Citric acid production and applications of submerged fermentation

Submerged fermentation is a type of fermentation in which the microorganisms are suspended in a liquid medium. The liquid medium also contains various other nutrients and growth factors in the necessary proportions in a dissolved or a particulate solids form.

The main application of submerged fermentation technique is in the extraction of metabolites (secondary metabolites) which are needed to be in liquid form for use.

APPLICATIONS OF SUBMERGED FERMENTATION

• The primary application of submerged fermentation is in the extraction process of metabolites (mostly secondary metabolites) that find applications in their liquid form.
• Citric acid is one of the most important metabolites as the production volume of it is high, for the production of antibiotics like penicillin.
• Submerged liquid fermentations are traditionally used for the production of microbially derived enzymes like cellulolytic enzymes.

CITRIC ACID PRODUCTION

• Citric acid is widely distributed in plant and animal tissues.
• It is an intermediate of the Krebb’s cycle, by which carbohydrate gets converted to CO2, in nature.
• Citric acid can be produced on the industrial scale by employing submerged state fermentation as the fermentation method.

Type of bioreactor used for submerged fermentation: 

1. Stirred tank bioreactor
2. Airlift fermenter.

Selection of strain and storage:

• Various criteria should be checked for the selection of production strains such as:

-High citric acid yield.

-Stability of the strain.

-Adequate amount of sporulation, etc.

Microorganisms used for the production of citric acid:

-Species of Penicillium and Aspergillus.

–Aspergillus niger is used as the principal fungus for citric acid production as it can produce large quantities of citric acid while growing on a carbohydrate medium. 

• Maintenance of the culture of the selected strain is the next important step in citric acid production and is done so by the storage of spores.

Steps used to carry out fermentation to ensure abundant production:

-High sugar concentration.

-Limited nitrogen/phosphorus concentration.

-Very low concentration of heavy metals like iron and manganese.

Submerged fermentation process:

-The strain used for the submerged fermentation of citric acid is Aspergillus japonicus.

-The organism shows sub-surface growth.

-Citric acid is produced within the culture solution.

-Using submerged fermentation for the production of citric acid is economical as compared to other fermentation methods.

Uses of citric acid:

• It Is extensively used in the production of carbonated drinks.
• It is used in plasticizers.
• It is used as a chelating and sequestering agent.
• Used in the pharmaceutical and food industries as an acidulant.

ADVANTAGES 

The advantages of submerged fermentation include:

• The duration of the process is short, therefore saves time.
• The overall cost of the process is low and the yield of products is high, making it a very economical process.
• The process of purification and processing of the products is far simpler compared to other processes.
• The cost of handling is low and the handling of the fermenter is easy therefore it reduces the labour involved.

LIMITATIONS

• The overall volumetric productivity of this process is low.
• The effluent that is generated during the process is high in quantity.
• The equipment that is used is expensive and complex.
• The products that are obtained by using this process may be of low concentration.

Article by– Shaily Sharma (MSIWM041)

Sources:

https://en.wikipedia.org/wiki/Fermentation

https://microbenotes.com/submerged-fermentation

FLUORESCENCE MICROSCOPY

Fluorescence microscopy is an essential tool in molecular and cellular biology. It is a technique that allows one to study and visualize the cellular structures and dynamics of tissues and organelles, and macromolecular assemblies inside the cell. It was devised in the early twentieth century by various scientists like Köhler, Lehmann, Reichert and others.

The wide utilization of fluorescent proteins since their discovery have revolutionized the applications and use of the microscope in biological studies.

A fluorescence microscope uses the property of fluorescence to generate an image. It uses a high-intensity light source that excites the fluorescent molecule that may be inherently present in the sample to be studied or may be artificially labelled with a fluorescent molecule. The fluorescent molecule is called the fluorophore which is usually present in the fluorescent dye. 

Therefore, one could say that any microscope that works on the same basis to study the properties of organic or inorganic substances is a fluorescent microscope.

A fluorescence microscope is a type of optical microscope that uses fluorescence (ability of a substance to emit light on excitation) and phosphorescence (ability of a substance to continue emitting light even after the removal or withdrawal of the excitation factor). It may use these properties instead of or in addition to the properties of scattering, absorption, reflection and attenuation. 

The setup for the microscope may be simple as in an epifluorescence microscope or it may have a complicated design like that of a confocal microscope. A confocal microscope uses optical sectioning to provide a better resolution of the fluorescence image.

Principle:

Fluorescent substances are the substances that absorb light of a particular energy and wavelength and then emit light of a longer wavelength and lesser energy.  

This phenomenon of fluorescent substances can be applied to the working of the fluorescent microscope. Fluorescent dyes (also called fluorochromes or fluorophores) are molecules that have the ability to absorb excitation light at a given wavelength, and then emit light of a comparatively longer wavelength after a delayed time interval.

In practical use, the sample is stained with a fluorescent dye and then illuminated with a blue light. The blue light (short wavelength) is absorbed by the fluorophores of the fluorescent dye, and the green light (which has longer wavelength) is emitted. This change is called the Stokes shift.

The light source that is used in fluorescent microscopy is a high intensity mercury arc lamp. The lamp emits white light when then passes through a device called an ‘exciter filter’. (as shown in the figure) This device filters the emission light to reveal the location of the fluorophores. It allows only the blue component of white light (white light comprises of coloured light of all wavelengths) to pass through and prevents the passage of light of other colors.

.

The dichroic mirror is used to reflect the blue light and allows the green light to pass. The angle of the mirror is fixed in such a way that the blue light is reflected towards the specimen placed below. It allows the passage of green light.

Finally, when the light reaches the ‘barrier filter’, it blocks out or removes all the remnants of the residual blue light from the specimen which may not have been ideally reflected by the dichroic mirror.

Thus, enabling the observer to perceive the glowing green portions of the specimen against the jet-black background of the dark field condenser that is used. The portions of the specimen that have not been stained remain invisible to the eye and this is how fluorescence microscopy provides a sharp image for the observation of the fine and intricate components of the sample to be studied.

Components:

The essential components of the fluorescence microscope are:

• Fluorescent dyes (fluorophore): Chemical compounds that have the ability to re-emit light upon excitation. Examples include; nucleic acid stain like DAPI and Hoechst, phalloidin etc.
• Light source: This is provided by a bright mercury vapor arc lamp, xenon lamp or LEDs with a dichroic excitation filter, lasers etc.
• Heat filter: The lamp produces infrared rays which generate considerable heat. No other major uses of the heat filter exist.)
• Exciter filter: The light undergoes cooling and passes through the exciter filter which allows the passage of the shorter waves which play a role in excitation of the fluorochrome dye coated sample on the slide and does not allow the other wavelengths to pass through.
• Dichroic mirror: An accurate colour filter/mirror which selectively allows the passage of light of a particular wavelength and reflects the others. 
• Condenser: A dark field condenser is usually used because it provides a dark background and it is easy to detect even mild fluorescence exhibited by the sample
• Barrier filter: It removes all the remnants of the exiting light and is situated in the body tube of the microscope between the objectives and the eye piece. 

Applications: 

• Identify structures in fixed and live biological samples in microbiological studies.
• Used in food chemistry for the assessment of the structural organization and spatial distribution of the components of food.
• Used for the study of mineral like coal and graphene oxide in minerology.
• Used in the textile industry for analysis of fibre dimensions. 

Article By- Shaily Sharma (MSIWM041)

References:

Fluorescence Microscopy (nih.gov)

Immunofluorescence staining – PubMed (nih.gov)

Fluorescence Microscopy – Explanation & Labelled Images (microscopeinternational.com)

Vaccine

QUIZ ON VACCINE

Que 1. Vaccine is prepared by

     a. weakened microorganism              b. toxins

        c. Surface protein                           d. all of the above

Que 2. The term vaccine and vaccination are coined by-

         a. Edward jenner                             b. Louis Pasture

         c. Robert Koch                                    d. Alexander Fleming

Que 3. Vaccine stimulates-

          a. T-cells                                               b. B-cells

          c. None of the above                      d. Both A and B

Que 4. Edward Jenner uses _______ to confer immunity against smallpox

          a. Polio virus                                     b. Cowpox virus

         c. HIV virus                                          d. Influenza virus

Que 5. _____ vaccine containing live organism which is weakened in the lab so that it cannot cause disease and activate the immune system against the antigen.

         a. Live attenuated                   b. Killed or Inactivated

         c. Subunit                                d. DNA

Que 6. Microorganism causing diseases are killed by the means of chemicals, heat or radiation. These are more stable and safer than live vaccines reason is that the dead microorganism cannot mutate back to cause diseases. Such type of vaccine is known as

         a. Live attenuated vaccine                  b. Killed or Inactivated vaccine

        c. Subunit vaccine                               d. DNA vaccine

Que 7. In ________ vaccine only the part which server as antigen and stimulate the immune system is used to prepare vaccine.

         a. Live attenuated                   b. Killed or Inactivated

         c. Subunit                                d. DNA

Que 8. The _____ vaccine is the DNA sequence used as vaccine.

         a. Live attenuated                   b. Killed or Inactivated

         c. Subunit                                d. DNA

Que 9. Examples of live attenuated vaccine-

        a. Mumps vaccine                   b. Measles vaccine

        c. Chickenpox                         d. All of the above

Que 10. Examples of DNA vaccine

        a. West nile virus                     b. Herpes virus

        c. Both A and B                      d. None of the above

ANSWERS

1. (D), 2. (A), 3. (D), 4. (B), 5. (A), 6. (B), 7. (C), 8. (D), 9. (D), 10. (C)

For detail study click on the link– Vaccine

PHYTOHORMONES

By: N. Shreya Mohan (MSIWM042)

Plant hormone, also known as phytohormones are signal mediated molecules produced by plants commonly controlling plant growth aspects such as defense mechanisms, stress tolerance, metabolism, reproduction and size. Hormones are elated within the plant by utilizing four types of movements. For localized movement, cytoplasmic streams within cells where delayed diffusion of ions and molecules between the cells are utilized. Vascular tissues are used to commute hormones from one part of the plant to another. These include phloem that move sugars from the leaves to the roots and flowers, and xylem that moves water and mineral solutes from the roots to the foliage of the plant respectively.

We will ponder over the major 5 plant hormones systematically and briefly-

• AUXINS– This hormone particularly takes part in cell enlargement, cell growth bud formation. It was the first hormone to have been discovered among the “big 5”. In collaboration with other hormones, auxins promote control the growth of stems, roots etc. It is primarily produced in certain parts of plants that are actively growing such as the stem. Auxins act in such a way that it inhibits growth of buds lower than the stem. This phenomenon is the apical dominance. In seeds, they promote certain protein synthesis, to which they develop after inside the flower after pollination resulting in fruit production. The most common found of auxin in plants are indole-3-acetic acid

• GIBBERELLIN- These set of chemicals are produced naturally by the plants and fungi too. It was first discovered by Japanese researchers where they noticed a certain chemical compound caused by a fungus called Gibberella fujikuroi that produced abnormal growth and falling over in rice plants. The chemical causing this was isolated and named Gibberellin ever since. They play a vital in plant life by making the stems longer by elongating the nodes between the stems. They are also required by the pollen during the process of fertilization.

• ABSCISIC ACID- Also known as the ABA. The chemical is usually abundant in chloroplast, thereby produced in the leaves of the plants particularly when the plant is under stress. It acts as a plant growth inhibitor and affects bud and seed dormancy. Without ABA, the seed would grown in warm temperatures during winters and can get killed if frozen. Therefore, plants start off as a seed with high concentrations of ABA. During water stress, ABA plays a pivotal role in plants. The water deficient plant sends a signal in via the root to the leaves, causing the ABA precursors to act and move back to roots, which ultimately closes the stomata from further transpiring.

• CYTOKININS- These group of chemical compounds are responsible for shoot formation and cell division. They are also responsible for mediating auxin transport throughout the plant. They also help delay senescence. They were initially named kinins as they were isolated from yeast. Cytokinin counter the apical dominance as created by auxins. They, with conjunction with ethylene promotes abscission of leaves, flowers.

• ETHYLENE-It is readily found in fast and dividing cells. They have very little solubility because they are gaseous in nature thereby diffusing easily from the plant. The concentration of ethylene depends on the amount of it diffusing and leaving the plant. The main role of ethylene is fruit ripening.

REFERENCES-

Plant Growth Hormones

https://en.wikipedia.org/wiki/Plant_hormone

STEM CELLS

                     By: N. Shreya Mohan (MSIWM042)

Stem cells are a group of undifferentiated cells that will differentiate into various types cells and proliferate indefinitely. They originate from cell lineages. They exact opposite to the progenitor cells, which does not proliferate indefinitely. In mammal, typically about 50-150 cells combine to form the inner cell mass (ICM) during the blastocyst stage of the embryonic development. These cells are stem cells too, having the ability to differentiate into various cell of the body. But this process is characterized by differentiating into there germ layers (layers that differentiate and give rise to tissues, organs). The three germ layers are ectoderm, endoderm and mesoderm particularly clear in gastrulation stage. These can systematically be isolated and cultured invitro during the stem cell stage and they are known as embryonic stem cells (ESCs). Parallelly, adult stem cells are found in particular areas such as the bone marrow or gonads. Their purpose, unlike the ESCs is to replenish the lost cells of the body, most common stem cells are the hemopoietic stem cells, which replenish blood and immune cells. Mesenchymal stem cells maintain bone cartilage and fat cells. The term “stem cell” was given by Theodor Boveri and Valentin Hacker during the 19^th century. The properties of the stem cell were given by Ernst McCulloh and James Till. We will ponder over the properties too-

• Self-renewal- The ability of the cell to undergo numerous cycles of division and cell growth is known as cell proliferation. This should be done while the undifferentiated state.
• Potency- The capability and power of the cell to differentiate into specialized cell types. Whether it be totipotent, pluripotent, multipotent or unipotent.

Potency refers to the potentiality to be able to differentiate into respective cell types. We will dive into a brief cognizance of each of the potent type:

1. Totipotent- Also known as omnipotent, these stem cells into embryonic as well as cells which are not embryonic. These cells have the ability to make a complete, functional organism. Cells include the product between the fusion of sperm and egg and the cells made after the first few divisions.
2. Pluripotent- They are the known “ancestors” of totipotent cells. They can differentiate into almost all cells, specifically, the cells derived from the three germ layers.
3. Multipotent- These cells will differentiate those type of cells that are closely related to each other.
4. Oligopotent- These cells will only differentiate into particular cells, such as the myeloid stem cells or the lymphoid cells.
5. Unipotent- These cells do not differentiate into any cell, but they do have the property of self-renewal (unlike the progenitor cells).

Stem cell therapy- a boon or a bane?

A filed with high scope, stem cell therapy is being used to treat diseases. Bone marrow replacement is one of a stem cell research which has proven effective in clinical trials. One good advantage of getting treated under this is that they lower symptoms of the disease to some extent. This leads to reduced intake of drugs required to supress the disease. One con is that the patients may require immunosuppression because the patient undergoes radiation before the transplant to remove the existing cells. This can prove detrimental chronically. For ESCs, ethics come into play as few argue that killing a new lifeform is considered unethical. 

Therefore, we should look into several parameters and be aware for it run without hurdles because stem cells have a lot of scope in the future.

 REFERENCES-

https://en.wikipedia.org/wiki/Stem_cell

https://www.medicalnewstoday.com/articles/323343

BIOTECHNOLOGY

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

In the emerging field of science and technology, Biotechnology is Growing and Developing field, where new ideas and Experiments and research make this field unique and diversifying. 

Biotechnology: The branch of science which uses technology with living system is biotechnology. Biotechnology uses modern system of modification of biological systems. There are many disciplines that belong to the field of Biotechnology. The development of various methods, approaches and research in this field gives a new way of approaching science and its outcomes.

Biotechnology an accidental history: 

Even though we consider biotechnology to be a modern science, but it was way 1000 years back when the methods and approaches where used by our ancient people, Around 7000 years ago there was accident use of bacteria to make vinegar by Mesopotamia. Before 2,300 years Theophrastus thought that brad beans left magic in soil, but later it was concluded that some bacteria’s could fix nitrogen which enriched the soil. Development of gene banks is not a new concept, In1495 BC Queen Hatshepsut of Egypt used the concept of collecting specimens of plants which produced Frankincense (hardened gum-like material from trunk of the Boswellia sacra tree). Fermentation was always an ancient method which evolved with upcoming generation. In 19^th century Sir Louis Pasteur discovered the fermenting beer using yeast. Gregor Mendel the father of genetics, was the one who believed that mathematics can be used with biology, but since his ideas and concepts were new and people considered it unbelievable was never awarded during is period. 

Evolution of Biotechnology :

• 6000 BC :Babylonians used yeast in beer industry 
• 320 BC :  Aristotle coined the theory of inheritance from father 
• 1630 : William Harvey explained sexual reproduction
• 1673:  Anton van Leeuwenhoek developed Microscope., identified that these microorganism
• 1859: Charles Darwin  Proposed Natural selection
• 1863 : Pasteurization discovered by Pasteur
• 1863 : Pasteurization discovered by Pasteur
• 1870: Mitosis discovered by Walter  Flemming
• 1880: Louis Pasteur discovered weak stain of Cholera
• 1902: Sutton discovered that segments get transferred from Chromosomes
• 1906: Salvarsan was discovered by Paul Ehrlich 
• 1907: Mutation theory by Hunt Morgan 
• 1909:Wilhelm Johannsen Coined word genotype and phenotype
• 1912:William Lawrence Bragg Discovered application of X-Rays
• 1926: The Theory of gene by Morgan
• 1928: Transforming principle by Fredrick Griffith
• 1941: George Wells Beadle and Edward L Tatum proposed one gene one enzyme theory 
• 1944: Selman Abraham Waksman discovered streptomycin as antibiotic
• 1945–1950: Animal tissue culture developed
• 1947: transposable elements  by Barbara MacClintock
• 1950: Chargaff rule
• 1953: Double helix model by Watson and crick
• 1957:Crick and Gamov studied ‘central dogma
• 1972: First recombinant DNA molecule
• 1973: Ames test
• 1990: Human Genome Project commencement 
• 1993: Kary Mulis developed PCR

Biotech Industries: 

1.  Genentech Inc. : This Company produced somatostatin in a bacteria in 1977
2.  Eli Lily : produced insulin using site directed Mutagenesis
3. Chiron crop: developed recombinant vaccine for hepatitis
4. Calgene Inc. : tomato polygalacturonase DNA used to synthesize antisense RNA
5. Novo Nordisk : focus mainly on Diabetes and hormone replacement therapy
6. Regeneron : Aims to develop largest gene sequencing
7. Alexion : develop immune-regulatory drugs
8. Biomarin  : Develop drugs for lysosomal storage disorder
9. Alkermes : Treatment for central nervous disorder
10. Ionis : Develop  RNA-based therapeutic products

 Top Indian Biotech Industries 

1. Biocon Limited:  Manufacture biotechnology products
2. Serum Institute of India: Worlds largest vaccine manufacturer
3. Panacea Biotec : 3^rd largest Biotech company

Scope of Biotechnology : 

Since, Biotechnology shares an integrated value with many other disciplines of science , it holds a very key and vital role in the field of science. The various fields associated with biotechnology is as follows 

“Biotechnology is the new brightest star in the field of techniques and Biology”- E Mithu  

DIABETES

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

Diabetes is a metabolic disorder characterized by hyperglycaemia which results in a lack of insulin secretion, insulin action, or both the conditions. Metabolic abnormalities are caused due to a low level of resistance to insulin. The effect of symptoms can be classified based on the type and duration of diabetes. Diabetes has also been associated with many metabolic disorders such as acromegaly and hypercortisolism for example insulin resistance has been observed in patients with acromegaly in the liver. Hypercortisolism (Cushing syndrome) produces visceral obesity, insulin resistance, dyslipidaemia which leads to hyperglycaemia and reduces glucose tolerance. Besides, diabetes been associated with metabolic disorders, clinical convergence between type 1 diabetes (T1D), and type 2 diabetes(T2D) is also observed. T2D patients develop a progressive decline in total beta-cell mass. Thus there are many interlinked complications due to diabetes.

According to the report by WHO 2019, 10 main issues demand attention one of them is noncommunicable diseases such as diabetes, cancer, and heart disease. These are collectively responsible for 70% of deaths worldwide. According to the National Health Portal, the Government of India, nearly 5.8 million deaths occur due to noncommunicable diseases in India (WHO 2015). As per data provided by Directorate General of Health Services Ministry of Health & Family Welfare, Government of India (MoHFW) 2016-2017, 2.24 core persons were screened for Common noncommunicable diseases like diabetes, hypertension, cardiovascular disorders, and common cancers. From this, 9.7 % was diagnosed to be diabetes, 12.09% was diagnosed to be hypertension, 0.55% was diagnosed to be cardiovascular disease and 0.17% was with common cancers.

Events occurred from discovery of Diabetes to development of various drugs 

YEAR	EVENTS
1552 BC	HESY-RA documented urination as symptom of mysterious disease
133 AD	Araetus of Cappodocia coined the word diabetes
1675	Thomas Willis coined the word mellitus
1776	Dobson confirmed presence of excess sugar in patients
1800	Discovered chemical test for presence of sugar in urine
1700’s and 1800’s	Physician began to realize dietary changes help manage diabetes
1857	Claude Bernard confirmed that the diabetes occur due to excess glucose production
1870’s	During Franco Prussian war French physician Apollinaire Bouchardat proved that the diabetes patients symptoms improved due to war related food rationing
1889	Oskar Minkowski and Joseph Von Mering extract obtained from dogs pancreas
Early 1900	Development of oat cure, potato therapy, starvation diet.George Zuelzar injected pancreatic extract to control diabetes
1916	Boston scientist Elliott Joslin wrote book “ The Treatment Of Diabetes Mellitus “
1922	Frederick Banting discovered insulin to treat diabetes and won Nobel Prize in medicine 1923
1978	Production of recombinant human DNA insulin
1996	For the treatment of type 22 diabetes Thiazolidinediones (TZDs) were introduced.
2005	The  amylin analogue known as pramlintide, which was approved by the FDA
2008	Colesevelam approved for type 2 diabetes by FDA
2009	Bromocriptine approved for diabetes
2013	Canagliflozin  is the first SGLT- 2 inhibitor  approved by FDA  [Sodium Glucose Co-Transporter 2 Inhibitors], Dapagliflozin approved in 2014 by FDA

(Source: Saudi Med et al., 2002, John et al., 2014)

Diagnosis of Diabetes: 

There are several methods used for the diagnosis of Diabetes Mellitus. According to American Diabetes Association (ADA) the most standard diagnostic criteria is as follows 

1. Hemoglobin A1c (HbA1c)
2. Fasting Plasma Glucose (FPG)
3. Oral Glucose Tolerance Test (OGTT)

 Hemoglobin A1c (HbA1c):

The average level of blood sugar over past two to three months can be diagnosed using hemoglobin A1c test. The main advantage of this type of diagnosis is that there is no need of fasting. A1c is measured using percentage The standard referred by ADA for normal person is less than 5.7%.

 Diagnosis of Diabetes by checking Hemoglobin A1c (HbA1c)

	Hemoglobin A1c
Normal	Less than 5.7%
Prediabetes	5.7% to 6.4%
Diabetes	6.5% higher

Fasting Plasma Glucose (FPG):

It is used to check fasting blood sugar levels. The patient should fast for 8 hours before the test. It is mainly done during morning. For normal person the FPG is lower than 100mg/dl.

Diagnosis of Diabetes by checking Fasting Plasma Glucose (FPG)

	FPG
Normal	100mg/dl or less
Pre diabetes	100 mg/dl to 125 mg/dl
Diabetes	126 mg/dl or high

Oral Glucose Tolerance Test (OGTT)

This method is used to diagnose blood sugar level before and after 2 hours of a sweet drink. For normal person the OGTT is less than 140mg/dl

 Diagnosis of Diabetes by checking Oral Glucose Tolerance (OGTT)

	OGTT
Normal	140mg/dl or less
Pre diabetes	149 to 199mg/dl
Diabetes	200 mg/dl or high

LYMPHOCYTE CULTURE

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

Cells circulating in body and part of immune system are known as lymphocytes. There are mainly two main types of lymphocytes, which includes B Cells and T cells. 

Peripheral Blood Lymphocyte culture: 

PBLC is a technique used in Cytogenetics used for karyotyping analysis. The most convenient method to study chromosomes and by using cell culture technique it can be helpful for clinical and research purposes.  The powerful mitogen for human T cell is PHA (Phytohaemagglutinin).PHA is a plant hormone, which is obtained from Red Kidney bean. The function of PHA is to bind to T Cell membrane, which stimulates metabolic activity.  It is important to obtain cells at metaphase plate, since the study of Chromosome is possible and also easy to stain the chromosome at metaphase stage. During metaphase chromosomes are attached to spindle fibres.

Procedure for PBLC

• Cell culturing should be done in a laminar Hood to avoid contamination. 
• 1 ml of Venous blood should be collected 
• Take 7 ml of RPMI1640 ( reconstituted with 10% fetal calf serum) in sterile culture tube
• Add 0.5 ml blood in culture tube
• Add 0.1 ml of PHA ( Previously thawed) 
• Incubate this tube in sterile CO2 Incubator at 37°C FOR 69 hours
• Add 40 µL of Colchicine at 69:00 hours
• Incubate for 30minutes at 37°C
• After Incubation centrifuge at 2000 rpm for 15 minutes
• Remove supernatant and add 5 ml of Hypotonic solution
• Mix gently and Incubate in waterbath for 37°C for 20 minutes
• Add ice cold fixative (1:3 acetomethanol) and flush thoroughly
• Centrifuge at 2000rpm for 15 minutes and remove supernatant
• Repeat this above two steps for 3-4 times
• Mix final suspension of 1 ml of fixative and make drop preparation in cold wet slide
• Fix on flame and stain with 4% Giemsa
• Observe and Scan for well spread metaphase plate in microscope

Function of each reagent

1. PHA: it binds to membrane of T Cell and stimulate metabolic activity and cell division
2. Colchicine: Suppress mitosis and cell division, but there is no interruption of chromosome multiplication
3. Hypotonic solution : Used for swelling of cells
4. Aceto-methanol: Helps in hindering cells at metaphase stage 

AFFINITY CHROMATOGRAPHY

BY: SHAILY SHARMA (MSIWM041)

Chromatography is a technique which is widely used for the separation of mixtures based on the physio-chemical differences between the stationary and the mobile phase of the chromatographic apparatus. The sample to be separated is dissolved in the mobile phase, which passes over the stationary phase (the immobile/fixed surface) and carries out the process of separation of molecules. 

Most chromatographic processes employ and rely on these physio-chemical differences between the stationary and mobile phases for the process of separation of molecules. For example, gel permeation chromatography, ion exchange chromatography etc.

However, an exception is affinity chromatography. Unlike gel permeation and ion exchange chromatography, affinity chromatography exploits the tendency or capacity of certain biomolecules to bind specifically and non-covalently to other molecules called ligands (i.e., “bio-specificity of molecules)

PRINCIPLE:

One of the most familiar concepts for anyone involved in the studies of biochemistry is “bio-specificity”. 

Specificity is a molecular recognition mechanism which operates through structural and conformational complementarity between the biomolecule and its substrate. It is known that a given enzyme only bind/react with its specific group of substrates and not with any other. 

This bio-specificity may not always be limited only to enzymes. Other examples of this kind of biomolecular interactions include;

• A given hormone binds only to its specific glycoprotein (known as the hormone receptor).
• Antibodies specifically bind to only a given antigen which is shaped/has a confirmation specific to that antibody.

To understand this principle better with respect to affinity chromatography, let us suppose that an enzyme is to be purified from a mixture of thousands of proteins.

•  Let the substrate analogue (molecule resembling the substrate but not capable of reaction) specific for this enzyme (which is to be separated) be coupled to the stationary phase (let’s say the stationary phase for this particular example be e.g., agarose). 

• All other molecules, which have no specificity for the ligand and are incapable of binding will pass down and out of the column, (as seen in the second diagram in the picture.)

• Lastly, once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength, or polarity conditions which would completely alter the strength of binding of the enzyme to the substrate and help in the elution of the desired product.

COMPLICATINS THAT ARISE DURING AFFINITY CHROMATOGRAPHY:

There are some complications which arise during affinity chromatography, they may be due to the nonspecific adsorption of sample components other than the desired one on to the matrix. Usually, when this happens, ionic and hydrophobic interactions are involved. 

This complication may be taken care of by judicious choice of operating conditions (e.g., pH, temperature, or ionic strength) in such a way that the physical conditions exclusively favour the binding of the desired molecule to the substrate and not any other undesired molecule.  

Another type of complication arises when one uses ligands, which interact with more than one macromolecule present in a given mixture.

IMPORTANT VARIALBES INVOLVED:

1. The type of matrix that is used.
2. The type of ligand used.
3. The conditions of binding and elution of the sample from the matrix.

A general discussion of these points has been given below:

Properties of the supporting matrix:

1. The matrix that is used in the process must be chemically inert to other molecules in order to minimize the rate of non-specific adsorption.
2. The matrix should possess good flow properties.
3. The matrix should be able to remain stable even at varying levels of pH and temperature, ionic strengths and denaturing conditions.
4. It should be highly porous to provide a large surface area for the attachment of ligand.

Ligand Selection:

1. The ligand used should be capable of forming moderately strong interactions with the desired macromolecule. 
2. The ligand that is to be bound must possess functional groups that may be modified to form covalent linkages with the supporting matrix. 

Ligand attachment:

• The covalent coupling of the ligand to the supporting matrix occurs in two steps;

1. Activation of the matrix functional groups, and
2. Covalent attachment of the ligand to the matrix.

• The chemical methods that are used must be relatively mild so as to ensure no/minimal damage to the ligand or the matrix.

APPLICATIONS OF AFFINITY CHROMATOGRAPHY:

1. Affinity chromatography has been widely used for the purification of varied number of macromolecules which are capable of showing ligand specific interactions like enzymes, hormones, antibodies, nucleic acids, membrane receptors etc.
2. Affinity chromatography has also been used for the purification of cells as this technique is known to affect the viability of cells less than other chromatographic techniques. Cells that have been purified using this technique include fat cells, T and B lymphocytes, spleen cells, lymph node cells etc.
3. An extension of affinity chromatographic principles is using magnetic gels. The gel beads contain a magnetic core that is chemically coupled to a protein ligand. The suspended cells are then allowed to interact with the microspheres. When a magnetic field is passed, the cells of interest move towards the poles of the magnets thereby getting separated. The cells can be collected by removing the magnetic field. 

Immunoglobulin negative thymocytes and neuroblastoma cells are separated using this technique.

Sources:

Biophysical Chemistry principles and techniques – Upadhyay and Nath

INTRODUCTION TO VIRUSES

BY: SHAILY SHAMA (MSIWM041)

HISTORY:

• Throughout the course of modern human history, the source some viral infections such as smallpox, polio, and the Spanish flu have been quite unknown to humans. They have been diseases which have had deadly effects on humanity. All that was known about these diseases was that they spread via person-to-person contact.

• In the second half of the1800’s, Louis Pasteur postulated that the disease rabies was caused by a “living thing” which is in all probability, smaller than bacteria itself. This postulation of his led him to develop the first vaccine against rabies in 1884. 

• The initial discovery of the light microscope held some promise with respect to the observation of the agents causing such sever diseases however, it was found that the microscope could only be used to observe bacteria, protozoa and fungi. The size of virus was smaller than these agents and therefore, could not be observed under a microscope.

• In the 1890’s, D. Ivanovski and M. Beijerinck showed that a disease caused in plants was cause by the tobacco mosaic virus which served as the first properly substantial revelation related to viruses.

• This discovery was then followed by two other scientists, Friedrich Loeffler and Paul Frosch who isolated the virus that caused foot and mouth disease in cattle. 

POSITION OF VIRUSES IN THE BIOLOGICAL SPECTRUM:

Viruses are extremely unique entities which are capable of infecting almost every single type of cell known including bacteria, algae, fungi, plants, animals and even protozoa. 

Questions regarding the nature of viruses like their origination, their state of existence (alive or non-living), their distinct biological characteristics etc. are still very dominant in the scientific world.

Some ideas that have been addressed are:

• Viruses are considered to be the most abundant microbes on earth, in terms of number.
• Viruses are considered to be obligate intracellular parasites that are incapable of dividing or multiplying unless they invade a specific host cell. They multiply by taking over the hosts genetic and metabolic machinery of the cells of the host.
• They are said to have been in existence for billions of years and have arisen from the loose strands of genetic materials released by the cells. (This is one widely accepted theory. However, it too has faced some criticism.)

PROPERTIES OF VIRUSES:

1. Viruses are obligate parasites of protozoa, bacteria, fungi, animals, plants, and algae.
2. They have an ultramicroscopic size range from 20nm up to 450 nm in diameter.
3. They have a very compact and economical structure. They are not cellular in nature.
4. They are inactive macromolecules outside the host cell and only get activated inside the host cells.
5. Viral nucleic acid can be DNA or RNA but never both together.
6. Their basic structure consists of a protein coat (the capsid) surrounding the nucleic acid core.
7. They lack the enzyme and machinery for basic metabolic processes and synthesis of proteins.
8. Virus multiply by taking over the host machinery and genetic material.

THE GENERAL STRUCTURE OF VIRUSES:

SIZE RANGE:

• Viruses represent the smallest infectious agents in the biological world. (with a few exceptions)
• They lie within the ultramicroscopic size range with sizes usually less than 0.2 micrometre. They are so small that one requires an electron microscope to detect or examine their structure.
• The animal size range may vary from parvoviruses (which are around 20 nm in diameter) to megaviruses and pandoraviruses (which are up to 1000nm in width) that may be as big as small bacteria.
• Some viruses which are cylindrical may be relatively long (around 800nm) but have a very narrow diameter (around 15nm).
• Negative staining using an opaque salt in combination with electron microscopy can be used for the observational studies of viruses.

STRUCTURAL COMPONENTS OF A VIRUS:

Viruses have a crystalline appearance due the occurrence of regular, repeating molecules. A number of purified viruses even form large aggregates and crystals when subjected to special treatments. 

The general plan of all viruses or the general architecture is quite simple. Almost all viruses contain a protein coat or the capsid which encloses the viral genome which may be a DNA or an RNA sequence. Apart from this, viruses only contain those parts which are needed to invade and take control over the hosts cellular machinery. 

Some important terms are:

• Capsid: The outer covering or the shell of the virus that surround the central nucleic acid core of the virus.
• 
• Nucleocapsid: The outer shell along with the nucleic acid core is called the nucleocapsid.
• Naked viruses: Viruses that do not contain a nucleocapsid layer are called naked viruses.
• Enveloped viruses: Some virus classes possess an additional covering which is external to the capsid which is called an envelope. This envelope structure is usually a piece of the hosts cell membrane. These types of viruses are called enveloped viruses.

THE VIRAL CAPSID:

The capsid layer of the virus, when magnified immensely, shows the appearance of small, prominent, geographic structures. These structural subunits of the capsid are called capsomeres. 

These capsomeres are capable of self-assembling into the finished capsid structure. Depending on the shape and assembly of the capsomeres, the resulting structure can be of two types; 

1. Helical: Helical capsids have rod shaped capsomeres that bind together to form a structure similar to hollow discs (like a bracelet). During the process of formation, these discs link together to form a continuous helix.

2. Icosahedral capsids: An icosahedron is a three-dimensional, 20-sided figure with 12 evenly spaced corners. Some viruses also show such an arrangement in this shape.
3. Complex viruses: Such viruses may have a specific head, a neck and other structures specific for the invasion of host cells. The most common examples of such viruses is phage viruses.

THE VIRAL ENVELOPE:

Enveloped viruses, when released from the host cells sometimes carry forward a piece of the hosts cell membrane with them in the form of an envelope. Although it is derived from the host, the envelope is different in the virus because the normal proteins of the host get replaced with the viral proteins. 

VIRAL GENOME:

At the center of the viral structure, within the capsid lies the viral genome which may be single or double stranded. The genetic material may be RNA or it may be DNA but it is never both even in viruses. The genome may be a few hundred to thousands of base pairs long. 

Sources:

Foundations in Microbiology – Talaro, Kathleen P