
preformed liquid density gradients such as CsCl for DNA banding
and isolation of plasmids, nucleoproteins, and viruses; NaBr and
NaI for fractionation of lipoprotein; Per coll, Ficoll, Metrizamide,
Dextran for separation of whole cells and sucrose solution for the
separation of DNase, RNase and Protease.
The two subtypes of density gradient centrifugation are rate-zonal
and isopycnic centrifugation.
Rate-zonal centrifugation
On top of a density gradient, the sample is overlaid as a small
zone. Depending on their mass, particles travel under centrifugal
force at various speeds. Size and mass are the main determinants
of how quickly particles settle. As the band of particles descends
through the density medium, zones with particles of comparable
size develop as the faster sedimenting particles pass the slower
ones.
Isopycnic centrifugation
Particles are separated exclusively based on their density in an isopycnic separation, also
known as buoyant or equilibrium separation. It is necessary for the gradient medium to
have a higher density than the particles that need to be separated.
Particles migrate under the influence of centrifugal force from a uniformly mixed sample
and density gradient until their densities are equal to those of the surrounding medium.
After centrifugation, particles of a certain density settle until their density equals that of
the gradient media (i.e., the equilibrium position).
Analytical Centrifugation
It aims to collect information to characterize the spun sample (sedimentation velocity,
viscosity, concentration, etc.), determine the relative molecular weight of the solutes,
purity of biomolecules, detect conformational changes of protein structure, etc.
Types of Centrifuges
Benchtop or Tabletop centrifuges
• They can be handy for tiny labs with limited space because of their
diminutive size.
• These are compact and are frequently employed in research and clinical
laboratories.
• A tabletop centrifuge is furnished with a lid that covers the apparatus
used to run the centrifuge and a rotor with racks for the test tubes.
Gas centrifuges
• These are used to separate molecules based on their masses and to separate gases based on their isotopes.
• Specifically, they are employed in the extraction and separation of uranium-235 and uranium-238.
Haematocrit centrifuge
• Haematocrit centrifuges operate between 7000 and 15000 rpm.
• The main purpose of hematocrit centrifuges is to calculate the volume-based erythrocyte percentage in blood. It is used to
produce plasma for photometric analysis of the bilirubin concentration of neonatal blood.
Microcentrifuge
• They have a very small footprint and take up minimal room on the workstation because of their highly compact form.
• These work well with small tubes (up to 2.0 ml) and are frequently employed in biological applications.
• They are used to microfilter small amounts of aqueous samples and hold pelleted nucleic acids, proteins from solutions, and
other substances.
Refrigerated Centrifuges
• These centrifuges run at their top speeds while keeping a constant temperature.
• It is used to analyze DNA, RNA, PCR, and antibodies because its temperature range is between -20 and -40 degrees Celsius.
• They are frequently used to collect sedimenting materials quickly, including yeast cells, chloroplasts, and more.
High-Speed Centrifuges
• A high-speed centrifuge is a type of centrifuge that can work at somewhat faster rates ranging between 15,000 and 30,000
revolutions per minute.
• High-speed centrifuges contain a device for regulating both the temperature and speed of the operation for the critical
analysis of delicate biological molecules.
• These centrifuges employ three rotors: fixed angle, swinging bucket, and vertical.
Low-speed centrifuges
• These are frequently used in laboratories for routine particle sorting operated at a maximum speed of 4000-5000 rpm.
• There are few instances of temperature regulation, and they are often operated at room temperature.
• These centrifuges employ swinging bucket and fixed-angle rotor types.
Continuous flow centrifuges
• It enables the centrifugation of large quantities of samples without influencing sedimentation rates.
• They also have greater capacities, which saves time by eliminating the need to load and unload the sample repeatedly as is
necessary with standard centrifuges.
Ultracentrifuges
• The ultracentrifuge is a highly developed and sophisticated centrifuge that can separate tiny molecules that conventional
centrifuges can’t separate at a fast rate.
• Ultracentrifuge rotor speeds can range from 60,000 to 150,000 rpm.
• They run samples in groups or as continuous flow systems and are larger.
Types of Ultracentrifuges
Preparative Ultracentrifuges
• Preparative ultracentrifuges are centrifuges
that are used to isolate and separate particles
inside an experiment using centrifugation.
• When a run is prepared for an ultracentrifuge,
the contents of the tubes are examined after
the centrifugation procedure, as opposed to
the centrifuge for analysis, which examines
during the centrifugation.
Analytical Ultracentrifuges
• Analytical centrifuges are ultracentrifuges used
to examine different particles inside the
specimen.
• It is utilized to perform a qualitative
examination of macromolecules in solution.
• These are fitted with sensing devices that track
the spin and movement of the constituents in
real time to calculate the sedimentation
coefficient.
Applications of Centrifuge
• Centrifuges are employed in chemistry, biology, biochemical, Limitations of Centrifuge
and clinical laboratories, such as testing the sedimentation • Separating light particles (with almost negligible mass)
rates of various blood cells. is exceedingly difficult.
• These are utilized in dairy industries to separate cream (fat) • A centrifuge, despite its kind, is a very sophisticated
from milk, and this process is known as churning. electrical device that requires specialist servicing in
• Giant centrifuging machines are used in water treatment, times of malfunction, making device maintenance
where it spins the mud and sludge out of the water to extremely difficult and expensive.
produce cleaner water. Likewise, solid matter is separated • High energy usage because it has so many power-
from freshly drilled-out petroleum in oil rigs. consuming features such as temperature control and
• Moreover, big spinning wheels are used to simulate a high- rotor spinning.
gravity environment to practice for the pilots. Hence, these • Due to their high rotation rates per minute,
are important in aeronautics and space. centrifuges produce noise disruptions. Since it uses
• Centrifugation is used to produce biological products and rotations, vibrations are unavoidable, which will cause
bulk drugs and perform biopharmaceutical analysis of drugs. noise pollution and prevent its use in some areas.
• It is applied in removing water from lettuce after washing it • Most often, a backup machine is required to step in
in a salad spinner and separating chalk powder from water. when the primary equipment breaks down.
• It is useful for separating the isotopes for nuclear weapon • Extensive use of polymers
programming.
CELL DISRUPTION
• Cell disruption is the process of obtaining intracellular fluid
via methods that open the cell wall.
• The overall goal in cell disruption is to obtain the
intracellular fluid without disrupting any of its components.
• The method used may vary depending on the type of cell
and its cell wall composition.
• Irrespective of the method used, the main aim is that the
disruption must be effective and the method should not be
too harsh so that the product recovered remains in its active
form.
• Cell disruption methods can be categorised into mechanical
methods and non-mechanical methods.
• Mechanical methods are divided into solid shear methods
and liquid shear methods.
• Non-mechanical methods can be divided into physical
methods, chemical methods and enzymatic methods.
Mechanical Methods of Cell Disruption
Mechanical methods are those methods that required some sort of force to separate out intracellular protein without adding
chemical or enzyme
1. Mortar & pastel/grinding
2. Blender
3. Bead beating
4. Ultra sonication
5. Homogenization
Mortar & Pestle
It involves the grinding of the cells such that they are disrupted.
This does not have to be in suspension and is often done with plant samples frozen in liquid nitrogen.
When the material has been disrupted, metabolites can be extracted by adding solvents.
Blenders
The use of blenders which employ high speed can be used to disrupt cell walls.
It is the same process used by centrifugation, which separates or concentrates materials suspended in a liquid medium.
Bead beating
Glass or ceramic beads are used to crack open cells
The kind of mechanical shear is gentle enough to keep organelles intact.
It can be used with all kinds of cells, just add beads to an equal amount of cell suspension and vortex.
Ultrasonication
Ultrasonic homogenizers work by inducing vibration in a titanium probe that is immersed in the cell solution.
A process called cavitation occurs, in which tiny bubbles are formed and explode, producing a local shockwave and disrupting cell
walls by pressure change.
This method is very popular for disruption of plant and fungal cells.
Homogenization
• Liquid-based homogenization is the most widely used cell disruption technique for small volumes and cultured cells.
• Cells are lysed by forcing the cell or tissue suspension through a narrow space
• Homogenizers use shearing forces on the cell similar to the bead method.
• Homogenization can be performed by squeezing cells through a tube that is slightly smaller than beads beating
Non-Mechanical Methods of Cell Disruption
Nonmechanical methods are further divided into three classes which are following :
A. Physical methods
1. Freeze-Thaw
It is suitable when working with soft plant material and algae.
Disruption is achieved via a series of freezing and thawing cycles.
Freezing forms ice crystals, which expand upon thawing, and this ultimately causes the cell wall to rupture.
2. Microwave/ Thermolysis
Microwave (along with autoclave and other high temperature methods) are used to disrupt the bonds within cell walls, and also
to denature proteins.
However, uncontrolled amount of heat can easily denature or damage target proteins and subtances.
3. Osmotic Shock
Through the process of osmosis, water can be moved into the cell causing its volume to increase to the point that it bursts.
The method however, can only work with animal cells and protozoa, since they do not have cell walls.
4. Electric Discharges
It is also possible to achieve cell disruption via electrical discharges in mammalian and other cells that are bounded by plasma
membranes only.
B. Chemical methods
•They are often used with plant cells (and sometimes in combination with shearing).
•Organic solvents such as toluene, ether, benzene, methanol, surfactants, and phenyl ethyl alcohol DMSO can be used to
permeate cell walls.
•EDTA can be used specifically to disrupt the cell walls of gram negative bacteria, whose cell walls contain lipopolysaccharides
that are stabilized by cations like Mg2+ and Ca2+.
•EDTA will chelate the cations leaving holes in the cell walls.
C. Enzymatic methods
Another strategy to achieve cell lysis is to use digestive enzymes which will decompose the microbial cell wall.
Different cell types and strains have different kind of cell walls and membranes, and thus the used enzyme depends on
microbe. For example, lysozyme is commonly used enzyme to digest cell wall of gram positive bacteria. Lysozyme hydrolyzes β-
1-4-glucosidic bonds in the peptidoglycan.
The cell wall of yeast and fungi differs significantly from the cell wall bacteria. One commonly used enzyme mixture for
degradation of cell wall of yeast and fungi is Zymolyase.
It has for example β-1,3 glucanase and β-1,3-glucan laminaripentao-hydrolase activities (Zymolyase | Yeast lytic enzyme).
In addition, the enzymes that are commonly used for degradation of cell wall of yeast and fungi include different cellulases,
pectinases, xylanases and chitinases.
Enzymes such as beta(1-6) and beta(1-3) glycanases, proteases and mannase can also be used to disrupt the cell wall.
Significance of Cell Disruption
• Cell disruption is an essential part of biotechnology and the downstream processes related to the manufacturing of biological
products.
• It is necessary for the extraction and retrieval of the desired products, as cell disruption significantly enhances the recovery
of biological products.
