Cryopreservation

Cryopreservation is a technique of preserving and storing viable biological samples in a frozen state over extended periods of time. The preservation of animal cell has become dependent upon using low temperature to render the cell metabolically inert. The term ‘cryopreservation’ refers to ‘a method of cold storage.’ The optimum temperature for preserving animal germplasm is liquid nitrogen temperature of -196 degrees Celsius. Temperatures from -70 to -80 degree Celsius may be sufficient enough to maintain cell viability for a few months. Cryobiology is the study of the effects of extremely low temperatures on biological systems such as cells or organisms.

Definition

Cryopreservation is the process of cooling and storing cells, tissues and organs at very low temperatures to maintain their viability. Spermatozoa were the first mammalian cells to be cryopreserved successfully (Polge et al 1949).

Cryopreservation methods

The two most commonly used cryopreservation methods for animal germplasm are slow programmable freezing and vitrification.

Slow programmable freezing – In slow freezing, cells in a medium are slowly cooled to below freezing point. Slow cooling is needed in order to increase osmotic strength, which causes an efflux of water from the cells. Machines are used to freeze biological samples  using programmable sequences i.e. ‘freezing down’ a sample to better preserve it for eventual thawing before it is frozen or cryopreserved in liquid nitrogen. Biological samples such as oocytes, skin, blood products, embryo and stem cells are preserved by slow programmable freezing machines.

Vitrification – The term ‘vitrification’ refers to any process resulting in ‘glass formation’(‘arrested liquid state’) the transformation from a liquid to a solid in the absence of crystallization (solid-liquid).  Vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution becomes an amorphous ice- it vitrifies.

Stroage temperature

Cells can be stored for varying lengths of time at temperatures between -70 to -196 degrees Celsius. For short –term storage (few weeks) cells can be preserved at -70 degrees Celsius in standard mechanical freezers. For long-term storage, cells can be preserved at liquid nitrogen containers.

Liquid nitrogen containers

Liquid nitrogen containers are basically robust, heavily insulated vessels into which liquid nitrogen is poured at regular intervals in order to maintain the required temperature. Cells can be stored in either vapour phase – containers at -120 degrees Celsius or liquid phase containers at -196 degrees Celsius of liquid nitrogen. Storage at -120 degrees Celsius does not significantly reduce cell viability. But indefinite storage of cells requires liquid phase containers.

Cryoprotective agents (CPAs)

Glycerol and DMSO are the most commonly used cryoprotective agents. Fetal bovine serum (FBS) is employed in mammalian cryopreservation solutions, but it is not a cryoprotective agent. Dextrans, sorbitol, trehalose, polyethylene glycols, starches, sugars, and polyvinylpyrrolidone provide considerable cryoprotection in a variety of biologic systems (Mazur 1981).  Salts, such as magnesium chloride, have been reported to be cryoprotective agents (Karow and Carrier 1969). Cryoprotectants protect slowly frozen cells by one or more of the following mechanisms: suppressing high salt concentrations; reducing cell shrinkage at a given temperature;reducing the fraction of the solution frozen at a given temperature and minimizing intracellular ice formation. Combinations of cryoprotectants may result in additive or synergistic enhancement of cell survival (Brockbank and Smith 1993, Brockbank 1992). Intracellular cryoprotectants of low molecular weights permeate cells e.g. glycerol, dimethyl sulfoxide. Extracellular cryoprotectants with relatively high molecular weights do not penetrate cells e.g. polyvinylpyrrolidone, hydroxyethyl starch.

Advantages of cryopreservation

Germplasm cryopreservation of the sperm, eggs and embryos contributes directly to animal breeding programmes. Germplasm cryopreservation also assist the ex situ conservation for preserving the genomes of threatened and endangered species. Cryopreserved sperm, oocytes and embryos are used for artificial insemination and embryo transfer in the livestock industry. Cryopreservation also has enormous applications in the artificial propagation of widely diverse aquatic organisms. Cryopreservation of  sperm and embryonic cells has been successful in a number of teleosts and invertebrate species.  The establishment of germplasm banks using cryopreservation can contribute to conservation and extant populations in the future. Cryopreservation provides a continuous source of tissues and genetically stable living cells for a variety of purposes including research and biomedical processes. Cryopreservation reduces the risk of microbial contamination or cross contamination with other cell lines. Cryopreservation reduces the risk of morphological or genetic changes. It also reduces costs of maintenance of cells, tissues or organs.

Safety considerations

The main dangers come from explosions of ampoules or cryotubes. The technician must wear appropriate protective clothing, protective goggles, face mask, insulated gloves etc. Careless handling of cold containers can also cause burns, so it is important to use handling tongues. Nitrogen gas is colorless, odourless and tasteless. It cannot be detected by the human senses. So liquid nitrogen can be used only in well ventilated areas.

Stages in the cryopreservation

Selection of cell lines -à cultivation of cells------à Screening of cells -----à preparation of cells-----à freezing of cells ---à Evaluation of viability.

Preparing cells for cryopreservation

Cells are prepared by trypsinization (0.25% w/v trypsin) to detach adherent cell from flask surfaces. Cells in suspension are centrifuged at around 100xg for 5to 10 minutes and the pellet is suspended in a small volume (1-2 ml) of storage medium.

Storage medium

The cryopreservation media generally consists of a base medium, protein source and a cryoprotective agent. The cryoprotective agent protects the cells from mechanical and physical stress and reduces water content within the cells. Cryoprotective agent minimizes the formation of cell-lysing ice crystals. The formation of ice crystals may disrupt the cell membrane leading to the death of the cells. The composition of the medium used to suspend cells contains a cryoprotectant (usually glycerol or dimethyl sulphoxide, DMSO) and a high protein concentration (serum 20% v/v) cryprotectant = 7-10 % (v/v); serum – 20% (v/v).

Freezing

Since freezing is stressful, the rate of cooling from 0 to 50 degrees Celsius must be slow and controlled. A fall of about 1 degree Celsius per minute is optimal. Rapid chilling results in thermal shock and leads to cell death or injury.  Once a temperature of -50 degrees Celsius is reached, the cells must be cooled rapidly to the final holding temperature.

Thawing of frozen cells

Cells retrieved from storage must be thawed rapidly to ensure maximum survival. The ampoule should be plunged into a beaker of water at 37 degrees Celsius. The cell suspension can be transferred drop wise into a container holding about 20 ml of pre-warmed growth medium supplemented with 10% fetal calf serum.

Conclusion

Cryopreservation of gametes, embryos and embryonic cells has become of immense value in animal biotechnologies which provide an important tool for protecting the endangered species and genetic diversity. Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. The establishment of cryobanks to utilize the cryopreservation worldwide would be a significant and promising task in the future.

Ultracentrifugation

Centrifugation is the most widely used technique for understanding cellular and subcellular structures. During centrifugation, solid particles experience a centrifugal force, which pulls those outwards i.e. away from the centre. The velocity with which a given solid particle moves through a liquid medium is related to angular velocity. In other words, centrifugation a procedure involves the use of centrifugal force for the sedimentation of components of a mixture. More dense components of the mixture move away from the axis of the centrifuge, while less dense components of the mixture move towards the axis. The sedimentation process is accelerated by the centrifugal field.

Definition of centrifuge

Centrifuge is an apparatus that rotates at high speed and by centrifugal force separates substances of different densities. In other words a centrifuge is a device for separating particles or macromolecules (e.g. cells, sub-cellular components, proteins, nucleic acids) from a solution according to their size, shape, density, viscosity of the medium and rotor speed.

Definition of ultracentrifuge

Ultracentrifuge is a high-speed centrifuge for separating microscopic and sub-microscopic materials to determine the sizes and molecular weights of colloidal and other small particles.

Principle of centrifugation

The principle of centrifugation is that an object moving in a circular motion at an angular velocity w is subjected to an outward force F through a radius of rotation r in cms expressed as F=w²r . F is frequently expressed in terms of gravitational force of the earth (RCF). The operating speed of the centrifuge is expressed as revolutions per minute ‘rpm’.

The velocity of the moving particle is expressed in the form of sedimentation coefficient (S) v = S (w²r). Sedimentation coefficient is characteristic constant of a molecule and is a function of size, shape and density. The rate of sedimentation can be increased by raising the revolutions per second.

General types of centrifuges

Low – speed centrifuge –(desk top or clinical centrifuges)- maximum speed 3000rpm. It is used for separating serum form blood or separation of RBC etc.

High speed centrifuge – 25,000 – 30,000rpm. It functions at low temperature of 0-4 degree Celsius. It is used for cell fractionation i.e. separation of organelles.

Ultracentrifuge – High speed at 75,000 rpm and refrigerated.  It is used for the separation of cell organelles.

Analytical centrifuge - it has a built in optical system to measure the sedimentation characteristics of macromolecules.

Components of a centrifuge

The principal components of a centrifuge are a rotor to hold sample tubes and an electric motor to spin the sample. There are 4 types of rotors: Swinging bucket rotor (horizontal orientation), fixed angle (30 degree to the axis of rotation), vertical tube (centrifuge tube orients at an angle of 90 degree).

Types of ultracentrifuges

There are two types: analytical and preparative models.

    Analytical ultracentrifuge – It consists of rotors and tubes called cells. The instrument is designed to allow the person to follow the progress of the substances in the cells, while centrifugation is in progress. By estimating sedimentation velocity during centrifugation, the molecular weight can be determined.

Preparative ultracentrifuge – It is used for the purification of the components of macromolecules and all determinations are made at the end of centrifugation. The instrument does not have a monitoring device. Centrifugation is accomplished by rotors which are either swinging bucket type or fixed angle rotors. Swinging bucket rotors in which the buckets become horizontal, while in motion. In fixed angle rotors, the tubes of the centrifuge are set at fixed angles and the rotor moves in a specified plane at all times.

Description of ultracentrifuge

The ultracentrifuge consists of a fixed angle rotor of aluminium or titanium revolving at high speed about an axis in an evacuated chamber. The solution containing macromolecules is taken in the cell having quartz windows.  The cell is almost filled with the solution and sealed to withstand the pressure developed in the intense centrifugal field. A beam of light is allowed to pass through the solution, which then falls on a detector – normally a photographic plate.  Since light passing through an area of the sample is proportional to the molecules present in that region, the darkening produced in the photographic plate indicates the concentrations at various depths of the centrifuge tube.

The ultracentrifuge can be useful in two different methods: sedimentation equilibrium and sedimentation velocity methods.

In sedimentation equilibrium method, equilibrium is attained between the rate of settling down of the molecules and at the rate at which they diffuse back because of the thermal motion and Brownian movement under action of gravity. This  method takes several days to complete because of the low centrifugal forces (10,000-100,000g).

In sedimentation velocity method, higher centrifugal forces (up to 500,000 times the gravity)  are applied to accelerate sedimentation. This method starts with a well defined boundary or layer of solution near the center of the rotation and follows the movement of this layer toward the outside of the cell as a function of time. When a solution containing uniformly distributed solutes is centrifuged at high speed (55,000rpm), the particles migrate outwards from the centre of rotation, forming a well defined boundary between the solvent portions with or without particles.

Analytical Ultracentrifugation (AUC)

All analytical techniques require the use of an ultracentrifuge and can be classified into differential centrifugation and density gradient centrifugation. The density gradient centrifugation is further subdivided as zonal and isopycnic centrifugation.

Differential centrifugation is a technique commonly used by biochemists. A tissue sample such as liver is homogenized at 32 degrees in a sucrose solution that contains a buffer. The homogenate is then placed in a centrifuge and spun at constant centrifugal force and at constant temperature. After sometime a sediment forms at the bottom of the centrifuge tube called pellet and the overlying solution is called supernatant. The overlying solution is then placed in another tube, which is then spun at higher speeds. 

Applications – Differential centrifugation is used to determine the number of components and number of species; detection of impurities, molar mass of species; kinds and stoichiometry of chemical reactions present in solution including association with ligands, self- association etc. Materials analyzed include macromolecules such as proteins, polysaccharides, nucleic acids; small molecules such as drugs, ligands, gases and large aggregates such as viruses and organelles.

Density gradient centrifugation

The separation is  done in a medium having different density gradients. The selection of gradient medium is an important prerequisite. The gradient medium should not affect the cell sample. The medium should be easily sterilizable, recoverable and non-corrosive. Most common media includes sucrose, glycerol, sorbitol etc. There are discontinuous and continuous density gradients. In discontinuous density gradient medium, the density increases one layer to another. This medium is useful in the separation of whole cells, sub-cellular organelles or lipoproteins. In continuous density gradient medium, the density decreases from the bottom of the solution to the meniscus. This medium is useful in the separation of ribosomes, viruses, proteins and enzymes.  Density gradient centrifugation can be of two types such as rate zonal centrifugation and isopycnic centrifugation.

Rate zonal centrifugation – centrifugation is carried out at a very low speed for a short time so that the particles settle down. However centrifugation should be stopped before the particles of any zone settle at the bottom e.g. separation of nucleic acids, ribosomal subunits.

Isopycnic centrifugation – Isopycnic means “of the same density.” Isopycnic = equal density and separation is on the basis of different densities of the particles. Molecules are separated on equilibrium position, not by rates of sedimentation. The particles of solution move according to their buoyant densities and become static at a place, where the density is greater than their own. This requires a very long time centrifugation and high speed. As an example, the pellet obtained by centrifugation of the tissue homogenate at 10,000g is suspended in increasing densities of sucrose solution and centrifuged for several hours at 40,000rpm. Now the individual organelles move to the region of their own equilibrium density and remain at the specific regions. This method is useful in the separation of proteins, intracellular organelles and nucleic acid fraction.

Applications of density gradients

Density gradients are widely used to separate and purify, on a preparative scale, a variety of cells, organelles and macromolecules such as nucleic acids or proteins. Gradients are required for analytical experiments for example to measure the apparent buoyant densities or sedimentation coefficients of particles; to estimate the size, conformation or turnover rates of proteins and nucleic acids; and to investigate the  effects of chemical, physical or biochemical treatment of the sample material.

Scientific research skills

Research can be defined as a careful and systematic investigation in some field of knowledge, undertaken to establish facts or principles. Scientific research is a continued search for scientific knowledge and understanding by scientific methods. Research is aimed at obtaining the information to test specific hypotheses.

6- basic steps in scientific method 

 1)Defining the problem by reviewing the relevant knowledge. Based on these activities, hypotheses are stated, questions are formed and experiments are designed. 2)Planning the research to collect the data required to evaluate the hypotheses or answering the research questions. 3)Carrying out the experiment to obtain the desired data. 4)Analyzing the results so that conclusions can be drawn.5) Interpreting the results so that practical applications can be made and 6)Reporting the results in a way that all relevant audiences will benefit from the knowledge obtained.

Methods of scientific research

There are three methods of scientific research such as descriptive method, experimental method and statistical method.

1. Descriptive methods 

They provide a description of the thing being studied. This method is very crude and it may be some combination of words and numbers. It can be a natural observation, systematic observation and by developing tests. Greeks first employed natural observation to record what they see. This method is unsystematic and time consuming. The research using systematic observation can be carried out by checklist technique, questionnaire method and public opinion.

2. Experimental methods

In this method, the experimenter changes or varies something by  keeping other conditions  as constant as possible and  looks for some effect of the changes or variations on the thing being studied.
Experimental design – it must be planned with great care to control various factors/ variables.

Variables – a variable is something that varies. It can be quantitatively measured. Variables fall into two classes: independent and dependent variables. An experiment must have at least one independent and one dependent variable. An experiment may also have more than one independent and more than one dependent variable. In a graph, the horizontal axis (abscissa) depicts the independent variable and the vertical axis (ordinate), the dependent variable.

1.       An independent variable is a variable that the experimenter selects and manipulates. In other words the variable that is purposely changed is an independent variable. Each change of a variable is known as a level of independent variable. Each Experimenter also selects the dependent variables.

2.       The dependent variable is a variable that changes as a result of changing the independent variable.

Constants – the various factors in an experiment that do not change.

Controls – the main point of doing an experiment is to compare control factors with experimental factors otherwise it is difficult to tell what is going on.

The scale – it is a measuring device which consists of a sequence of interchangeable units beginning with zero.

Ranking – it is the arrangement of the units of a measurable quality in the order of amount.

Rating – it is the arrangement of items into ascending or ordered classes.

3. Statistical methods

Statistical methods focus on the significance of differences, sampling error and probability. Correlation refers to a co-relationship between two sets of scores.  Reliability refers essentially to repeatability. Validity refers to what the test is supposed to be measuring.

Scientific reasoning

It refers to a body of techniques for investigating a phenomena and acquiring knowledge. It consists of systematic observation, measurement and experiment. Scientific methods requires intelligence, imagination and creativity. This method is an ongoing cycle of formulating, testing and modifying hypotheses.

Types of research design

Exploratory research (huh?) is conducted to generate or gather basic knowledge. It is performed to clarify relevant issues, uncover variables or simply to collect more information.

Descriptive research (who, what, where, how) is conducted to provide further insight into the research problem by describing the variables of interest.

Causal research (if...then) is done to provide information on cause and effect relationships.

Importance of scientific research

•  Develop new methods to conduct scientific research.
• Increase the sum total of information/ philosophies in various fields of science.
• Develop and apply new devices to conduct research.
• Increase the general availability of new materials and certain services.

Parts of a research article

Abstract –a brief overview of the article.
Introduction – the purpose of the present study.
Review of literature – present relevant literature related to the present scientific study and justifying the choice of the present study which is uncovered till date.
Methodology - define new terms and describe the instruments and procedures.
Result – report the findings with tables and figures.
Conclusions – support the present findings with relevant literature.
References - cited literature may reflect the investigator’s knowledge on the subject and validity of the present study with reference to relevant published information.
"Man is the interpreter of nature, and science is the right interpretation."

Systems Biology - concepts, properties and insights

 Systems biology sees an organism as a living system and not as a machine. Living organisms, societies and ecosystems are all systems. Living systems form multi-leveled structures. Each level consists of several subsystems. The molecules in the subsystems combine to form organelles, which in turn combine to form cells. The cells form tissues and organs, which themselves form larger systems like digestive or nervous systems. They altogether present a ‘stratified order’ of organization from molecules to human beings. Then people form families, tribes, societies and nations.

What are natural systems?

All the natural systems are integrated wholes, whose specific structures arise from the interactions and interdependence of their parts. Most natural systems are hierarchical: they are composed of smaller sets of systems made up of smaller interacting parts. The properties of a system cannot be reduced to those of its parts. A living system is capable of adaptation and evolution. In the evolutionary process, there is a progressive increase of complexity, coordination and interdependence. The living systems exhibit self-maintenance which includes the processes of self-renewal, healing, homeostasis and adaptation. They also show self-transformation and self – transcendence, a phenomenon that expresses itself in the processes of learning, development and evolution.

The living organisms have an inherent potential for reaching out beyond themselves to create new structures and new patterns of behavior. The stability of a living system is continually tested by its fluctuations. When a system is disturbed, it has the tendency to maintain its stability by means of negative feedback mechanism. The stability of a living system is never absolute.

Example of a natural system

Planet earth is an integrated system consisting of 4 main subsystems. The lithosphere consists of rocks and minerals that form the solid body of the earth. The atmosphere has a layer of air surrounding the earth’s surface. The hydrosphere forms water on and near the earth’s surface. The biosphere is a layer of living organisms of which human beings form a part.

What is a system?

The word ‘system’ is defined as a group of interrelated, interacting or interdependent constituents forming a complex whole. The components work together to perform a function e.g., a computer.

There are three propositions in the definition: 1.the system is made up of component parts; 2.the parts work together; 3. The whole thing serves some purpose.

A system is a set elements that are orderly and interrelated to make a functional whole e.g., social system

Boundaries are the borders that separates one entity from another e.g., skin. Homeostasis is the tendency of a system to maintain a relatively stable, constant state of balance.

Systems view – looks at the world in terms of relationships and integration.

Systems thinking –it is process thinking.

Systems approach – emphasizes basic principles of organization.

Concept of holon and holarchy

Holon implies wholeness. A holon is something that is simultaneously a ‘part’ and a ‘whole’. The term holon was coined by Arthur Koestler from the Greek ‘holos’ meaning whole (all) and the suffix ‘-on’ meaning an individual part. This concept describes something that is whole in itself and, simultaneously a part of a larger system. So the main characteristic of a holon is the duality of being both an autonomous whole, while also being a part of a larger whole. This larger whole does only exist by the combination and the interactions of the composing parts holons. In other words a holon is a whole to those parts beneath it in a hierarchy but a part to those wholes above it. For example we can see an organization as a holon, because it is made up of smaller systems (e.g., people), but it is also a part of a larger system ( e.g., the community or economy).
A holarchy is conceived of as a hierarchical structure of holons. Our body, society, nature, the earth and the universe are all examples of so-called holarchies. The holarchy is the grouping of parts to create larger wholes where wholes are greater than the sum of the composing pats and each part in itself is also a whole.
                              Organisms-->organs-->tissues-->cells--->molecules.

Objective of systems biology

The world of nature is often very complex. To understand this complexity, scientists try to envisage ‘systems model’. Models in science tend to be simplified representations of reality that can be explained mathematically and through the use of graphics.

                                 Inputs----àSystem----àoutputs

Common characteristics of systems

1.    All systems have some structure and organization.

2.    They are all some extent generalizations, abstractions or idealizations of the real world.

3.    They all function in some way.

4.    There are, therefore functional as well as structural relationships between the units.

5.    Function implies the flow and transfer of some material.

6.    Function requires the presence of some driving force or source of energy.

7.    All systems show some degree of integration.

Properties of biological systems

1.    A biological/ natural system is intrinsically dynamic in nature. They show a high degree of internal flexibility and plasticity. It is this flexibility that enables living organisms to adapt to new circumstances.

2.    The functioning of organisms is guided by cyclical patterns of information flow known as feedback loops. When a system breaks down the break down is usually caused by multiple factors that may amplify each other through interdependent feedback loops.

3.    A living organism is a self-organizing system, which means that its order in structure and function is not imposed by the environment but is established by the system itself.

4.    Living organisms are open systems which mean that they have to maintain a continuous exchange of energy and matter with their environment to stay alive.

5.    Living organisms have a high degree of stability with dynamic properties. The stability consists in maintaining the same overall structure in spite of ongoing changes and replacements of its components.

6.    The phenomenon of self-organization is dynamic with self-renewal and self-transcendence. Self-renewal is the ability of living systems continuously to renew and recycle their components, while maintaining the integrity of their overall structure. Self-transcendence is the ability to reach out creatively beyond physical and mental boundaries in the process of learning, development and evolution.

7.    Fluctuations play a central role in the dynamics of self-maintenance. All variables of the system oscillate between a wide range of upper and lower limits so that the system is in a state of continual fluctuations, even when there is no disturbance. Such a state is known as homeostasis.  It is a state of dynamic and transactional balance. Negative feedback is only one aspect of self-organization through fluctuations.

Types of systems

Systems can be divided into open and closed systems depending on the inputs and outputs of energy.Traditionally systems can be 4 types like morphological, cascading, and input-output and control systems.

Black box system-we only understand inputs and outputs

Grey box system – we understand internal working

White box system – we understand individual components, their flows and storages.
Thermodynamic systems classification
An open system exchanges matter and energy with its environment.
A closed system exchanges only energy with its environment.
An isolated system exchanges neither matter nor energy with its environment.

Key traits of environmental systems

1.    Openness – it exchanges matter and or energy with other systems.

2.    Integration – refers to the strength of the interactions among the parts of the system and

3.    Complexity- how many kinds of parts a system has.

   Traits of an ecosystem

   An ecosystem is basically an energy processing and nutrient-regenerating system. Plants and animal populations within the system represent the subsystems. Inputs into the systems are both biotic and abiotic. The abiotic inputs are energy and inorganic matter. Radiant energy influences temperature, moisture, seasonality and photosynthesis. Inorganic matter consists of all nutrients like water, carbon dioxide, oxygen and so forth that affect the growth, reproduction and replacement of biotic material and maintenance of energy flow. The biotic inputs include other organisms that move into the ecosystem.

Electrophoretic techniques for Bio-medical Researchers

Meaning of electrophoresis

Electrophoresis is a separation technique that is based on the movement of charged particles in an electric field. The term ‘electrophoresis’ was coined from the Greek word ‘phoresis’, which means ‘being carried’. Electrophorosis literally means ‘to carry with electricity’

Discovery of electrophorosis

In 1807,Russian physicist Alexander Reuss identified the migration of colloidal particles in an electrical field. In 1879, Hermann von Helmholtz generalized the experimental observations into an equation of electrophoretic principles. In 1930, Swedish chemist Arne Tiselius described the first electrophoretic system. He received the Nobel prize for chemistry in 1948 for his work.

Definition of electrophoresis 

Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field.

Principles of electrophoresis

Electro-migration 

At any given pH , the electrically charged molecules may exist in solution either as cations (+) or anions (-).Negatively charged molecules move to the anode(+).Positively charged molecules move to the cathode(-).Highly charged molecules move faster towards the electrode of opposite charge than those with lesser.
Electro-osmosis

The movement of entire fluid near wall of capillary in one direction!
anode (+ve) -> cathode (-ve)
Electromotive force(EMF) 

Electrophoresis is based on electromotive force(EMF) that is used to push or pull the molecules through the gel matrix. By placing the mixture of molecules in wells in the gel and applying an electric current, the molecules will migrate through the matrix. The separated molecules take directions based on the total electric charges.
Principle of velocity of migration of separated molecules

Velocity of migration of the molecules, v=E.q / f
Where E=electric field in volts/cm; Q=the total electric charge on the molecule; F=the frictional coefficient which is a friction of the mass and shape of the molecule.

Electrophoretic Theory

Two laws are relevant to the use of power supplies for electrophoresis of macromolecules: Ohm’s law and second law of electrophoresis.

 Ohm’s Law - Current (I)=Voltage (V)/Resistance (R)

Ohm’s Law states that current is directly proportional to the voltage and is inversely proportional to the resistance. Resistance of the system is determined by the buffers used, the type and configurations of the gels being run, and the total volume of all the gels being run.

      Second Law of electrophoresis - Watts (W)=Current (I) x Voltage (V)

     The Second Law states that power or watts (a measure of the heat produced) is equal to the               product of the current and voltage.  Since V=I x R, this can also be written as Watts=I² x R.

Factors influencing rate of migration of ion

The migration of ions in an electric field depends on the net charge of the molecule, size and shape of the molecule, buffer pH, strength of electrical field, properties of support media and Temperature of the operating system.

Buffers for electrophoresis

Barbitone buffer – (around 8.0 pH)- serum protein separation , poor resolution, weak buffer.
Phosphate buffer ( around 7.0 pH) - Enzyme separation,low buffering capacity.- high conductivity
Tris – borate – EDTA buffer (TBE) -(pH around 8.0) - Nucleic acid Separation,Good resolution , high buffering capacity , low conductivity.
Tris – acetate – EDTA buffer (TAE)- (pH around 8.0) - Nucleic acid separation, high resolution , high buffering capacity , low conductivity.
Tris – glycine buffer -(pH more than 8.0)- Protein separation, high buffering capacity , low conductivity

Support media for electrophoretic run

Paper – poor conductor of electricity absorbate proteins, non - transparent poor resolution.
Agar- flow of solvent electro endosmosis, vary thickness , transparent poor resolution
Cellulose acetate strip- tailing of bands poor resolution non-absorbing.
Starch- form opaque gels non-absorbing high resolution
Agarose -highly transparent porous – high resolution east preparation
Acrylamide – stable , non –reactive highly transparent.

Kinds of electrophoretic techniques

Zonal electrophoresis - Consden,Gordon and Martin in 1946 introduced this technique. Sample is applied as a narrow band. Separation occurs discrete bands. Numerous support media –paper, cellulose acetate, agar gel starch gel and acrylamide gel can be used.
Paper electrophoresis - This technique was introduced by Durrum (1950), Flynn and Mayo (1951). A small volume of the sample is placed evenly along a line drawn across a strip of Whatmann paper previously soaked in buffer. The ends of the paper are soaked in buffer solutions. Passage of electricity cause separation.
Starch gel electrophoresis - Starch matrix is suitable for isoenzymes . Partially hydrolysed potato starch is used. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining.
Cellulose acetate electrophoresis - Kohn (1957-1961) introduced this technique. Strips of cellulose acetate are used.Better resolving power. No absorption of proteins. No trailing. Excellent separation of plasma proteins, transparent.
Gel electrophoresis - Electrophoresis through agarose or polyacrylamide gels is a standard method used to separate, identify and purify nucleic acids. Gel electrophoresis involves the use of a gelatinous material such as agarose, acrylamide, starch or cellulose acetate as the matrix. The gel acts as a support medium for the sample. Gels are used to separate samples containing proteins or DNA.

     Starch Gel -- swollen potato starch granules.

     Agarose Gel is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool. 

      Polyacrylamide Gels -Polyacrylamide gel is made chemically by acrylamide (the monomer) and bisacrylamide (the cross-linker) catalyzed by initiator (amonnium persulfate or  riboflavin)  and accelerator (TEMED). Acrylamide can be polymerized into any desired shape :

•        Tube Gels -- polymerize in glass tubing ==> cylindrical shape

•        Slab Gels -- polymerize between glass plate 

Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes.

•        The separation medium is a gel made from agarose, which is a polysaccharide derivative of agar.

•        The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon charge, size and shape.

•         These characteristics,together with buffer conditions, gel concentrations and voltage, affect the mobility of molecules in gels.

  Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis 
 SDS- PAGE is a most widely used technique for analysis and characterization of proteins and nucleic acids.

•        Sample preparation – The protein sample is heated at 100⁰C in a dilute solution sodium dodecyl sulfate .This breaks down all native quaternary, tertiary, and secondary structures. Then b-mercapto ethanol is added to cleave the disulfide bonds.

•        Gel preparation – the polymerization is initiated by ammonium per sulfate or riboflavin. N-tetramethyl ethylene diamine (TEMED) catalyses the formation of free radicals from     persulfate which in turn initiate polymerization. Gels ranging from 3 to 30% acrylamide concentration can be made and can be used for the separation of molecules up to 1x10⁶ datons.

•        Sample application –about 2 µg of the sample is loaded in each well. Over loading of samples decrease the resolution of bands.

•        Marker dyes – to follow the sample tracking a marker dye e.g. bromophenol blue gives color. After run the gel was stained with the dye coomasie blue and photographed.

Applications of PAGE –

1.    PAGE is used to estimate molecular weight of proteins and nucleic acids.

2.    PAGE is used to determine the subunit structure of proteins.

3.    PAGE is used to purify isolated proteins.

4.    PAGE is used to investigate various liver and kidney diseases by analyzing human serum proteins.

5.    PAGE is used to monitor the changes in protein content in body fluids.

q Continuous - discontinuous  gel systems –

q  Continuous  system--gel and tank  buffers are the same, single phase  gel; examples are PAGE, agarose,  and starch gels.

q Discontinuous system--gel and  tank buffers are different, two phase gel (stacking gel); example  is PAGE.

Discontinuous polyacrylamide gel electrophoresis –DISC-PAGE -

Two gel systems – a stacking gel and a running gel

Several buffer systems

·         Cathode -  Tris – glycine 8.6 pH

·         Wells – Tris – Cl 6.5 pH

·         Stacking gel – Tris – Cl 6.5 pH

·         Separating gel – Tris – Cl 8.7 pH

·         Anode  - tris – glycine 8.

Generation of voltage discontinuity

Uses of electrophoresis techniques 

•         Human DNA can be analyzed to provide evidence in criminal cases, to diagnose genetic diseases, and to solve paternity cases.

•         Samples can be obtained from any DNA-containing tissue or body fluid, including cheek cells, blood, skin, hair, and semen.

•        A person’s “DNA fingerprint” or “DNA profile” is constructed by using gel electrophoresis to separate the DNA fragments from several of its  highly variable regions.

•        Conservation biologists use DNA profiling to determine genetic similarity and kinship among populations or individuals.

Chromatographic techniques for Bio-medical researchers

Meaning of chromatography

Chromatography is the science of separation techniques . The technique is used to fractionate mixture of gases, liquids  or dissolved solids. The name chromatography (Greek: chroma means color and graphein means writing) literally means writing in color. In other words writing out the ‘signature ‘ of a mixture in color.

Definition of chromatography

Chromatography is defined as  a physical separation technique used to separate macromolecules  based on differences in their structure and / or composition i.e.their size, shape or charge (Heftmann 1992 ). Chromatography is a dynamic separation system which partitions chemical substances between two phases (Biphasic system)-a stationary phase (SP) and a mobile phase (MP). Chromatographic – like separation processes occur in nature

–for e.g. migration of water through soil  results in purification of water. Chromatography is a science which studies the separation of molecules

Discovery of chromatography

The Russian botanist Mikhail S. Tswett (1872-1919) found that pigment composition became separated when plant pigment (chlorophyll) together with petroleum ether went through calcium carbonate layer. Chromatography is a method  in which the components of a mixture are separated on an adsorbent column in  a flowing system (Tswett,1906)

Biphasic systems of chromatography 

 Stationary phase consists of small solid particles with micro porous surface. Mobile phase can  be a gas or a liquid which carries the components of a mixture. The rate of movement of a given component of a mixture depends on the degree of solubility  in the solvent system. More soluble substances travel more slowly down the column than the less soluble.

Rf   value : relative front 

The relation of the distance traveled by compound to that of the solvent front is called Rf value. Parameters influencing Rf value includes temperature, solvent system, direction of flow and type of paper.
Rf value   = Distance   traveled  by the solute

                        --------------------------------------

                   Distance traveled by the solvent   

Kinds of chromatographic techniques            

Column chromatography
A mixture of components dissolved in a solvent is poured over a column of solid adsorbent . The column is eluted with the same or a different solvent. The stationary phase is solid. The mobile phase (the eluent) is liquid.

Paper chromatography

        The paper adsorbs water from the atmosphere of the developing chromatogram. The water is the stationary phase. The eluting solvent is the mobile phase.

Chromatographic  principles   of   separation

1- Elution development - The components of the mixture are separated into zones by the passage of one or more solvents through the column. This technique is most widely used in GC, GLC, LLC and LSC.
2. Gradient elution - A gradual change in composition of the eluting solvent is used to achieve separation of compounds of widely varying affinities for the stationary phase. The solvent composition gradient may be linear with increasing or decreasing concentration, pH, polarity or ionic strength.
3. Frontal analysis - No solvent is used for irrigation. The solution itself is added continuously.
4. Displacement analysis - The components in the mixture are adsorbed on the column. The irrigation of the column is carried out with the solution of another substance, having a higher preferential adsorption on the column than that of the components of the mixture sought.

Research  applications  of chromatography

There are two kinds of research applications .i.e. analytical and preparative applications. Analytical application is to determine the chemical composition of a biological sample. Preparative application is to  purify and collect one or more components of a biological sample.

Significance of chromatographic methods

•         They serve to resolve and  identify the separated components of a mixture.

•         Very small quantities of substances could be analyzed qualitatively and quantitatively.

•         The equipment is very simple except HPLC

•         No special skill is required for performing the method

•         The results are remarkably reproducible.

Histological techniques for Bio-medical Researchers

Histotechnology

It is the preparation of tissues for microscopic examination. It is an effective diagnostic tool in clinical pathology. Histological  preparations  reveal normal tissue structure, tissue abnormalities  and cancerous conditions.

Branches of histotechnology

—  Histology- the microscopic study of the normal tissues.

—  Histopathology – the microscopic study of  tissues affected by disease.

—  Histochemistry – the techniques provide information on the chemical composition of parts of tissues.

—  Cytochemistry – the techniques provide information on the chemical composition of parts of cells.

Steps in the processing of tissues

1.    Fixation – preservation of tissues in its original condition.

2.    Dehydration – removal of water from tissues.

3.    Clearing – infiltration of paraffin solvent.

4.    Embedding – infiltration of paraffin wax.

5.    Microtomy – preparing thin slices of tissues.

6.    Staining – colouring of tissues.

7.    Mounting – arranging tissues on slides.

     Meaning of fixative 

A fixative is described as a chemical substance which will preserve the shape, structure, relationship and chemical constituents  of tissues and cells after death.

Purpose of fixing agents

1.    To kill and preserve living tissues.

2.    To stabilize the tissue and cell structure for subsequent treatments( wax embedding, sectioning, mounting).

3.    To prepare tissue for staining and optical contrast.

4.    To harden the tissue for section cutting

Requirements of a good fixative

1. Penetrate the tissue and cells rapidly and evenly.
2. Prevent autolysis and bacterial decomposition.
3. Preserve tissues in their natural state and fix all chemical cell components ( proteins, carbohydrates, fats etc.,)
4. Preserve cell volume.
5. Avoid excessive hardness of fixed tissue.
6. Allow enhanced optical differentiation by staining.
7. Make the cellular components insoluble to liquids used in tissue processing.
8. Be nontoxic and non-allergenic
9. Providing iso-osmotic conditions to the tissues.

General principles of fixation

Amount of fixing fluid should be approx. 10 to 20 times more than the volume of tissue held in a container  with a required fixation time. Temperature has an important effect. A lower temperature retard fixation –reduce autolytic reaction. A higher temperature will decrease the required for fixation but will increase autolysis.

Methods  of  Fixation 

—  Fixation by heat  - this denatures and coagulates proteins resulting in some distortion

                              but is useful in fixing smears.

—  Cryostat (freezing) fixing - it does not denatures proteins and minimizes distortion.

                               useful in locating particular chemicals - histochemistry

—  Fixation by chemicals - chemical fixatives are used.

Simple fixatives or primary fixatives or unmixed fixatives

Formalin-  non-coagulant fixative,acidic, cheap,easy to prepare, relatively stable

Mercuric chloride-Coagulant fixative,—  black precipitate in tissues

Glacial acetic acid - Protein precipitant,Colourless solution, Pungent smell

Ethyl alcohol-  Colourless liquid, Reducing agent

Osmium tetroxide- Strong oxidizing agent, Expensive, poor penetration

Potassium dichromate - Strong fixative,Fix lipids

Trichloro acetic acid -  Protein precipitant

Picric acid -  Protein precipitant,—  Used as saturated solutions

Micro-anatomical fixatives

—  10% formal saline

—  10% neutral buffered formalin

—  Heidenhain’s  Susa

—  Formal-sublimate

—  Formal-saline sublimate

—  Zenker’s fluid

—  Helly’s fluid

—  Bouin’s fluid

—  Gendre’s fluid

Physico-chemical features of fixatives

—  Degree of ionization

—  Oxidation-reduction potential

—  Reactions with proteins, lipids, carbohydrates

—  Rate of penetration

—  Shrinkage or swelling

—  Degree of hardening

—  Methods of washed out

—  Effect on staining

—  Compatibility with other fixatives

Paraffin wax technique of tissue blocks

—  Dehydration-the alcohol method

                                  -the acetone method

                                  - the dioxane (diethylene dioxide) method

—  Clearing- de-alcoholisation -clearing agents- xylene, benzene, toluene, chloroform.

—  Embedding- blocking – out in wax-wax impregnation

—  wax-wax impregnation- It can be cold wax infiltration and melted wax infiltration.

—  Complete wax infiltration is essential for the production of good sections.

—  Hard tissues requires a higher melting point wax.

—  Number wax changes and time in each wax change, depend upon the density and size of the tissue.

—  Embedding media – wax, gelatin, celloidin, polyester wax.

Microtomes—

The microtomes cut the tissue at a pre-determined uniform thickness. These instruments are designed for the accurate and serial cutting of thin slices of tissue. Several models are available – sliding, rotary, rocking ultra- thin microtomes.

Processing of paraffin – embedded tissues

Fixing tissues-> Post-fixation treatment->initial dehydration->complete dehydration-->Dealcoholization->wax impregnation->wax embedding->trimming the block->microtomy->drying sections-->de-paraffinization->hydration- down grading->staining->dehydration- up grading->de-alcololization->Mounting-->observing in microscope.

—Staining

Staining is used to obtain contrast between the constituent parts of a tissue section. The depth of colouration is affected by chemical affinity, density, and permeability. Certain stains are metachromatic –i.e. they are capable of imparting one colour to certain constituents and another to others.