Tag: MicroScopia IWM

GEL PERMEATION CHROMATOGRAPHY

by MicroScopia IWM, posted in Practical notes

BY: SHAILY SHARMA (MSIWM041)

Chromatography, broadly, is a technique to separate two phases which are mutually immiscible. The phases are brought in contact with each other and one of these phases is stationary while the other phase is a mobile phase which moves over the surface of the stationary phase or percolates it. The interactions of the sample mixture which are established after the movement of the mobile phase over the stationary phase lead to the separation of the desired compound present in the mobile phase based on the differences in the physio-chemical properties of the two mediums.

There are various techniques used in the process of chromatography like plane chromatography, paper chromatography, thin layer chromatography, column chromatography etc. 

Based on these techniques, further different types of chromatographic procedures have been curated which include adsorption chromatography, partition chromatography, gel permeation chromatography, ion exchange chromatography and affinity chromatography. 

GEL PERMEATION CHROMATOGRAPHY:

Gel permeation chromatography is a chromatographic procedure that separates molecules based on their molecular size. This method has various other names such as molecular sieve, gel filtration and molecular exclusion chromatography.

This method is widely used due to its many advantages, some of which are:

  1. It is a very gentle technique that permits the separation of labile molecules.
  2. It is a technique in which the solute recovery is almost 100%.
  3. The reproducibility of this technique is high.
  4. The technique is not very time consuming and is relatively inexpensive.

PRINCIPLE: 

The principle of gel permeation chromatography is relatively very simple.

A long column filled with gel beads or porous glass granules is allowed to attain equilibrium with a solvent which is suitable for the separation of the desired compound or molecule. 

Assuming that the mixture to be separated contains molecules of varying sizes, when this mixture will be passed through the column containing the beads;

  • The larger molecules pass through the interstitial spaces between the beads and do not enter the spaces inside the beads. This occurs because the diameter of the molecule is larger than the pore size of the beads.
  • Therefore, the larger molecules are able to pass down the column with little or no resistance.
  • The small molecules however, have a size which is smaller than the diameter of the pores of the beads and therefore these molecules enter the beads and reach the end of the column after a longer period of time due to the resistance that is created by the passage of the molecules through the beads.See the source image

The degree of retardation (or the extent of the time taken by the molecule to reach the bottom) is proportional to the time it spends inside the pores of the gel which is a function of the molecules pore size and the molecules size.

The molecules which have a diameter equal to or smaller than the pore size, do not enter and are said to be excluded. Therefore, the exclusion limit of a gel can be defined as the molecular weight of the smallest molecule which is not capable of entering the pores. For e.g. linear polysaccharides and fibrous proteins have a much lower exclusion limit as compared to the globular proteins of comparable molecular weight. 

TYPES OF GELS USED:

A good gel filtration medium should possess a few properties or characteristics like:

  • The material of the gel should be chemically inert.
  • It should contain a small number of ionic groups.
  • The material of the gel should have a wide variety of particle and pore sizes.
  • It should have a uniform particle and pore size.
  • The material of the gel should be of high mechanical rigidity.

Some examples of the types of gels used in gel permeation chromatography include sephadex, polyacrylamide, agarose, styragel etc.

TECHNIQUE/PROCESS OF GEL PERMEATION CHROMATOGRAPHY:

This chromatographic technique can be performed wither by column or by thin layer chromatography techniques. 

The initial step of the process is:

  1. Preparation of the beads:

Prior to use, the gels used in the column must be converted to their swollen form by either soaking them in water or using a weak salt solution. The greater is the porosity of the gel beads, more will be the time taken for them to attain equilibrium and reach their maximum size.

If porous glass granules are used, the gel beads need not be hydrated at all. 

  1. Preparation of the column:

The gel beds, in their swollen form, are mounted or supported on a column on a glass wool plug or nylon net and the previously swollen gel is added in the form of slurry. The preparation is then allowed to settle. Air bubbles must be removed by connecting the column to a vacuum pump. It must be noted that the level of the liquid must not go lower than the top of the bed. See the source image

  1. Addition of the sample:

The sample must be added from the top of the column using a funnel and the volume of the sample that must be added depends on the size and the type of the gel that is used in the preparation of the stationary phase. 

  1. Collection of the sample:

The eluant used is steadily added and the effluent can be collected in various various fractions in separate tubes.

  1. Detection of sample: 

The most common detection methods include the collection and analysis of the fraction and continuous methods in which the UV absorption, refractive index or radioactivity is measured.

APPLICATIONS:

  1. Gel permeation chromatography is mainly and widely used for the separation of biomolecules and purification. Biomolecules like proteins, hormones, enzymes etc. can be separated using this technique.
  2. This technique is especially useful for the separation of 4S and 5S tRNA.
  3. This technique is used for the separation of salts and small molecules from macromolecules.
  4. Gel permeation chromatography is also chiefly used for the detection of molecular weights of macromolecules. 

Sources:

Biophysical Chemistry Principles and Techniques by Updhyay and Nath

ION EXCHANGE CHROMATOGRAPHY

by MicroScopia IWM, posted in Industrial microbiology

BY: SHAILY SHARMA (MSIWM041)

Chromatography is a separation and purification technique which is used for the separation of solutes in a mixture, biomolecules etc. on the basis of distribution of the sample to be separated between stationary phase (phase which is not mobile and is usually mounted against a support like a chromatographic column) and the mobile phase (which is continuous/is poured or passed over the stationary phase)

Ion Exchange Chromatography is a method used for the separation as well as purification of ionically charged biomolecules like proteins, polynucleotides, nucleic acids etc. 

This technique finds a wide array of applications in the scientific world because of its simplicity and high resolution. 

  • PRINCIPLE:

The process of ion exchange can be defined as the reversible exchange of the ions present in a solution, with the ions electrostatically bound to the inert support medium.

The main factor which governs the process of ion exchange chromatography is the electrostatic force of attraction present between the ions. This electrostatic force of the ions depends on their relative charge, radius of the hydrated ions and the degree of non-bonding

interactions.

Usually, ion exchange separations are carried out in columns packed with an ion-exchanger. The ion-exchanger is a support medium which is inert and insoluble. The medium may be capable of covalently binding to positive (anion exchanger) or negative (cation exchanger) functional groups. The ions which do bind to the exchanger electrostatically are called counterions. 

The conditions of separation can be manipulated in such a way that some compounds are electrostatically bound to the ion exchanger while some are not, therefore, helping in the separation of the desired compound.

The sample which contains the sample to be separated is allowed to percolate through the exchanger for a certain amount of time that will be sufficient for the equilibrium of the ions to be achieved. 

E–   Y–  E–   X+ + Y+

In the equation mentioned above, 

  • E–  : Charged cation exchanger.
  • Y+ : Counterion of the opposite charge associated with the exchanger matrix.
  • X+ : Charged molecule bearing a charge similar to the counterion to be separated. This molecule is capable of exchanging sites with the counterion as shown above.

Once the exchange of the counterion with the sample has been achieved, the rest of the uncharged and like charged species is washed out of the column.

The ions that did bind can then be eluted out by either percolating the medium with increasing concentration of Y+ (works by increasing the possibility that the Y+ will replace the X+ in the above-mentioned equation due to it being present in a higher concentration). The elution can also be carried out by increasing the pH of the solvent and hence converting X+ to an uncharged species.

Simply put, once the sample containing the specific ions to be separated it passed through the ion exchanger column, the sample ions (which act as counterions to the ions of the exchanger column) bind with the ions on the exchanger column and form associations. However, the ions of the same charge as the exchanger column, present in the sample solution, repel each other and, therefore, do not bind and pass through the column.

The principles which have been mentioned above also apply to other macromolecules such as proteins and nucleic acids which are capable of showing the presence of both positive and negative ions. The type of molecules can bind to both anionic and cationic exchangers. 

  • TYPES OF ION EXCHANGE RESINS:

The two main types of materials used to prepare ion exchange resins are:

  1. Polystyrene, and
  2. Cellulose

Polystyrene resins are prepared by the polymerization reaction of styrene and divinyl benzene. These resins are very useful for separating compounds with a small molecular weight. 

Cellulose based resins have a much greater permeability to macromolecular polyelectrolytes as compared to polystyrene resins and they also possess a much lowers charge density. 

Based on the type of charge carried by these ion exchangers and their strength, the ion exchange resins can broadly be classified into four types:

Strong cationic exchange resins:Weak cationic exchange resins:
Sulphonated polystyreneSulphopropyl cellulose Condensed acrylic acidCarboxymethylcellulose
Strong anionic exchange resins:Weak anionic exchange resins:
Polystyrene with -CH2NMe3ClDiethyl (2 hydroxypropyl)quaternary amino celluloseDiethylaminoethyl celluloseDiethylaminoethyl agarose
  • CHOICE OF BUFFERS:

The buffer is that component of the chromatographic column that helps in the maintenance of the pH. The choice of these buffers is usually dictated by the compounds to be separated and whether the ion exchange is anionic or cationic.

  • Anion exchange chromatography should be carried out with cationic buffers.
  • Similarly, cation exchange chromatography should mostly be carried out with only anionic buffers for satisfactory separation and results. 

      Examples of some buffers used in this technique are:

  • Ammonium acetate
  • Ammonium formate
  • Pyridinium formate
  • Ammonium carbonate etc.
  • APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY:
  1. The most significant application of ion exchange chromatography is in amino acid analysis.
  2. This technique is used to determine the base composition of nucleic acids.
  3. Ion exchange chromatography is used as a method of purification of water. Water is completely deionized using this technique.
  4. This technique is used for the ultra-purification of metal ion free reagents.
  5. It can also be used for the separation of a varied number of vitamins, biological amines, organic acids as well as bases. 

Source:

Biophysical Chemistry principles and techniques – Upadhyay and Nath

Overview of infections of respiratory tract and its pathogens

by MicroScopia IWM, posted in Medical microbiology/Immunlogy

By: Shaily Sharma (MSIWM041)

  • A brief overview of the infections of the respiratory tract and its pathogens:

The respiratory tract along with the gastrointestinal tracts is one of the major connections between the interiors of the body and the outside environment.

The respiratory tract is the pathway is that pathway of the body through which fresh oxygen enters the body and removes the excess carbon dioxide which is not needed by the body. 

  • Anatomy of the respiratory system:
  • Broadly, the respiratory system of humans can broadly be divided into two distinct areas; the upper and the lower respiratory tracts.
  • The parts that consist the lower respiratory tract are:
  1. Trachea
  2. Bronchi, and
  3. Bronchioles
  • The respiratory pathway begins with the nasal and the oral passages. These passages serve to humidify the air that is inspired. These pathways extend past the nasopharynx and the oropharynx to the trachea and then to the lungs.
  • The trachea is the organ that divides into the bronchi, which then further subdivides into the bronchioles. The bronchioles are the smallest branches of the trachea which finally terminate into the alveoli.
  • Approximately 300 million alveoli are said to present in the lungs. These mainly serve as the primary, microscopic, gas exchange structures of the respiratory tract.

See the source image

  • The lungs (along with the respiratory system) and the heart lie in the thoracic cavity. 
  • The thoracic cavity has three partitions that are separated from one other by the pleura (the pleura majorly cushions the lungs and reduce the friction which may develop between the lungs, rib cage and the chest cavity. It is a two layered membrane covering the lungs.)
  • The lungs occupy the right and the left pleural cavity while the mediastinum (the space between the right and the left lungs) is occupied by the esophagus, trachea, large blood vessels along with the heart.
  • Pathogenesis of the respiratory tract:
  • The success of an organism to cause disease is mainly dependent on the organism’s ability to cause disease (pathogenesis), and
  • The human hosts ability to prevent the infection (strength of the host’s immune system)
  • The host factors that help in non-specifically protect the respiratory tract from infection are:
  1. Nasal hair
  2. Convoluted passages and the mucous lining of the nasal turbinate
  3. Secretory IgA and non-specific antibacterial substances (like lysozyme) in respiratory secretions
  4. The cilia and the mucous lining of the trachea and reflexes such as coughing and sneezing. 
  • In addition to the non-specific hosts defenses, normal flora of the nasopharynx and the oropharynx help in the prevention of colonization of the upper respiratory tract. 

Microorganism factors:

Organisms possess certain traits that promote colonization leading to infection in the host. The factors that influence the respiratory tract infections are –

  1. Adherence: 
  • The potential of a microorganism depends, in one way or the other, on its ability to establish a stable contact/foothold on the surface of the host by the process of adherence. 
  • The ability of microorganisms to adhere to the host surface is dependent on two factors:
  1. Presence of normal flora, and
  2. Overall state of the host.
  •  Surviving or growing on host tissue without causing harmful effects is called colonization. 
  • Most etiologic agents must first adhere to the mucosa of the respiratory tract to some extent before they can cause harm.
  • Example: Streptococcus pyogenes possess specific adherence factors and its gram-positive cell wall contains lipoteichoic acids and M proteins. Many gram-negative bacteria like Enterobacteriaceae, Pseudomonas spp., Bordetella pertussis, adhere by the means of proteinaceous fingerlike projections called fimbriae. 
  • Viruses possess either a hemagglutinin or other proteins that mediate that epithelial attachment.
  1. Toxins: 
  • Certain microorganisms are considered to be etiologic agents of disease because they possess virulence factors that are expressed in every host. 
  • Example: Corynebacterium diphtheriae. 
  • Some strains of Pseudomonas aeruginosa also produce toxins which are similar to the toxins of Diphtheria.
  •  Bordetella pertussis which is the causative agent of whooping cough produces toxins that play a role in inhibiting the activity of phagocytic cells and damaging the cells of the respiratory tract.
  1. Microorganism growth: 
  • Pathogens cause disease by merely growing in the host tissue, interfering with normal tissue function and attracting host immune effectors, such as neutrophils and macrophages.
  • Example: S. pyogenes, M. tuberculosis, Mycoplasma pneumoniae, etc.
  1. Avoiding the Host Response: 
  • Certain respiratory tract pathogens possess the ability to evade host defense mechanisms.
  •  S. pneumoniae, H. influenza, K. pneumoniae and others possess polysaccharide capsules that serve both to prevent engulfment by phagocytic host cells and to protect somatic antigens from being exposed to host immunoglobulins.
  • Organisms of the respiratory tract and agents that cause diseases: 

Pathogens may or may not cause the respiratory infection but can be present as a part of normal flora.

  •  Some of the pathogens that exist and results in the respiratory infection are referred to as true pathogens. 
  •  Some of the pathogens that are present in the body but never cause an infection until and unless they are met with the favorable conditions are called

as opportunistic pathogens.  

  • Possible pathogen: they are the pathogens that are likely to cause respiratory

infections.

  • Example: Actinomyces spp., Haemophilus influenzae, Enterobacteriaceae, etc.See the source image
  •  Rare pathogen: pathogens that may cause a respiratory infection are rare

pathogens. Example: Coxiella burnetti, Brucella spp., Salmonella spp, etc.  

  •  Definite respiratory pathogen: pathogens that always cause respiratory infections are called as definite respiratory pathogens.
  • Example: Bordetella pertussis, Blastomyces dermatitidis, Legionella spp., etc.
  • Different types of agents that cause respiratory diseases are bacteria, fungi or

viruses.  

  •  Bacterial agents: the bacterial agents that cause respiratory infections are

Mycoplasma spp., Streptococcus pneumoniae and Neisseria meningitides. 

  • Fungal agents: the fungal agents that cause respiratory infections are Candida

albicans, Cryptococcus neoformans and Histoplasma capsulatum.  

  •  Viral agents: the viral agents that cause respiratory infections are human

metapneumovirus, adenovirus, enteroviruses, and herpes simplex virus. 

  • Major respiratory diseases are caused by M. tuberculosis, S. pyogenes and

K.pneumonia.

Sources:

text=The%20presence%20of%20normal%20flora,harmful%20effects%20is%20termed%20colonization.

DETECTION OF PEPTIDE HORMONES

by MicroScopia IWM, posted in Biochemistry, Medical microbiology/Immunlogy

BY: Reddy Sailaja M (MSIWM030)

HORMONES

A hormone is a signaling molecule secreted by endocrine glands in response to physiological stimuli in multi cellular organisms. These hormones circulate in blood and reach its destination to exert specific function. Hormones help maintain physiological and behavioral functions in the organisms.

Hormones are classified into three main classes:

  • Steriod hormones – Lipid soluble and move across plasma membrane of the targeted cells.
  • Peptide hormones – Water soluble and act through cell surface receptors present on the targeted cells.
  • Aminoacid derivatives

Table 1: Three classes of hormones

Peptide hormones

Peptide hormones are made up of small amino acid chains called, peptides. Peptide hormones are synthesized in the cells from amino  acids based on mRNA sequence that is derived from DNA template within the nucleus.

Peptide hormones can’t navigate across plasma membrane of the cell. Hence, they exert their function by binding to the receptor present on the cell surface of the target cell that in turn trigger signal transduction and cellular response. Some peptide hormones like parathyroid hormone-related protein, angiotensin II etc interact with intracellular components within cytoplasm or nucleus by an intracrine mode of interaction.

Some of the examples of peptide hormones are as follows:

  • Adrenocorticotrophic hormone
  • Thyroid stimulating hormone
  • Vasopressin
  • Angiotensin II
  • Antrial natriuretic peptide
  • Calcitonin
  • Follicle stimulating hormone
  • Insulin
  • Growth hormone
  • Parathyroid hormone
  • Prolactin

Table 2: Main peptide hormones – details and functions

Detection of peptide hormones

As peptide hormones circulate in blood to reach their destination, serum generally acts as the source of detection and measurement.

The following are the main detection methods used to detection peptide hormones:

  1. Sandwich ELISA technique:
  2. In sandwich ELISA method, two antibodies are used to detect hormone of interest. One of the antibodies is attached to the solid support on the micro titer plate, called as capture antibody.
  3. Second antibody labeled with a signal molecule (enzyme or radioisotope or chemilumiscent) acts as detector.
  4. When the analyte containing mixture is loaded onto the micro titer plate, capture antibodies bind to the analyte via epitope that is present on the analyte surface and catch hold of analytes.
  5. When the detector antibodies are added to the plate, they bind to different location on the analyte.
  6. In the enzyme based reaction, when substrate is added, it reacts with enzyme that is attached to the detector body and shows response in the form of color change.

Figure 1: Sandwich ELISA to detect analytes in the blood

  • Radioimmunoassay (RIA):
  • RIA is an in vitro detection technique that detects and measures antigens (like hormones and other foreign substances) in the blood. RIA technique is discovered by Berson and Yalow in 1960 to analyze insulin levels in blood.
  • RIA method is based on the radioactivity measurement associated with antigen-antibody interactions in the reaction.
  • A known antigen that is radiolabelled is incubated with antibody at known concentrations.
  • When the analyte containing solution is added to the labelled antigen-antibody mixture, antigen of interest replaces labelled antigen and bind to the antibody.
  • More the antigen of interest present in the solution, more labelled antigen will be displaced and replaced with antigen of interest.

Figure 2: Radioimmunoassay

  • Enzyme multiplied Immunoassay Technique (EMIT):
  • EMIT is more easy and equivalent detection method, both qualitatively and quantitatively to measure wide-range of analytes from the serum.
  • EMIT is based on the principle that the amount of analyte present in the solution is directly proportional to the inhibition of enzyme-substrate reaction complex.
  • In this technique, initially a known analyte is labelled with an enzyme and antibody specific to drug is allowed to bind drug-enzyme complex. This results in inhibition of enzyme activity.
  • When the solution containing analyte is added to the above mixture, the analyte releases the antibody from the drug-enzyme complex, thereby increasing enzymatic activity.
  • Therefore, enzyme activity is proportional to the analyte present in the sample added and is measured by absorbance value changes of the enzyme.

Figure 3: Enzyme multiplied Immunoassay Technique

  • Immunoradiometric assay (IRMA)
  • IRMA utilizes radiolabeled antibodies to detect analytes of interest.
  • In this technique, antibody is directly labeled with radioisotopes rather than using two antibodies as in other immune assays.
  • When an analyte containing solution is added to micro titer plateradiolabeled antibodies bind to the specific epitopes of the anlytes and forms the antigen-antibody complex.
  • I125 and I131 radioisotopes used in general for this assay.
  • Unbound radiolabeled antibodies are removed from the plate by second wash during the process.

Figure 4: Immunoradiometric assay

The other peptide hormone detection methods are listed below:

  • Ultrafiltration
  • Chromatography
  • Time resolved fluorescence
  • Mass spectrometry
  • Two site immunometric technique

EXTRACTION OF PLASMID DNA

by MicroScopia IWM, posted in Biotechnology

BY: K. Sai Manogna (MSIWM014)

In 1952, Joshua Lederberg invented the word ‘plasmid.’ It was initially formed from bacteria; plasmids are extrachromosomal genetic elements that can reproduce independently in most Archae, Eukarya species, and Eubacteria. Plasmids are double-stranded circular DNA molecules that are different from the chromosomal DNA of the cells. 

The genetic material found within the chromosomal DNA guides the structure and function of a bacterial cell. In some instances, plasmids are usually not required for the host bacterium to survive. Although not necessary, by encoding functions that may not be defined by the bacterial chromosomal DNA, plasmids significantly contribute to bacterial genetic diversity and plasticity. Antibiotic tolerance and the expression of proteins, for example. The plasmid is also encoded by antibiotic resistance genes, allowing the bacteria to survive in an environment containing antibiotics, thereby providing the bacterium with a competitive advantage over species susceptible to antibiotics. Plasmids may be altered as a means to express the protein of interest for e.g., production of human insulin using recombinant DNA technology. 

 As a mechanism for gene-cloning and as a vehicle for gene-expression, plasmids have been central to modern recombinant DNA technology. 

Isolation of Plasmids 

In molecular biology, bacterial plasmid DNA isolation is a crucial technique and an integral step in many processes, such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations need the isolation of high purity plasmid DNA. In all molecular biology procedures, cloning, such as digestion with restriction enzymes, PCR, transfection, in vitro translation, blotting, and sequencing, purified plasmid DNA can be used for immediate usage. 

In molecular biology, alkaline lysis is used to separate plasmid DNA or other cell components, such as proteins, by splitting open the cells. Primarly, bacteria containing the plasmid of interest are allowed to grown and then lysed with an alkaline lysis buffer composed of a sodium dodecyl sulphate (SDS) detergent. Cellular debris is extracted, and the plasmid is separated and filtered through a series of steps, including agitation, precipitation, centrifugation, and the removal of the supernatant. 

Principle:

Purification of plasmid DNA from bacterial DNA using alkaline lysis is based on the differential denaturation of plasmid and chromosomal DNA. Disruption of the cellular structure to produce a lysate, separation of the plasmid from the chromosomal DNA, cell debris, and other insoluble material are the essential steps of plasmid isolation. With a lysis buffer solution, bacteria are lysed. 

Materials and Equipment:

Refrigerated centrifuge

Vortex

Microwave oven

pH meter 

Orbital shaker 

Micropipettes

Autoclave

LB plate with Bacterial colonies

1.5 ml micro-centrifuge tubes

Autoclaved distilled water

Microtips

Microfuge tubes 

Chemicals:

1. Lysis Solution (Solution I)

2. Denaturation solution (Solution II)

3. Neutralizing solution (Solution III)

4. TE Buffer

5. RNase

6. Phenol: Chloroform: isoamyl alcohol

7. 70% Ethanol

8. Isopropanol 

Preparation of Stock solutions:

  1. Solution I (Lysis solution): 50mM Glucose, 25mM Tris-Hcl (pH 8.0), 10mM EDTA (pH 8.0). Store at 4oC.
  2. Solution II (Denaturation solution):1% SDS, 0.2N NaOH (pH 12.0). Freshly prepared and store at room temperature.
  3. Solution III (Neutralizing solution): 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of water. Store at 4oC.
  4. TE Buffer: 10mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0)
  5. RNase (1mg/ml)
  6. Phenol: Chloroform: Isoamyl alcohol -(25:24:1).
  7. Washing buffer
  8. Elution buffer

Biological material:

Overnight grown culture of E.coli.

PROCEDURE

Harvesting of the cells: 

  1. The single bacterial colony is transferred into a 5 ml LB medium containing appropriate antibiotic, incubated for 16 hrs at 37oC with vigorous shaking. 
  2. About 1.5 ml culture is transferred into a microfuge tube and centrifuged at 6000 rpm for 5 min at 4oC and discard the supernatant. 

Isolation of Plasmid by alkyl-lysis method:

1. The bacterial pellet is re-suspended in 100μl of ice-cold solution-I by vigorous vortexing. This is essential to ensure that the bacterial pellet is wholly dispersed in the solution I. 

2. Add 200μl of freshly prepared solution II. Mix the contents by inverting the tubes rapidly 4 to 5 times. A transparent and viscous solution ensured complete lysis. Do not vortex. Incubate on ice for 3 min. 

3. Add 150μl of ice-cold solution III. Mix by gently inversion for about 10 seconds to disperse the solution III through the vicious bacterial lysate. Further incubate the tubes on ice for 5 min. 

4. Centrifuge at10,000 rpm for 5 minutes at 4oC and transfer the supernatant to a fresh tube. 

5. To this add equal volumes of phenol-chloroform, mix by vortexing. After centrifuging at 10,000 rpm 5 minutes at 4oC, transfer the aqueous phase to a fresh tube. 

6. Precipitate the double-stranded DNA by adding an equal volume of isopropanol, mix gently, and allow the mixture to stand for two minutes. Then centrifuge at 10,000 rpm for 10 minutes at 4oC. 

7. Remove the supernatant by gentle aspiration. 

8. Then wash the pellet with 1 mL of 70% ethanol (twice). Allow the DNA pellet to air dry for 10 minutes. 

9. Re-dissolve the DNA pellet in 50μl of TE (pH 8.0). 

Isolation of PLASMID by spin column method:

1. Inoculate a single colony of bacterial cells from the petri plate using a sterile inoculation loop to 5ML of LB broth and allow them to grow overnight at 37°C at 200 rpm.

2. From the pre-inoculum transfer, 1.5 ML of bacteria-containing cells into 2mL centrifuge tubes and centrifuge the cells at 8000 rpm. for one minute. Then the supernatant was discarded.

3. Re-suspend the cells in 200 µL of resuspension solution. Then mix the cells thoroughly by pipetting up and down or vortexing it.

4. To this, add 200 µL of lysis solution and mix the tubes by gently inverting up and down.do not vortex it. They were then incubated for five minutes.

5. To this, add 350 µL of the neutralizing solution and, for mixing, invert the tubes 4 to 6 times. Then Centrifuge these tubes at 10,000 rpm for 10 minutes.

6.    Binding column preparation: Take a new centrifuge tube and insert a new binding or spin column into it. To this, add 500 µL of column preparation solution and spin at 10,000 rpm for one minute. Then discard the solution that was collected in the centrifuge tube.

7. Then, transfer the clear lysate to the binding column and centrifuge at 10,000 rpm for one minute. Discard the flow-through that was remained in the centrifuge tube.

8.    Washing: In this step, add 750 µL of washing solution to the binding column and spin the centrifuge tube for one minute. Then discard the solution that remained in the centrifuge tube. (Do the washing step twice).

9. Then, change the spin column into the new centrifuge tube. Then spin the column for one minute and incubate at room temperature for 2 – 5 minutes.

10. ELUTION: In this step, the spin column is transferred to the fresh collecting tube. Then add 70 µL of elution buffer to the spin column and spin for one minute at 10,000 rpm. Label the vial as elution I.

11. Transfer the same spin column to the fresh collecting tube and add 40 µL of elution buffer to the spin column and spin for one minute at 10,000 rpm. Label the vial as elution II.

12. Then discard the spin column, and the vials labelled as elution I and II are stored at -20oC or -80oC.

Preparation of agarose gel electrophoresis:

  1. First, take a gel casting tray, then clean the tray with ethanol and seal the edges with transparent tape and place the comb.
  2. Then 0.8% of agarose solution was prepared using 1X TBE. When the temperature was lowered, add a little amount of Ethidium bromide to it and mix well.
  3. After mixing, pour the agarose solution into the gel casting trays and then allow for polymerization. Make sure with the absence of air bubbles while pouring the solution. After polymerization, remove the comb without breaking the gel. 
  4. Submerge the agarose gel into the electrophoresis tank containing 1X TBE buffer.

Electrophoresis of isolated plasmid DNA.

1. Take 3 – 4 µL of eluted sample and add 2 µL of sample buffer.

2. Load 5-6 µL of a sample into each well along with a DNA marker or ladder.

3. Run the electrophoresis and allow the plasmid DNA to run in 1X TBE buffer at a constant voltage. Keep track of the dye front.

4. Then disconnect the power electrophoresis tank and remove the gel using gloves. Place the gel in the Gel Documentation System or Transilluminator and visualize the DNA bands in the UV light.

5. Quantify the concentration of DNA from the DNA marker or ladder used.

Role of chemicals used:

Spin column: The desired nucleic acids should be bound to the column after centrifuging the lysate through the silica membrane, and impurities such as protein and polysaccharides should be in the flow-through. While plant samples are likely to contain polysaccharides and pigments, the membrane may be slightly brown or yellow in blood samples. The washing steps will remove such impurities. Usually, there are two washing measures, but this varies depending on the type of sample. A low concentration of chaotropic salts to eliminate residual proteins and pigments will also be part of the first wash. In order to extract the salts, this is often accompanied by an ethanol wash. Columns contain a resin of silica which binds to DNA/RNA selectively. By its capability to bind silica in the presence of high concentrations of chaotropic salts, the DNA of interest can be isolated. 

Washing: With an alcohol-based wash, these salts are then removed, and the DNA is eluted using a low-ionic-strength solution such as TE buffer or water. Dehydration and the formation of hydrogen bonds that compete against poor electrostatic repulsion seem to be driving the binding of DNA to silica. Therefore, a high salt concentration will help push DNA adsorption to silica, and the DNA will be released at a low concentration. 

Elution buffer: The Elution buffer is initially used to wash away unbound proteins and release the ligand’s desired protein at a higher concentration. The elution buffer must work rapidly without altering the desired protein’s function or activity. This enables to stand within the membrane for a few minutes before centrifugation for optimum DNA elution.

Glucose: It helps in maintaining osmolarity and keeps the cells from bursting.

Tris HCL and EDTA: This helps in chelating divalent metal ions such as magnesium calcium and destabilizes the cell wall, inhibiting DNases’ action.

NaOH and SDS: In the lysis buffer or solution II, sodium hydroxide, and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS) are present. SDS is used to solubilize the cell membrane. NaOH helps in the breakdown of the cell wall, but more significantly, it interferes with the hydrogen bonding between the DNA bases, turning the cell’s double-stranded DNA, including the genomic DNA and the plasmid, into single stranded DNA.

RNase: It helps incomplete digestion of unwanted RNA from the plasmid sample.

GALACTOSE TO GLUCOSE SYNTHESIS

by MicroScopia IWM, posted in Biochemistry

BY: Ria Fazulbhoy (MSIWM031)

Introduction

Galactose is a sweet tasting monosaccharide sugar which is a carbohydrate, which is an aldohexose molecule. Galactose is a very important molecule in the body as it is needed for the synthesis of lactose, glycolipids, glycoproteins as well as proteoglycans. It is also known as “brain sugar” as it is useful in formation of the glycolipids which occur in the brain and the myelin sheaths of the nerve cells. Galactose can be hydrolysed from interstitial disaccharide lactose, which is known as the ‘sugar of milk’. (lactose = glucose + galactose). Glucose and galactose are stereoisomers of each other, differing in stereochemistry at carbon 4.

Conversion of galactose to glucose

Galactose can be readily converted to glucose in the liver. This is done in the following steps:

  1. The first step is the conversion of galactose to galactose-1-phosphate on the action of galactokinase. It is a phosphorylation reaction, in which ATP acts as the donor of the phosphate group.
  2. Galactose-1-phosphate reacts with UDP glucose i.e. uridine diphosphate glucose (UDPGlc) to form uridine diphosphate galactose (UDPGla). Glucose-1-phosphate is also formed and given out in this step.

This reaction is catalysed by an enzyme called galactose-1-phosphate uridyl transfer

  1. After this, UDP galactose which is formed is converted to UDP glucose by an epimerization reaction.

This is catalysed by UDP-4 glucose epimerase.

  1. The epimerization reaction will involve the oxidation and reduction of carbon 4, using NAD+ as coenzyme.
  2. Finally, from the so formed UDP glucose, glucose is released in the form of glucose 1 phosphate

NOTE: The epimerization reaction is freely reversible, thus glucose can convert back to                 galactose as and when required. This is the reason that galactose is not a dietary essential.

To yield lactose in the mammary gland, UDPGal condenses with glucose. This reaction is catalysed by lactose synthase

Galactosemia is a disease related to this pathway:

  • Galactosemia is the disruption of galactose metabolism.
  • Classic galactosemia is the most common type, in which a deficiency of galactose 1 phospahte uridyl transferase activity is inherited.
  • Due to this, galactose 1 phosphate is accumalated and as a consequence, the inorganic phosphate of the liver is depleted.
  • This results in the ulitimate liver failure and mental detoriation.
  • Infants who are afflicted seriously fail to thrive. They go through symptoms of vomitting and diarhhea after consuming dairy products, enlargement of the liver, and jaundice (which can progress to cirrhosis)
  • Cataract, lethargic developement and retarded mental developement is also very common amongst children. Cataract is observed due to deposition of a galactose metabolite, galactitol, in the lenses of the eyes. Galactitol is osmotically active, due to which water diffuses in the eyes and instigates cataract formation.
  • Other side effects include increase in level of blood galactose level, which is then found in urine.
  • Females can also display ovarian failure.
  • A stirct limitation of galactose consumption in diet can greatly reduce these symptoms.
  • An important diagnostic criterion for this the absence of transferae in the red blood cells.

Conclusion:

Thus, galactose is a very important carbohydrate present in the body which can be easily converted to glucose ( in the form of glucose-1-phosphate) for energy. This is a  reversible reaction, as glucose and galactose are stereoisomers. This is why galactose is not an essential need for the body.

Deficiency in the enzymes that carry out this conversion may result in a vast range of symptoms under galactosemia.

BACTERIAL WATERBRONE PATHOGENS

by MicroScopia IWM, posted in General microbiology

BY: K. Sai Manogna (MSIWM014)

The potential for spreading through drinking water is the emerging pathogenic bacteria of concern outlined here, but they do not correlate with the existence of E. Coli or with other measures of the consistency of drinking water widely used such as coliform bacteria. There are no satisfactory microbiological markers of their existence in most cases. To understand the real nature and dimension of the diseases caused by water polluted with these bacteria and the ecology of these pathogens, further studies are required.

Mycobacterium Avium Complex (Mac):

The complex Mycobacterium avium (Mac) consists of 28 serovars of two species: Mycobacterium avium and Mycobacterium intracellular. With the discovery of disseminated infection in immunocompromised individuals, especially people with HIV and AIDS, the Mac species’ significance was recognized. MAC members are deemed to be opportunistic human pathogens. A wide range of environmental sources, including coastal waters, rivers, lakes, streams, wetlands, springs, soil, piped water supplies, plants, and house dust, have defined Mac species. Mac species have been isolated from the delivery systems of natural water and drinking water in the USA. The ubiquitous existence of Mac organisms stems from their ability under varied conditions to thrive and evolve. Mac species can proliferate at temperatures up to 51°C in water and expand over a broad pH range in natural waters. These mycobacteria are incredibly resistant to the use of chlorine and other chemical disinfectants in drinking water care. Standard drinking-water treatments may not remove Mac species but may substantially reduce the numbers present in the source water to a level that poses a negligible risk to the general public if it is running satisfactorily. In delivery systems, the entryway for these mycobacteria is through leaks. For their continued presence in distribution systems, the growth of Mac organisms in biofilms is probably significant.

Slow-growing mycobacteria can be present in the surface biofilm at densities higher than 4,000 per cm2, producing a potentially high exposure level. The signs of Mac infections result from either respiratory or gastrointestinal colonization, potentially spreading to other places in the body. Exposure to Mac species may occur through the consumption of contaminated foodstuffs, the inhalation of air containing contaminated soil particles, or through touch or ingestion, aspiration or aerosolization of the organisms containing drinking water.  Unlike gastrointestinal pathogens, where E. No appropriate indicators have been identified to signal increasing Mac species concentrations in water systems, and coli can suggest possible presence.

Helicobacter pylori:

As a significant etiologic agent for gastritis, Helicobacter pylori has been cited and has been involved in the pathogenesis of duodenal ulcer and peptic disease and gastric carcinoma. Most people who are infected by this pathogen, however, remain asymptomatic. Using methods based on history, H. There has been no isolation of pylori from environmental sources, including water. Molecular methods have, on the other hand, been useful in detecting the pathogen.

Fluorescence in situ hybridization (FISH) has been successfully used to detect this pathogen in drinking water delivery systems and other water bodies. To detect the presence of H, a polymerase chain reaction was also used. Pylori DNA in drinking water, especially biofilm-associated. In biofilms for drinking-water, H. Pylori cells lose culturability rapidly and enter a viable but non-culturable state. Cells persist for more than one month in these biofilms, with densities exceeding 106 cells per square cm. It remains unclear how the organism is transmitted. Nevertheless, the fact that it has been oral-oral or fecal-oral transmission is demonstrated by recuperation from saliva, dental plaques, stomach, and fecal samples. Water and food tend to be of less immediate significance, but they can still play a significant role in improper sanitation and hygiene.

Aeromonas Hydrophyla:

Over the past years, A. Hydrophila has received attention as an opportunistic pathogen for public health. The elderly, children under the age of five, and immunosuppressed persons may play a significant role in intestinal disorders. Gram-negative, non-spore-forming, rod-shaped, facultative anaerobic bacilli belonging to the Aeromonadaceae family are Aeromonas hydrophila. Even though the dominant species is typically hydrophila, whereas other aeromonads, such as A.Sobria, and A.Caviae were isolated from human feces and water sources. Species of Aeromonas, including A. Hydrophila, in the field, are ubiquitous. It is also segregated from food, potable water, and aquatic ecosystems. Concentrations of Aeromonas spp. in safe rivers and lakes Typically, 102 colony-forming units (CFU)/mL are around. In general, groundwater contains less than 1 CFU/mL. It was noticed that drinking water immediately leaving the treatment plant contained between 0 and 102 CFU/mL. Drinking water can show higher concentrations of Aeromonas in delivery systems due to the growth of biofilms. With Aeromonas spp. growth was observed between 5° – 45° C.

A. Hydrophila is immune to standard treatments with chlorine and is likely to live within biofilms. Ingestion of infected water or food or touch of the organism with a break in the skin are the typical routes of infection suggested for Aeromonas. A potential source of pollution for human beings may be drinking or natural mineral water. There was no recorded person-to-person transmission.

INDUSTRIALLY IMPORTANT MICROORGANSIMS

by MicroScopia IWM, posted in Industrial microbiology

BY: Reddy Sailaja M (MSIWM030)

Microorganisms are minute creatures from time immemorial and omnipresent in all kinds of ecosystems on earth. Microorganisms are of different types: Bacteria, Fungi, Protozoa, Algae and Virus. These are not visible through naked eye and require microscope for their structural and functional evaluation.

Industrial microbiology deals with the application of various microorganisms for industrial processes that are beneficial to mankind.

Characteristics of industrially important microorganisms include:

  • Non pathogenic and non-contagious
  • Easy and rapid growth on industrial scale
  • Need of inexpensive medium for culture and growth
  • Production of spores
  • Easy inoculation
  • Desired product should be produced rapidly on large scale
  • capability to genetic manipulation, if required

Major industrial products produced by microorganisms:

  • Pharmaceutical drugs
  • Vaccines
  • Organic acids and solvents
  • Steroids
  • Dairy products
  • Enzymes
  • Beverages
  • Antibiotics
  • Amino acids
  • Vitamins

Figure 1: Major applications of microorganisms

Beverages:

Yeast species was widely used from thousands of years to produce beverages like wine, brandy, whiskey etc. Yeasts fall under kingdom Fungi and are eukaryotic, single celled organisms. Yeasts species, Saccharomyces cerevisiae  was allowed to grow on malted cereals and fruit juices to produce ethyl alcohol.

Beyond yeast, bacteria – Acetobacter, Lactobacsp. B. bucheri, etc and fungi – Pichia fermentans, Cyberlindnera mrakii etc are usually used in wine production

Antibiotics:

Discovery of Penicillin, an antibiotic by Alexander Fleming in 1928 is a significant step in medical microbiology history in the 20th century. Antibiotic is a bioactive substance produced by a microorganism that has the ability to either inhibit the growth or kill the other microorganism. Fungi are the main source of antibiotics.

Fungi of Actinomycetes group produce antibiotics like tetracyclin, streptomycin, actinomycin D etc. While, antibiotics like Penicillin, Cephalosporin etc are produced by filamentous fungi.

Organic acids and solvents:

Organic acids and solvents are produced from the microorganisms mainly for the pharmaceutical and other industrial needs. These compounds can be produced either from glucose or in the form of end products from pyruvate or ethanol.

Microorganisms that produce organic acids include:

Aspergillus niger – Citric acid

Acetobacter aceti – Acetic acid

Lactobacillus – Lactic acid

Salmonella – Formic acid

Escherichia coli – Butric acid and malic acid

Acetobacter xylinum – Ascorbic acid

Enzymes:

Enzymes, also called biological catalysts occur naturally in the living system and regulate biochemical reactions. As enzymes have wide applicability in both medical and non-medical fields, microorganisms are genetically modified to produce industrially important enzymes. Amylase was the first industrial enzyme to be produced in the year 1896. Amylase has the ability to regulate indigestion and other digestive system related disorders.

Enzymes are widely used in food preservation, food processing, leather and paper industries, detergents and scientific research and development sectors like molecular biology.

MicroorganismSubstrate for growthEnzyme producedApplications
Aspergillus niger, Penicillium sp.PectinPectinaseAlcohol production, clarification of fruit juices
Saccharomyces diastaticusStarchAmylaseAlcohol production, starch removal glucose syrups production
Bacillus sp.ProteinsProteaseBread, baked foods, waffles production
Aspergillus sp.LipidsLipaseFood and aroma enhancement, biofuel degradation
Cellulomonas sp.CelluloseCellulaseAlcohol and glucose production
Streptomyces sp.XylanXylanasePaper production, biofuel production
Actinomyces, Streptomyces sp.ChitinChitinaseFood additive, therapeutic agent, antifungal and antitumor

Table 1: Enzymatic application of microorganisms

Amino acids:

Amino acids are building blocks of proteins and have high demand as supplements in food and nutraceutical industries. These are also used as supplements in bakery and packed foods. Microorganisms utilize amino acids for their metabolism and growth. Microorganisms are stimulated to produce extra amino acids, such that the extra amino acids are excreted into the surrounding medium.

Lysin and glutamic acid are the prime amino acid supplements in the preparation of bread and other nutritional supplements. Glutamic acid in the form of Monosodium Glutamate is used as flavor enhancing compound in the packed foods.

Glutamic acid is being produced from Corynebacterium gluatmicum. Corynebacterium sp. also used in:

  • Steroid conversion – an important process in the development of pharmaceutical products.
  • Hydrocarbon degradation – breakdown of plastic and oils for environmental protection.

Other amino acid producing bacteria include:

L-alanine – Corynebacterium dismutans, Pseudomonas dacunhae, Escherichia coli

L-arginine – Serratia marcescens, Bacillus subtilis

L-aspartic acid – Escherichia coli

Vitamins:

Vitamins are critical for vital functions and a healthy life. As the human body unable to synthesize these compounds, it is necessary to be supplied in the diet in small amounts. Vegetables and meat are sources of vitamins.

Microorganisms are grown in bulk quantities for the commercial production of vitamins like – thiamine, riboflavin, folic acid, vitamin B12, ascorbic acid, beta-carotene etc.

Some microorganisms that produce vitamins include:

Beta-carotene – Blakeslea trispora, Phycomyces blakesleeanus

Riboflavin – Mycocandida riboflavin, Candida flareri, Clostridium buytilicum

Vitamin B12 – Pseudomonas denitrificans, Bacillus megaterium, Propionibacterium freudenreichii.

Pharmaceutical drugs:

Trichoderma polysporum produces ‘Cyclosporin A’ which is used as immunosuppressive agent during organ transplantation. Moscus purpureus produces ‘Statins’ that has the ability to reduce blood cholesterol levels.

Single cell proteins:

Microorganisms that are rich in protein content can be used as protein supplements for human and domestic applications and are called single cell protein (SCP).

Algal species, Spirulina is the most popular SCP being produced commercially as a protein supplement.

Other SCP producing microorganisms include:

Bacteria – Pseudomonas fluorescens, Lactobacillus, B.megaterium

Algae – Chlorella pyronoidosa, Chondrus crispus

Fungi – Aspergillus fumigates, A. niger, Rhizopus cyclopium

Yeast – S.cerevisae, C.tropicalis, C.utilis

Apart from the above applications, microorganisms have major applications in vaccine production, biofuel production and in treatment of malnutrition.

ARTIFICIAL INTELLIGENCE AND THE HEALTHCARE INDUSTRY TODAY

by MicroScopia IWM, posted in Biotechnology

BY: Ria Fazulbhoy (MSIWM031)

The healthcare industry is going through a massive shift in many ways and using methods. The new world of artificial intelligence and technology has left no stone unturned, including the field of biology. From personal assistants like Siri, Alexa and Google home to smart watches tracking your steps, heart rates and sleep patterns, and mobile applications directing fitness and mental health, this amalgamation of technology and biology has made its way into everyone’s life. This is just the beginning, and we have a long way to go in the exploration of applications of this unique innovation.

Various emerging Industry Applications:

  1. Medical imaging: Many companies today are involving Artificial Intelligence – associated platforms in the medical field for scanning devices in order to improve image clarity and clinical outcomes. This is done by reducing exposure to radiation (For Example: CT scans for liver and kidney lesions).
  1. Management of chronic diseases: Machine learning is being integrated into devices like sensors, to detect, monitor and automate the delivery of treatment. This reduces manpower and increases efficiency.
  1. Drug discovery: A number of applications are there which involve the use of AI with drugs. This includes designing of drugs, polypharmacology, drug repurposing, screening of drugs and chemical synthesis.
  1. Telemedicine: Electronic consultations, medicine management and analyzing medical records and other data of the patients drastically helps the doctors, nurses as well as the patients. Today’s technology has enabled telemedicine to be an emerging field and has helped millions across the globe, especially during the trying times of the Covid-19 pandemic.
  1. Robot assisted surgeries: Data from real surgical processes are collected by cognitive surgical robotics to improve on and improve the already existing approaches to surgery. This greatly helps with minimizing the patient’s treatment time and chance or probability of error.

These are a few upcoming trends amongst various others.

Noteworthy examples of Integration of artificial intelligence in healthcare:

  1. KENSCI

KENSCI is a company targeted towards AI for hospital risk prediction. Its main goal is to combine artificial intelligence and big data to predict financial, operational and clinical risk in hospitals. It takes data from different and existing sources, which can then predict who may get sick and also keeps hospital costs in check.

  • XTALPI (Cloud based digital drug discovery)

This unique technology combines cloud and quantum physics which results in the prediction of small molecules’ chemical and pharmaceutical properties which plays an extremely important role in drug design and development, and thus, drug discovery. This company has a polymorph prediction i.e., crystal structure prediction technology, which is able to predict complex molecular systems and structures within days, as compared to the normal time of weeks and months.

  • ATOMWISE 

Atom wise constitutes a neural network for clinical trials, known as Atom Net. This helps to identify differing biological activity of patients, including their characteristics. The technology can screen anywhere between 10 to 20 million genetic compounds every day and has been found to deliver results 100 times faster than other competitive pharmaceutical companies in the markets. Clinical trials are essential for new discovery of pharmaceuticals and vaccines, and this practice is helping us move toward a cure for serious diseases, including Ebola and multiple sclerosis.

  • DEEP GENOMICS

This is a Canadian based platform which aims at helping researchers find candidates that would be compatible with drugs that are mainly targeted towards neuromuscular and neurodegenerative disorders. Finding the right candidate can improve chances of successful clinical trials. This also aids in the revolutionizing stream of personal medicine.

CORONARY ARTERY DISEASE/ ISCHEMIC HEART DISEASE

by MicroScopia IWM, posted in Medical microbiology/Immunlogy

BY: Ria Fazulbhoy (MSIWM031)

How does it occur?

This disease is currently the leading cause of death, worldwide. The ischemic heart disease is also known as coronary artery disease (CAD), atherosclerotic heart disease and coronary heart disease (CHD).

The disease occurs due to the blockage of the arteries leading to inadequate blood supply to the heart due to a number of reasons like building of plaque, excess exertion, high levels of cholesterol, etc. While narrowing may be caused by a blood clot or blood vessel constriction as well, it is more often caused by plaque accumulation, called atherosclerosis.

In the blood, cholesterol particles begin to accumulate on the walls of the arteries that supply the heart with blood. Eventually, it can form deposits called plaques. These deposits narrow the arteries and obstruct the flow of blood eventually.

The amount of oxygen supplied to the heart muscle is decreased by this reduction in blood flow. The heart muscle cells die, which is called a heart attack or myocardial infarction, when the blood supply to the heart muscle is completely blocked (MI).

Risk factors that increase chances of the disease:

The risk of developing ischemic heart disease is raised by a variety of variables. Not all individuals with risk factors are going to get ischemic heart disease. There are risk factors for ischemic heart disease include:

  • Diabetes
  • Family history of the condition of the heart
  • High cholesterol in the blood
  • Blood Pressure High
  • Strong triglycerides in the blood
  • Obesity
  • Inactive physique
  • Smoking and other forms of tobacco

Symptoms:

People can experience ischemic heart disease symptoms either regularly or only occasionally, depending on the severity of the case. Some common symptoms experienced by patients include:

  1. Angina pectoris

When the heart muscle is deprived of enough oxygen, the pain endured is called angina pectoris. This is a clinical condition characterized by chest, chin, shoulder, back, or arm pain that is normally exacerbated by exercise or emotional stress and immediately relieved by rest or nitroglycerin use.

  1. Breath shortage: One can experience shortness of breath or severe exhaustion with exercise if the heart can’t pump enough blood to meet the needs of your body.
  1.  Heart attack. A coronary artery that is totally blocked can cause a heart attack. Crushing pressure in one’s chest and pain in your shoulder or arm, sometimes with shortness of breath and sweating, are the classic signs and symptoms of a heart attack.
  1.  Arrhythmia – abnormal heart rhythm: Inadequate blood flow to the heart or heart tissue damage can interfere with the electrical impulses of your heart, causing irregular heart rhythms.

Diagnosis:

  1. Electrocardiogram – ECG or EKG: The electrical activity, rate, and regularity of your heartbeat are calculated by
  2. Echocardiogram: A image of the heart is generated using ultrasound (special sound wave).
  3. Exercise stress test: Tracks your heart rate as you walk on a treadmill. This helps to decide how well the heart functions as more blood needs to be pumped.
  4. Chest X-ray:  In order to create an image of the heart, lungs, and other organs in the chest, x-rays are used.
  5. Coronary angiogram: Monitors blockage and passage of blood into the coronary arteries. In order to detect dye injected through cardiac catheterization, X-rays are used.
  6. Coronary artery calcium scan:  A computed tomography (CT) scan searches for calcium accumulation and plaque in the coronary arteries.

Treatment:

Treatment given to patients suffering from Ischemic heart disease can include drug therapy, regular exercise, quitting of smoking/ tobacco, healthy dietetic.

Medications used to treat the disease include:

  • Angiotensin-converting enzyme (ACE) that relax blood vessels and decrease blood pressure.
  • Angiotensin receptor blockers (ARBs) which help in lowering the blood pressure.
  • Anti-ischemic agents like ranolazine, for example (Ranexa)
  • Antiplatelet drugs that stop blood clots from forming
  • Beta-blockers that decrease the heart rate
  • Calcium channel blockers that lower the heart muscle workload