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Orbital Hybridization: sp1, sp2, and sp3 Hybridization,...
Continue ReadingWhat is Hybridization?
The concept of hybridization was introduced by scientist pauling. He defined hybridization as redistribution of the energy of orbitals of individual atoms to give new orbitals of equivalent energy. This new orbitals formed are called hybrid orbital.
Features of Hybridization
• Number of hybrid orbitals = number of atomic orbital that get hybridized.
• Hybridized orbitals have equal energy and shape.
• Hybridization takes place with the orbitals of valence shell.
• Hybridisation does not take place in the isolated atom.
Types of Hybridization
sp Hybridization
When one s and one p orbital in same main shell mix together to give new hybrid, orbital called sp hybridised orbital. Forms linear molecules with 180 angle.
Example: all compounds of beryllium such as BeF2, BeCl2
sp2 Hybridization
One s + two p orbitals of the same shell = 3 equivalent orbital , new orbitals formed are called sp2 hybrid orbitals.
Angle: 120
Shape: trigonal planar shape
Example: All the compounds of Boron such as BCl3
sp3 Hybridization
1 s orbital + 3 p orbitals of same shell = 4 new equivalent orbital. new orbitals formed are called sp3 hybrid orbitals.
Angle: 109°28’ with one another Shape Tetrahedral
Example: ethane (C2H6), methane.
sp3d Hybridization
3p orbitals + 1d orbital = 5 orbitals of equal energy. Angle.
Equatorial orbitals: 3 hybrid orbitals lie in the horizontal plane inclined at an angle of 120 Axial orbitals.
The remaining 2 orbitals lie in the vertical plane at 90 degrees plane Shape : trigonal bipyramidal geometry
Example: Phosphorus pentachloride (PCl5)
sp3d2 Hybridization
1s + 3p + 2d orbitals = 6 identical hybrid orbitals.
Shape: octahedron.
Angle: 90 degrees to one another.
Example: sulfur hexafluoride SF6
sp3d3 Hybridization
1s +3p+3d =5 orbital of same element mix and recast to form hybrid orbitals
Shape: pentagonal bipyramidal geometry
Example: IF7
Concept of Hybridization of Carbon Atom
sp Hybridization: When Carbon is bound to two other atoms with the help of two double bonds or one single and one triple bond.
Example: Hybridization of CO2.
sp2 Hybridization: When carbon atom bonding takes place between 1 s-orbital with two p orbitals then the formation of two single bonds and one double bond between three atoms takes place.
Example: Hybridization of graphite
sp3 Hybridization: When the carbon atom is bonded to four other atoms.
Example: Hybridization of CH4 (Methane)
How to Determine Hybridization?
Hybridization of Nitrogen in Ammonia, NH3
Step 1: Write the Lewis structure
The valency of nitrogen is 3.
Thus, it forms 3 bonds with three hydrogen atoms.
There is also a lone pair on nitrogen.
Step 2: Calculate number of sigma (σ) bonds
Nitrogen in ammonia is bonded to 3 hydrogen atoms.
Number of sigma bonds are = 3.
Step 3: Calculate number of lone pairs
Number of lone pairs on nitrogen atom = (v – b – c) / 2
= (5 – 3 – 0) / 2 = 1 lone pair
There are 5 balance electrons in nitrogen atom before bond formation.
Step 4: Calculate steric number of nitrogen atom
Steric number = number of σ bonds + number of lone pairs
Thus, 3 + 1 = 4
Step 5: Give hybridization and shape of molecule
Nitrogen in ammonia is sp3 hybridization.
The shape is pyramidal.
Hybridization and Shape of XeF4
Step 1: Calculate the number of sigma (σ) bonds
Number of sigma bonds formed by xenon = four as it is bonded to only 4 fluorine atoms.
The valency of fluorine = one.
Step 2: Calculate number of lone pairs
The number of lone pairs on xenon atom = (v – b – c) / 2 = (8 – 4 – 0) / 2
Step 3: Calculate the steric number of central atom
Steric number = number of σ-bonds + number of lone pairs = 4 + 2 = 6
Step 4: Allocate hybridization and shape of molecule
The hybridization is sp3d2.
Thus, Structure is based on octahedral geometry
Hence, Shape is square planar.
Hybridization and Shape of SO2
Step 1: Write the Lewis structure
Sulfur’s valency may be 2 or 4 or 6.
Oxygen’s valency = one.
Each oxygen makes two bonds with sulfur atom.
One is sigma bond and the second one is pi bond.
The total number of bonds formed by sulfur with two oxygen atoms = four.
Step 2: Calculate number of sigma (σ) bonds
The number of sigma bonds formed by sulfur atom = two
As it is bonded to two oxygen atoms.
Step3: Calculate number of lone pairs
The number of lone pairs on sulfur atom is = (v – b – c) / 2 = (6 – 4 – 0) / 2; Thus 1.
Number of valence electrons in sulfur is 6.
Total number of bonds including sigma and pi bonds is = 4.
Step 4: Calculate steric number of central atom
Steric number = no. of σ-bonds + no. of lone pairs = 2 + 1 Thus 3
Step 5: Give the hybridization and shape of molecule
The hybridization is sp2.
Structure is built on trigonal planar geometry with one lone pair.
Shape is = angular.
Steric Number
If steric number is 4, then it is sp3
If steric number is 3 then it is sp2
If steric number is 2 then it is sp
C1 – SN = 3 (three atoms connected), thus sp2
C2 – SN = 3 (three atoms connected), thus sp2
O4 – SN = 3 (1 atom + 2 lone pairs), thus sp2
O5 – SN = 4 (2 atoms + 2 lone pairs), thus sp3
C6 – SN = 4 (4 atoms), thus it is sp3
C7 – SN = 4 (4 atoms), thus it is sp3
N8 – SN = 4 (3 atoms + 1 lone pair), thus it is sp3
C9 – SN = 2 (2 atoms), thus it is sp
C10 – SN = 2 (2 atoms) thus it is sp
​Hybridization Citations
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Atomic Number: Definition, Examples, and Facts
Continue ReadingAtomic Number: Introduction
Atoms are the vital building blocks of all matter and are composed of protons, neutrons, and electrons. The atoms are electrically neutral, the number of positively charged protons is equal to the number of negatively charged electrons.
Subsequently neutrons do not affect the charge, the number of neutrons is not reliant on the number of protons and will differ even amongst atoms of the same element.
History of Atomic Number
In 1910, Henry Gwyn-Jeffreys Moseley gave the concept of atomic number that was evolved from historic research. While experimenting on several chemical elements with X-rays.
He observed the pattern formed by reflected rays. After this experiment, he discovered that the wavelength of the reflected X-rays decreased in a regular predictable pattern with the increase in atomic mass.
According to his hypothesis, the regular variation in wavelength from element to element was triggered by an increase in the positive charge on atomic nuclei in going from one element to the next-heavier element.
After the discoveries made by Moseley, another scientist named Dmitri Mendeleev in 1850 proposed a new considerate understanding of the periodic law.
Mendeleev said that the properties of elements differ in a regular and expectable pattern when the elements are organized according to their atomic masses.
His theory was somewhat correct as the periodic table on this basis had a most important flaw. Certain pairs of elements seem to be misplaced when arranged according to their masses.
Various difficulties disappear when the atomic number rather than atomic mass is used to construct the periodic table. An element`s chemical properties rest on the number and arrangement of electrons in its atoms.
The number of electrons in an atom, is determined by the nuclear charge. Thus, the number of protons in a nucleus governs the chemical properties of an element.
What is Atomic Number?
The atomic number is represented by the letter Z . It is defined as the number of protons in the nucleus of each atom of that element.
The one characteristic that makes each element unique compared to all other elements is the number of protons. The elements are different because of their atomic number.
For example, any atom with an atomic number of 8 (its nucleus contains 8 protons) is an oxygen atom, and an atom with a different number of protons would be a different element.
Figure below showing the periodic table that displays all of the known elements and is arranged in order of increasing atomic number. In the below figure of the periodic table, the atomic number is displayed above the elemental symbol.
Figure: The periodic table is classified elements by atomic number
For example, Hydrogen at the upper left of the table has an atomic number of 1. Each hydrogen atom has one proton in its nucleus. Subsequent on the table is helium, whose atoms have two protons in the nucleus. Lithium atoms have three protons, beryllium atoms have four protons, and so on.
Meanwhile atoms are neutral, the number of electrons in an atom is equal to the number of protons. Hydrogen atoms normally have one electron that occupies the space outside of the nucleus.
Helium having two protons, will have two electrons. The count of proton will always be equal to an atom’s atomic number. This value will not vary unless the nucleus decays or is been bombed.
Mass Number
Several experiments have shown that the majority of the mass of an atom is been concentrated in its nucleus that is composed of protons and neutrons.
The mass number is represented by the letter A. It is also defined as the total number of protons and neutrons in an atom.
Table below shows the data from the first six elements from the periodic table. If we take the example of helium here. The atomic number of helium is 2 so that means it has 2 protons in its nucleus. And the nucleus also contains 2 neutrons.
So, the mass number of helium is 4. So this concludes helium atom contains 2 electrons as the number of electrons is equal to the number of protons.
Another example taken here can be of Lithium that has three protons and four neutrons and here the mass number will be equal to seven.
Table: The atoms of the first six elements
Name Symbol Atomic Number (Z) Protons Neutrons Electrons Mass Number (A) Hydrogen H 1 1 0 1 1.01 Helium He 2 2 2 2 4.00 Lithium Li 3 3 4 3 6.94 Berylium Be 4 4 5 4 9.01 Boron B 5 5 6 5 10.18 Carbon C 6 6 6 6 12.01 Atomic Number Calculation
Therefore, the formula can be made from this, by knowing the mass number and an atomic number of an atom we can determine the number of neutrons present in that atom by subtraction.
Number of neutrons = Rounded Mass number – Atomic number
So, this shows how can we calculate the atomic number and mass number of a given element.
Atomic Number Citations
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Mitosis: Definition, Stages, Types, Diagram, and Facts
Continue ReadingMitosis: Introduction
Cell cycle is the sequential process taking place to regulate the growth of organism; cell divides to produce a genetic replica and enters the stage of cell growth.
Cell growth involves the synthesis of organic material and integrates information across its counter parts for synchronous development of the whole body.
The cell synthesis phase lasts till a cell reaches its maturity; on initiation the cell again divides to produce new cell and the process continues.
Cell cycle is a sequential development of cell between two cell divisions. The cycle is genetically controlled and are programmed in every cell and are specific for each region.
Varied species has variable time length of cell cycle decided by physiological and influences pertaining to their niche.
Two Phases of cell cycle are: Interphase and Mitotic Phase.
Interphase involves G1, synthesis and G2 phase; chromosomal replication and development is regulated by the phase; determines the quality and quantity of chromosome entering the daughter cells and a balance is maintained by the phase.
Karyokinesis and Cytokinesis division, segregation of chromosome and cell takes place during Mitotic Phase.
The notable feature of cell cycle is eukaryotic organisms though diverse and distinct have a common type of cell division over the Kingdom of Eukaryotes is a scientific wonder and research have emphasized that timing of a cell entering the cell cycle is essential in cell cycle regulation.
Phases of Cell Cycle
The cell cycle is common for all eukaryotic organisms; travelling through 2 major phases based on the cell division: Interphase and Mitotic Phase.
Interphase consists of 3 phases Gap 1 phase, Synthesis Phase and Gap 2 Phase.
Similarly, mitosis has four phases Prophase, metaphase, anaphase, and telophase.
The development of cells through these phases are influenced and facilitated by heterodimeric protein kinases – Cyclin and Cyclin Dependent Kinases.
Mitosis Phase
The changes in above phases are minimal or not clearly visible in microscopes whereas the changes in M Phase are easily detectable.
The phase has 4 parts in which the division takes place systematically and continuously.
The cell stages are easily visible in plant parts as the specialized dividing region – Meristem is prevalent in roots and shoots are continuously dividing providing a mechanical support and functional integrity to the plants.
The 4 phases are: Prophase, Metaphase, Anaphase and Telophase. Each phase has a distinctive change to be identified and Eukaryotic cells replicates in the same order in most of the organisms.
I. Prophase
The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division.
RNA synthesis is inhibited.
The nuclear envelope disintegrates.
The chromatin condensation during G2 phase completes forming Chromatids.
Chromatids are held together by centromere.
The formation of cytoskeleton is initiated along with microtubules.
Kinetochores are formed.
II. Metaphase
Nuclear membrane is eliminated completely chromosomes are completely condensed.
The cytoskeleton – spindle fibers develop and attach to the kinetochores.
This phase also signifies with equatorial alignment of chromosome.
The phase has a checkpoint where it makes sure all the chromatids are well connected to the kinetochores.
Feeble connection leads to unequal distribution of chromosomes in the daughter cells forming defective cell when not eliminated becomes lethal to the organism.
III. Anaphase
Chromosomal split forms daughter chromatids; travels to the opposite poles.
The chromosomes are V – Shaped as they are dragged to the opposite sites by the shortening of the spindle fibers.
So that daughter cells have an equal number of chromosomes to function regular cellular activities.
IV. Telophase
Microtubules disappear and chromosomes decondense to chromatin mass.
Nuclear envelope starts to form.
The disintegrated organelles form again with 2 nuclei and nucleoli.
This reappearance of the cell organelles and equal distribution of the cellular materials ensures each cell to function independently in the cell cycle.
These phases mark the Karyokinesis were the nucleus and other cell parts are newly formed.
V. Cytokinesis
Cytokinesis is the formation of daughter cells after mitosis; indicated by furrow which starts to differentiate two daughter cells grows gradually forming a cell plate while the organelles formed gets segregated.
Cell plate represents the lamella between 2 cell walls.
Mitosis Regulation
The cell cycle is completely regulated by Cyclins and Cyclin dependent protein kinases; belongs to serine – threonine sub classes; Phosphorylates and dephosphorylates the activating components to proceed through the cell cycle depending on the external cues.
The commitment to enter cell division is irreversible when a cell traverse from G1 phase to synthesis Phase is controlled by CYC – CDK complexes as all other phase to enter Mitotic phase.
Mitotic Phase requirements are dealt by the previous phases were DNA replication and chromosomal condensation along with mitotic transcriptional factors and other proteins are synthesized to ensure the cell division which is also supported by the phytohormones.
The main CYC – CDK complex in mitotic cycle is the CYCD-CDKA whose expression is essential for daughter cells to develop well depending on the nutrient provided.
The transition from G2 to M phase is mediated by CYCB – CDKB from G2 phase ensures a proper cell division; expressed from S phase by minimal accumulation gradually increases in quantity to transit from G2 phase to Mitotic phase.
Mitotic CYC – CDK complexes are synthesized during S phase but remains phosphorylated which induces inactivation of the compound.
Following DNA replication phosphatase cdc 25 is activated; dephosphorylation of threonine and tyrosine removes the inhibition from Wee 1 kinase promotes expression of Mitotic CDK – CYC inducing mitosis and forms 2 daughter cells.
Endoreduplication
In endoreduplication the cells undergo continuous division without the formation of cell wall (i.e.) CYTOKINESIS increasing the chromosomal number of the plant.
This is referred as ploidy and in plants; increases the yield and stability of the plant; is under debate.
Endoreduplication are predominant in endosperms. The protein expression producing the mitotic phase is inhibited by regulatory mechanism for the expression of endoreduplication.
The main feature in endoreduplication is that it initiates after a mitosis takes place.
Several factors are involved in the ubiquitination of the cyclin to ensure the cellular restriction to a particular function.
Mitosis Citations
- Chromatid cohesion during mitosis: lessons from meiosis. J Cell Sci . 1999 Aug;112 ( Pt 16):2607-13.
- Mitosis: towards a molecular understanding of chromosome behavior. Curr Opin Cell Biol . 1991 Feb;3(1):59-66.
- Mitosis in filamentous fungi: how we got where we are. Fungal Genet Biol . 1999 Jun;27(1):1-25.
- Abnormal mitosis in reactive astrocytes. Acta Neuropathol Commun . 2020 Apr 15;8(1):47.
- Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints. Chromosome Res . 2011 Apr;19(3):291-306.
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Plasmid: Definition, Vector, Types, and Transformation
Continue ReadingWhat are Plasmids?
A plasmid is a little, extrachromosomal DNA molecule inside a cell that is truly isolated from chromosomal DNA and can imitate autonomously.
They are most ordinarily found as little roundabout, twofold abandoned DNA molecules in microorganisms; notwithstanding, plasmids are once in a while present in archaea and eukaryotic organic entities.
In nature, plasmids frequently convey qualities that advantage the endurance of the creature and present specific benefits like anti-microbial obstruction.
While chromosomes are huge and contain all the fundamental hereditary data for living under ordinary conditions, plasmids are generally tiny and contain just extra qualities that might be helpful in specific circumstances or conditions.
Counterfeit plasmids are generally utilized as vectors in atomic cloning, serving to drive the replication of recombinant DNA arrangements inside have organic entities.
In the lab, plasmids might be brought into a cell by means of change.
Plasmids are considered replicons, units of DNA fit for imitating self-rulingly inside a reasonable host.
In any case, plasmids, as infections, are not for the most part named life. Plasmids are communicated starting with one bacterium then onto the next (even of another species) generally through formation.
This host-to-have move of hereditary material is one instrument of even quality exchange, and plasmids are viewed as a component of the mobilome.
Not at all like infections, which encase their hereditary material in a defensive protein coat called a capsid, plasmids are “stripped” DNA and don’t encode qualities important to encase the hereditary material for move to another host; nonetheless, a few classes of plasmids encode the conjugative “sex” pilus vital for their own exchange.
The size of the plasmid shifts from 1 to more than 200 kbp, and the quantity of indistinguishable plasmids in a solitary cell can go somewhere in the range of one to thousands under certain conditions.
History of Plasmid
The term plasmid was presented in 1952 by the American atomic researcher Joshua Lederberg to allude to “any extrachromosomal inherited determinant.”
The term’s initial utilization incorporated any bacterial hereditary material that exists extra chromosomally for essentially some portion of its replication cycle, but since that depiction incorporates bacterial infections, the thought of plasmid was refined over the long run to contain hereditary components that recreate autonomously.
Later in 1968, it was concluded that the term plasmid ought to be received as the term for extrachromosomal hereditary component, and to recognize it from infections, the definition was limited to hereditary components that exist only or transcendently outside of the chromosome and can duplicate self-governingly.
Different Categories of Plasmids
Plasmids might be grouped in various manners. Plasmids can be extensively characterized into conjugative plasmids and non-conjugative plasmids.
Conjugative plasmids contain a bunch of move qualities which advance sexual formation between various cells. In the unpredictable cycle of formation, plasmids might be moved starting with one bacterium then onto the next by means of sex pili encoded by a portion of the exchange qualities.
Non-conjugative plasmids are unequipped for starting formation, subsequently they can be moved distinctly with the help of conjugative plasmids.
A moderate class of plasmids are mobilizable, and convey just a subset of the qualities needed for move. They can parasitize a conjugative plasmid, moving at high recurrence just in its quality.
Plasmids can likewise be arranged into inconsistency gatherings. A microorganism can hold onto various sorts of plasmids, yet various plasmids can possibly exist in a solitary bacterial cell in case they are viable. Another approach to group plasmids is by work.
There are five principle classes:
Fertility F-plasmids, which contain tra qualities. They are fit for formation and result in the statement of sex pili.
Resistance (R) plasmids, which contain qualities that give resistance against anti-toxins or toxic substances.
Generally known as R-factors, before the idea of plasmids was perceived. Col plasmids, which contain qualities that code for bacteriocins, proteins that can kill different microbes.
Degradative plasmids, which empower the absorption of strange substances, for example toluene and salicylic corrosive.
Virulence plasmids, which transform the bacterium into a microorganism. for example Ti plasmid in Agrobacterium tumefaciens Plasmids can have a place with more than one of these useful gatherings.
Properties of Plasmid
There are two sorts of plasmid incorporation into a host microbes: Non-coordinating plasmids reproduce likewise with the top case, while episomes, the lower model, can incorporate into the host chromosome.
With the end goal for plasmids to duplicate freely inside a cell, they should have a stretch of DNA that can go about as a beginning of replication.
Oneself reproducing unit, for this situation, the plasmid, is known as a replicon. A commonplace bacterial replicon may comprise of various components, for example, the quality for plasmid-explicit replication commencement protein (Rep), rehashing units called iterons, DnaA boxes, and a neighboring AT-rich locale.
More modest plasmids utilize the host replicative proteins to make duplicates of themselves, while bigger plasmids might convey qualities explicit for the replication of those plasmids.
A couple of kinds of plasmids can likewise embed into the host chromosome, and these integrative plasmids are now and then alluded to as episomes in prokaryotes.
Plasmids quite often convey no less than one quality. Many qualities conveyed by a plasmid are advantageous for the host cells, for instance: empowering the host cell to get by in a climate that would some way or another be deadly or prohibitive for development.
A portion of these qualities encode attributes for anti-infection resistance or resistance to substantial metal, while others might create virulence factors that empower a bacterium to colonize a host and beat its safeguards or have explicit metabolic capacities that permit the bacterium to use a specific supplement, including the capacity to debase hard-headed or poisonous natural mixtures.
Plasmids can likewise give microbes the capacity to fix nitrogen. A few plasmids, in any case, have no perceptible impact on the aggregate of the host cell or its advantage to the host cells can’t be resolved, and these plasmids are called mysterious plasmids.
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Normally happening plasmids differ enormously in their actual properties. Their size can go from tiny, small scale plasmids of under 1-kilobase sets (Kbp) to extremely huge mega plasmids of a few mega base sets (Mbp).
At the upper end, little varies between a mega plasmid and a minichromosomal. Plasmids are for the most part roundabout, yet instances of direct plasmids are additionally known.
These straight plasmids require specific components to repeat their finishes. Plasmids might be available in an individual cell in changing number, going from one to a few hundreds.
The ordinary number of duplicates of plasmid that might be found in a solitary cell is known as the plasmid duplicate number, and is dictated by how the replication commencement is controlled and the size of the molecule. Bigger plasmids will in general have lower duplicate numbers.
Low-duplicate number plasmids that exist just as one or a couple of duplicates in every bacterium are, upon cell division, at risk for being lost in one of the isolating microscopic organisms.
Such single-duplicate plasmids have frameworks that endeavour to effectively convey a duplicate to both girl cells. These frameworks, which incorporate the parABS framework and parMRC framework, are regularly alluded to as the parcel framework or segment capacity of a plasmid.
The utilization of plasmids as a procedure in sub-atomic science is upheld by bioinformatics programming. These projects record the DNA succession of plasmid vectors, help to foresee cut destinations of limitation compounds, and to design controls.
Instances of programming bundles that handle plasmid maps are ApE, Clone Manager, GeneConstructionKit.
Plasmid Citations
- Plasmid Typing and Classification. Methods Mol Biol . 2020;2075:309-321.
- Plasmid copy number and plasmid stability. Adv Biochem Eng Biotechnol . 2004;86:47-82.
- Fitness Costs of Plasmids: a Limit to Plasmid Transmission. Microbiol Spectr . 2017 Sep;5(5).
- Isolation of plasmid DNA from bacteria. Methods Enzymol . 2013;529:135-42.
- Plasmid persistence: costs, benefits, and the plasmid paradox. Can J Microbiol . 2018 May;64(5):293-304.
- Plasmid Detection, Characterization, and Ecology. Microbiol Spectr . 2015 Feb;3(1):PLAS-0038-2014.
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Thin Layer Chromatography: Definition, Principle, and Types
Continue ReadingWhat Is Thin Layer Chromatography?
Thin Layer Chromatography is a strategy used to detach non-volatile blends.
The trial is directed on a sheet of aluminium foil, plastic, or glass which is covered with a thin layer of adsorbent material.
The material normally utilized is aluminium oxide, cellulose, or silica gel. After the example has been applied on the plate, a dissolvable or dissolvable blend (known as the portable stage) is drawn up the plate through fine activity.
Since various analytes climb the TLC plate at various rates, detachment is achieved. The versatile stage has various properties from the fixed stage.
For instance, with silica gel, an exceptionally polar substance, non-polar versatile stages, for example, heptane are utilized. The portable stage might be a blend, permitting scientific experts to calibrate the mass properties of the versatile stage.
After the trial, the spots are envisioned. Frequently this should be possible just by extending bright light onto the sheet; the sheets are regularly treated with a phosphor, and dull spots show up on the sheet where mixtures ingest the light impinging on a specific region.
Substance cycles can likewise be utilized to envision spots; anisaldehyde, for instance, structures shaded adducts with numerous mixtures, and sulfuric corrosive will burn most natural mixtures, leaving a dull spot on the sheet.
On finish of the partition, every segment shows up as spots isolated upward. Each spot has a maintenance factor (Rf) communicated as:
Rf = dist. covered by sample/dist. covered by solvent
The factors influencing retardation factor are the dissolvable framework, measure of material spotted, spongy and temperature.
Attention is one of the quickest, most economical, least difficult and simplest chromatography methods. Thin-layer chromatography can be utilized to screen the advancement of a response, recognize intensifies present in each combination, and decide the immaculateness of a substance.
Explicit instances of these applications include dissecting ceramides and unsaturated fats, location of pesticides or bug sprays in food and water, breaking down the color arrangement of strands in crime scene investigation, measuring the radiochemical immaculateness of radiopharmaceuticals, or distinguishing proof of therapeutic plants and their constituents.
Various improvements can be made to the first technique to mechanize the various strides, to expand the goal accomplished with TLC and to permit more precise quantitative examination.
This strategy is alluded to as HPTLC, or “elite TLC”. HPTLC commonly utilizes thinner layers of fixed stage and more modest example volumes, accordingly, diminishing the deficiency of goal because of diffusion.
Thin Layer Chromatography Principle
Like other chromatographic strategies, thin-layer chromatography (TLC) relies upon the division standard.
The partition depends on the general partiality of mixtures towards both the stages.
The mixtures in the portable stage move over the outside of the fixed stage.
The development happens so that the mixtures which have a higher fondness to the fixed stage move gradually while different mixtures travel quick.
Consequently, the detachment of the combination is achieved.
On fruition of the detachment interaction, the individual parts from the combination show up as spots at separate levels on the plates.
Their person and nature are recognized by reasonable discovery strategies.
Thin Layer Chromatography Procedure
Prior to beginning with the Thin Layer Chromatography Experiment, let us comprehend the various parts needed to lead the strategy alongside the stages in question.
Thin Layer Chromatography Plates – instant plates are utilized which are artificially inactive and stable. The fixed stage is applied on its surface as a thin layer.
The fixed stage on the plate has a fine molecule size and furthermore has a uniform thickness. Thin Layer Chromatography Chamber – Chamber is utilized to foster plates.
It is dependable to keep a consistent climate inside which will help in creating spots. Likewise, it forestalls the dissolvable vanishing and keeps the whole cycle sans dust.
Thin Layer Chromatography Mobile stage – Mobile stage is the one that maneuvers and comprises of a dissolvable blend or a dissolvable.
This stage ought to be sans particulate. The higher the nature of virtue the advancement of spots is better. Thin Layer Chromatography Filter Paper – It must be set inside the chamber. It is soaked in the portable stage.
Thin Layer Chromatography Experiment
The fixed stage that is applied to the plate is made to dry and balance out. To apply test spots, thin checks are made at the lower part of the plate with the assistance of a pencil.
Apply test answers for the stamped spots. Empty the portable stage into the TLC chamber and to keep up with equivalent mugginess, place a soaked channel paper in the versatile stage.
Spot the plate in the TLC chamber and close it with a cover. It is kept so that the example faces the versatile stage. Drench the plate for improvement.
Make sure to keep the example spots well over the level of the versatile stage. Try not to submerge it in the dissolvable. Stand by till the advancement of spots.
When the spots are created, take out the plates and dry them. The example spots can be seen under an UV light chamber.
Thin Layer Chromatography Applications
The subjective testing of Various prescriptions like narcotics, nearby sedatives, anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is finished by TLC.
Attention is incredibly valuable in Biochemical investigation like partition or disconnection of biochemical metabolites from its blood plasma, pee, body liquids, serum, and so forth.
Thin layer chromatography can be utilized to distinguish normal items like fundamental oils or volatile oils, fixed oil, glycosides, waxes, alkaloids, and so forth.
It is broadly utilized in isolating multicomponent drug details. It is utilized to clean of any example and direct examination is done between the example and the real example.
It is utilized in the food business, to isolate and distinguish colors, improving specialist, and additives.
It is utilized in the restorative business. It is utilized to consider if a response is finished.
Drawbacks Of Thin Layer Chromatography
Thin Layer Chromatography plates don’t have longer fixed stage. When contrasted with other chromatographic procedures the length of partition is restricted.
The outcomes created from TLC are hard to duplicate. Since TLC works as an open framework, a few factors, for example, mugginess and temperature can be ramifications to the ultimate result of the chromatogram.
As far as possible is high and in this manner assuming you need a lower location limit, you can’t utilize TLC. It is just a subjective investigation method and not quantitative.
Thin Layer Chromatography Citations
- Review of advances in the thin layer chromatography of pesticides: 2008-2010. Methods Enzymol . 2013;533:303-24.
- Review of advances in the thin layer chromatography of pesticides: 2008-2010. J Environ Sci Health B . 2011;46(7):557-68.
- Review of advances in the thin layer chromatography of pesticides: 2012-2014. J Environ Sci Health B . 2015;50(5):301-16.
- Sample Preparation for Thin Layer Chromatography. Adv Chromatogr . 2017;53:301-329.
- Thin layer chromatography/mass spectrometry. J Chromatogr A . 2011 May 13;1218(19):2700-11.
- Modern thin layer chromatography. J AOAC Int . Sep-Oct 2008;91(5):1142-4.
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What is Electron Microscopy? Principle, Types, and...
Continue ReadingWhat is Electron Microscopy?
Electron microscopy (EM) is a technique to obtain images of biological and non-biological specimens with greater resolution.
It is utilized in biomedical exploration to examine the nitty gritty design of tissues, cells, organelles and macromolecular complexes.
The high resolution of EM pictures results from the utilization of electrons as the origin of illuminating radiation.
EM is utilized simultaneously with ancillary techniques like thin sectioning, negative staining etc. to address explicit questions.
EM pictures give key detail on the basic structure of cell function and of cell disease.
Electron Microscopy Principle
An electron microscope utilizes an ‘electron beam’ to create the pictorial representation of the object and magnification is gotten by ‘electromagnetic fields’; in contrast to light or optical microscopes, in which ‘light waves’ are utilized to deliver the picture and magnification is acquired by an arrangement of ‘optical focal lenses’.
It has effectively been examined that, the more modest is the frequency of light, the more noteworthy is its resolving power.
The frequency of green light (=0.55µ) is 1, 10,000 times longer than that of electron beam (=0.000005µ or 0.05 Å; 1µ = 10,000 Å).
That is the reason, in spite of its more modest mathematical aperture, an electron microscope can resolve objects as little as 0.001µ (=10 Å), when contrasted with 0.2µ by a light microscope.
Subsequently, as compared to light microscope, the resolving power of an electron microscope is 200 times more prominent.
For instance, if light microscope produces magnification up to ×2000, then electron microscope produces up to ×400,000 magnification.
Types of Electron Microscope
There are two important types of electron microscope – the transmission EM (TEM) and the scanning EM (SEM).
I. Transmission Electron Microscope (TEM)
The transmission electron microscope (TEM) is utilized to observe thin specimens (tissue sections, molecules, etc.) through which electrons can pass producing a projection image.
The TEM does closely resemble in various angles with the conventional (compound) light microscope.
TEM is utilized, in addition to other things, to image the inside of cells, the construction of protein molecules (differentiated by metal shadowing), the association of molecules in infections and cytoskeletal filaments (ready by the negative staining technique), and the course of action of protein molecules in cell membranes (by freeze-fracture).
II. Scanning Electron Microscopy
Scanning electron microscopy relies upon the emission of secondary electrons from the surface of a specimen.
As a result of its incredible profundity of focus, a scanning electron microscope is the EM simple of a stereo light microscope.
It gives detailed pictures of the surfaces of cells and entire living beings that are not possible by TEM.
It can likewise be utilized for particle counting and size determination, and for measure control.
It is named a scanning electron microscope on the grounds that the picture is framed by scanning a focused electron beam onto the surface of the specimen in a raster pattern.
The interaction of the primary electron beam with the atoms close to the surface result in the emission of particles at each point in the raster.
These can be gathered with an assortment of detectors, and their overall number meant brilliance at every equivalent point on a cathode beam tube.
Since the size of the raster at the specimen is a minute than the survey screen of the CRT, the resulting picture is a magnified image of the specimen.
Fittingly prepared SEMs (with secondary, backscatter and X-beam detectors) can be utilized to analyse the topography and atomic distribution of specimens, and furthermore, for instance, the surface circulation of immuno-labels.
III. Reflection Electron Microscope (REM)
Likewise, TEM in REM, an electron beam is incident on a surface yet rather than utilizing the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is distinguished.
This technique is normally combined with reflection high-energy loss spectroscopy (RHELS) and reflection high energy electron diffraction (RHEED).
In addition to this, another variety is spin-polarized low-energy electron microscopy (SPLEEM), which is utilized for taking a gander at the microstructure of magnetic spaces.
IV. Scanning Transmission Electron Microscope (STEM)
The STEM rasters a focused incident probe all around a specimen that (likewise with the TEM) has been thinned to enable identification of electrons scattered through the specimen.
The high resolution of the TEM is subsequently conceivable in STEM.
The focusing activity (and distortions) happen before the electrons bombard the specimen in the STEM, yet thereafter in the TEM.
The STEMs utilization of SEM-like beam rastering works on annular dark-field imaging, and other logical techniques, yet in addition implies that picture information is obtained in serial instead of in parallel fashion.
Regularly TEM can be outfitted with the scanning option and afterward it can work both as TEM and STEM.
V. Scanning Tunneling Microscopy (STM)
In STM, at high voltage a conductive tip held is brought close to a surface, and a profile can be received depending on the tunneling probability of an electron from the tip to the example since it is an function of distance.
Disadvantages of Electron Microscopy
Electron microscopes are extravagant to assemble and keep up with, yet the funds and running expenses of confocal light microscope system currently covers with those of fundamental electron microscopes.
Microscopes intended to accomplish high resolutions should be housed in stable structures with uncommon administrations, for example, magnetic field cancelling systems.
The examples to a great extent must be seen in vacuum, as the molecules that make up air would scatter the electrons.
An exemption is liquid-phase electron microscopy utilizing either a closed liquid cell or environmental chamber, for instance, in the environmental scanning electron microscope, which permits hydrated examples to be seen in a low-pressure (up to 20 Torr or 2.7 kPa) wet environment.
For in situ electron microscopy of different techniques, gaseous samples have also been created.
SEM working in traditional high-vacuum mode normally image conductive specimens; in this way non-conductive materials require conductive covering (gold/palladium combination, carbon, osmium, and so forth).
It becomes possible to observe a non-conductive material without coating using the low-voltage mode of modern microscopes.
Minute, stable specimens like carbon nanotubes, diatom frustules and little mineral crystals (asbestos filaments, for instance) doesn’t require any special treatment prior being observed in the electron microscope.
Samples of hydrated materials, including practically all biological specimens must be ready in different manners to balance out them, diminish their thickness (ultrathin sectioning) and increases their electron optical contrast (staining).
These cycles might bring about artefacts, however these can for the most part be recognized by contrasting the outcomes got by utilizing drastically unique specimen arrangement methods.
Since the 1980s, examination of cryofixed, vitrified specimens has additionally gotten progressively utilized by researchers, further affirming the legitimacy of this technique.
Electron Microscopy Citations
- A primer on resolving the nanoscale structure of the plasma membrane with light and electron microscopy. J Gen Physiol . 2019 Aug 5;151(8):974-985.
- Electron microscopy of specimens in liquid. Nat Nanotechnol . 2011 Oct 23;6(11):695-704.
- Diagnostic Electron Microscopy of Viruses With Low-voltage Electron Microscopes. J Histochem Cytochem . 2020 Jun;68(6):389-402.
- Quantitative Cryo-Scanning Transmission Electron Microscopy of Biological Materials. Adv Mater . 2018 Oct;30(41):e1706681.
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Fluorescence In Situ Hybridization (FISH): History, limitations,...
Continue ReadingFluorescence In Situ Hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a laboratory technique for identifying and locating a particular DNA sequence on a chromosome.
The technique depends on exposing chromosomes to a little DNA sequence considered a probe that has a fluorescent molecule joined to it.
FISH is a molecular technique that is regularly used to distinguish and specify explicit microbial gatherings.
This technique can be utilized to determine, with the presence or nonattendance of a fluorescent signal, regardless of whether explicit genetic components exist in an example.
This can be valuable for determining if microorganisms have a specific gene present and additionally if that gene is being communicated under a given arrangement of conditions.
Fluorescent probes are intended to append to explicit genetic districts of microorganisms that will separate them from different gatherings.
At the point when these probes are applied a fluorescent microscope can be utilized to recognize the presence or nonappearance of individual microbial gatherings.
Another sister technique, called Flow-Cytometric Analysis (FCM), can likewise be completed when fluorescent labels are applied to microbial populaces.
Fluorescent signal is utilized to exclude or sort individual genotypes of gatherings of cells.
Fluorescence In Situ Hybridization (FISH) Principle
In this methodology, a fluorescent dye is connected to a purified piece of DNA, and afterward that DNA is incubated with the full arrangement of chromosomes from the originating genome, which have been joined to a glass microscope slide.
The fluorescently marked DNA finds its matching section on one of the chromosomes, where it sticks.
By looking at the chromosomes under a microscope, an analyst can find the region where the DNA is bound on account of the fluorescent dye appended to it.
This information consequently uncovers the area of that piece of DNA in the starting genome.
Fish Probe
In biology, a probe is defined as a single strand of either DNA or RNA that is complementary to a nucleotide sequence of interest.
RNA Probes
RNA probes can be intended for any gene or any sequence within a gene for representation of mRNA, lncRNA and miRNA in tissues and cells.
FISH is utilized by examining the cellular reproduction cycle, explicitly interphase of the nuclei for any chromosomal anomalies.
FISH permits the analysis of an enormous series of authentic cases a lot simpler to distinguish the pinpointed chromosome by creating a probe with an artificial chromosomal establishment that will draw in comparable chromosomes.
When a nucleic abnormality is identified the hybridization signals for each probe.
Each probe for the discovery of mRNA and lncRNA is made out of ~20-50 oligonucleotide pairs, each pair covering a space of 40–50 bp.
The specifics rely upon the specific FISH technique utilized.
For miRNA recognition, the probes utilize exclusive chemistry for explicit discovery of miRNA and cover the whole miRNA sequence.
Urothelial Cells Marked With Four Different Probes
Probes are regularly gotten from fragments of DNA that were separated, purged, and intensified for use in the Human Genome Project.
The size of the human genome is so enormous, contrasted with the length that could be sequenced straightforwardly, that it was important to separate the genome into fragments.
To save the fragments with their individual DNA sequences, the fragments were added into an arrangement of continually replicating microbes populaces.
Clonal population of microscopic organisms, every populace maintaining a single artificial chromosome, are put away in different labs all throughout the planet.
The artificial chromosomes (BAC) can be developed, extricated, and named, in any lab containing a library.
Genomic libraries are frequently named after the institution in which they were created.
A model being the RPCI-11 library, which is named after Roswell Park Comprehensive Cancer Center in Buffalo, New York.
These fragments are on the request for 100 thousand base-combines, and are the reason for most FISH probes.
Preparation and Hybridization Process: RNA
Cells, circulating tumor cells (CTCs), or formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections are fixed, then, at that point permeabilized to permit target availability.
It has likewise been effectively done on unfixed cells.
A target-specific probe, made out of 20 oligonucleotide pairs, hybridizes to the target RNA(s).
Separate however viable signal amplification frameworks empower the multiplex assay.
Signal amplification is accomplished through series of consecutive hybridization steps.
Toward the end of the assay the tissue samples are pictured under a fluorescence microscope.
Preparation and Hybridization Process: DNA
Scheme of the principle of the FISH Experiment to restrict a gene in the nucleus. Initial, a probe is developed.
The probe should be sufficiently enormous to hybridize explicitly with its objective however not really huge as to obstruct the hybridization cycle.
The probe is labeled straightforwardly with fluorophores, with targets for antibodies or with biotin.
Tagging should be possible in different manners, for example, nick translation, or Polymerase chain reaction utilizing labelled nucleotides.
Then, at that point, an interphase or metaphase chromosome arrangement is created. The chromosomes are firmly attached to a substrate, normally glass.
Repetitive DNA sequences should be hindered by adding short fragments of DNA to the sample.
The probe is then applied to the chromosome DNA and incubated for roughly 12 hours while hybridizing. A few wash steps eliminate all unhybridized or partially hybridized probes.
The outcomes are then visualized and quantified utilizing a microscope that is fit for exciting the dye and recording pictures.
In the event that the fluorescent signal is weak, amplification of the signal might be essential in order to surpass the identification limit of the microscope.
Fluorescent signal strength relies upon numerous variables, like, probe labeling efficiency, the type of probe, and the kind of dye.
Fluorescently labelled antibodies or streptavidin are bound to the dye molecule. These secondary components are chosen so they have a strong signal.
Probe Variation and Analysis
Single-Molecule RNA FISH
Single-molecule RNA FISH, is a strategy for detecting and quantifying mRNA and other long RNA atoms in a thin layer of tissue sample.
Fiber FISH
It is an another technique to interphase or metaphase arrangements, fiber FISH, interphase chromosomes are joined to a slide so that they are stretched up in an straight line, as opposed to being firmly coiled, as in regular FISH, or acquiring a chromosome region adaptation, as in interphase FISH.
Q FISH
Q-FISH combines FISH with PNAs and software of computer to evaluate fluorescence intensity.
Flow FISH
Flow-FISH utilizes flow cytometry to perform FISH naturally using per-cell fluorescence estimations.
MA FISH
Microfluidics-assisted FISH (MA-FISH) utilizes a microfluidic flow to raise DNA hybridization efficiency, declining cost of FISH probe consumption and diminish the hybridization time.
Mar FISH
Microautoradiography FISH is a technique to combine radio-labelled substrates with customary FISH to identify phylogenetic groups and metabolic activities concurrently.
Hybrid Fusion FISH
Hybrid Fusion FISH (HF-FISH) utilizes primary addictive excitation/emission combination of fluorophores to create extra spectra via a labelling process called as dynamic optical transmission (DOT).
FISH Application
Fluorescent probes of different colours can be utilized simultaneously for varying targets simultaneously to decide which part of population various individuals make up.
For example, DAPI that binds DNA vaguely, the blue DAPI stain show the size of the combined microbial populations and when contrasted and the differentially stained images of a similar population can offer scientists a thought of what extent of the entire each of the different domains are liable for.
Medical Application of FISH
At the point when the child’s developmental disability isn’t perceived, the reason for it can conceivably be determined using FISH and cytogenetic techniques.
Conclusion of certain disorders should be possible with the use of FISH like Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome.
In medicine, FISH can be utilized to create a diagnosis, to assess prognosis, or to assess remission of an illness, like cancer.
Fluorescence In Situ Hybridization (FISH) Citations
- Fluorescence in situ hybridization: past, present and future. J Cell Sci . 2003 Jul 15;116(Pt 14):2833-8.
- Use of Fluorescence In Situ Hybridization (FISH) in Diagnosis and Tailored Therapies in Solid Tumors. Molecules . 2020 Apr 17;25(8):1864.Â
- Fluorescence in situ hybridization in plants: recent developments and future applications. Chromosome Res . 2019 Sep;27(3):153-165.
- Fluorescence In Situ Hybridization Probe Validation for Clinical Use. Methods Mol Biol . 2017;1541:101-118.
- Fluorescence in situ Hybridization (FISH). Curr Protoc Cell Biol . 2004 Sep;Chapter 22:Unit 22.4.
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Optochin Susceptibility Test: Principle, Steps, and Importance
Continue ReadingOptochin Susceptibility Test
Optochin susceptibility test is usually done for identifying the species of Streptococcus pneumoniae.
Differentiating Streptococcus pneumoniae from the other species of Streptococci variants depends on demonstrating optochin susceptibility, bile solubility, reaction with a specific DNA probe, or detection of species-specific capsular polysaccharides.
Most of the clinical microbiology laboratories depends on the optochin susceptibility test.
Initially Optochin susceptibility was first described for differentiating pneumococci, from other α-hemolytic streptococci in the year 1915, but this test is being virtually unused in the laboratories in the mid-1950s.
Optochin is a chemical named as ethylhydrocupreine hydrochloride which is completely soluble in water.
Optochin is used to identify Streptococcus pneumoniae, and alpha hemolytic Streptococcus which is mostly susceptible to the chemical, Optochin.
Where as the species of alpha-hemolytic Streptococcal species are resistant to optochin.
This test helps in determining whether bacterium is either sensitive or resistant to the chemical.
This test is also widely used in the form of filter paper discs or impregnated with ethylhydrocupreine hydrochloride, and they are applied directly on the inoculated plates before incubating.
Optochin test is one of the tests which can be performed in shorter time than the other tests like bile solubility test.
Features of Optochin Susceptibility Test
Streptococcus pneumonia is most commonly found in the respiratory tract of the humans, as other pathogens like Streptococci, and it has a hemolytic pattern which helps in distinguishing it from the other alpha-Streptococci and the Lacto cocci.
Optochin is a chemical that is used in the presumptive identification of alpha-hemolytic Streptococcus pneumoniae, which is sensitive to optochin.
Where as the other species like alpha-hemolytic Streptococcus species are resistant to optochin.
Optochin is completely soluble in water.
This test is widely used in the form of filter paper disks.
Bowen and Jeffries in the year 1955 impregnated the disks with the reagent ethylhydrocupreine hydrochloride that are applied directly to inoculated plates before incubation to demonstrate the susceptibility of pneumococcus for identification.
Optochin Susceptibility Test Principle
Streptococcus pneumoniae is generally found in the respiratory tract and it also has its hemolytic pattern which is identical to other alpha-hemolytic Lacto bacilli and streptococci.
Streptococcus pneumoniae is sensitive to the chemical optochin, which is not able to form colonies in its presence and as and its change in surface tension and causes the cell membrane of Streptococcus pneumonia to lyse.
Optochin is a chemical which is quinine derivative and it has a capacity to soluble in water where all the alpha-hemolytic streptococcal species are resistant to this chemical.
Optochin susceptibility test is usually employing in identifying the streptococcus which is sensitive to the chemical optochin.
For determining the susceptibility of the organism, Streptococcus pneumonia, filter paper disks are impregnated with the chemical optochin and it is used in a disk diffusion test.
This test is usually performed on blood agar, which is a zone for creating inhibition of the lysis of the cell membranes in the Streptococcus pneumonia cells surrounding the disk which helps in determining the positive test.
This test is very easy to perform and it also economic and has a sensitivity of more than 95%.
Optochin Susceptibility Test Requirements
• 5% of sheep blood agar.
• Optochin disks- each disk should be impregnated with 5µg of optochin
• Incubator
• Sterile forceps
• Sterile inoculating loops
Optochin Susceptibility Test Procedure
 With a help of an inoculating loop, 2 to 3 streaks are drawn on a well isolated colonies of a pure culture medium, which is tested on 5% of blood agar.
 Optochin disks are placed on the inoculated surface of agar with the help of sterile forceps.
 Ensure whether the disks are adhered firmly to the surface of the agar by using a sterile forceps or loops.
 Then the plates are placed in an incubator at temperature of about 35 to 37ºC.
 Observe the growth on the surface of the blood agar plates and finally the inhibition of the diameter zone is measured including the diameter of the disk plate.
Optochin Susceptibility Test Results
Positive Results:
 If the zone of inhibition is detected as 14mm or greater than that and a diameter of 6mm disk, it results in the positive condition.
 If the zone of inhibition is formed lesser than 14mm the strain is identified as pneumococcus only if it is soluble in bile.
Negative Results:
 If there is no detection of any formation of zones, the test is negative and it indicates the absence of Streptococcus pneumoniae.
Optochin Susceptibility Test Limitations
 Usually, Streptococcus pneumoniae are isolated and they must be incubated in a carbon-dioxide enriched environment, as some isolates will grow poorly or it does not grow at all.
 Optochin susceptibility is one of the presumptive tests which is recommended as one of the important biochemical tests which is used to perform the complete identification process.
 Any zone of inhibition which is less than 14mm is not enough for performing the test of pneumococci, as the strain is important for identifying the pneumococcus with confirmation by a positive solubility test of bile or serology is performed.
Optochin Susceptibility Test Citations
- Assessment of the Optochin Susceptibility Test to Differentiate Streptococcus pneumoniae from other Viridans Group Streptococci. Clin Lab . 2021 Mar 1;67(3).
- Identification of Streptococcus pneumoniae: Development of a Standardized Protocol for Optochin Susceptibility Testing Using Total Lab Automation. Biomed Res Int . 2017;2017:4174168.
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Gelatin Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingGelatin Hydrolysis Test
Generally, gelatin is used to define an animal protein or a collagen which is a component of a vertebrate in a connective tissue. Gelatin is used as a solidifying agent in a food for a long day ago, Organisms that produces proteolytic enzyme, specifically gelatinases help in hydrolysing the gelatin into a polypeptide and individual amino acids.
During this process, gelatin losses its structure and converts in to liquid form. Robert Koch used gelatin in his culture in the form of a nutrient gelatin which is one of the oldest solid culture media.
Gelatin usually dissolves in a water at 50 degree Celsius and it exists as the liquid at a temperature of above 25 degree Celsius and it further solidifies and forms a gel like substance when it is cooled below 25ºC.
What is Gelatin Hydrolysis Test?
Gelatin hydrolysis test is also called as Gelatin liquefication test, as this test involves the process of liquefication of gelatin in the presence of an enzyme gelatinase.
Gelatinase is considered as one of the most important enzymes in various pathogenic organisms as it is produced extracellularly, and hydrolysis gelatin which is derived from the connective tissues of the vertebrates in the form of collagen.
This enzyme also works as a virulent factor which dissolves the connective tissues of the host and aids in producing invasive infections.
Gelatin is a protein that hydrolyses in the presence of an enzyme gelatinase by breaking down the complex structure into monomeric amino acids.
This test is being followed from early days in the form of presumptive test to identify the pathogenic organisms like Serratia, Pseudomonas, Flavobacterium and Clostridium.
Gelatin Hydrolysis Test Principle
Gelatin is a type of protein derived from the animal tissues, collagen and it forms a solid structure at low temperature. This protein is metabolised or degraded by a group of enzymes known as gelatinase.
Gelatinases are the proteolytic enzymes which liquifies the gelatin into polypeptides and individual amino acids.
The degradation of the gelatin into polypeptides, is followed by covering the polypeptides into amino acids.
Gelatinase is very important in bacteria as gelatin is comparatively a large polymer and thus it cannot be transported into the cell membranes, as gelatinase breaks this compound into the smaller peptides it can easily be transported into the cell and it is utilised by the bacteria.
In hydrolysis test, media containing gelatin is used and its hydrolysis is detected either by liquification of the media or by flooding the media with mercuric chloride, as mercuric chloride helps in precipitating the gelatin and make the hydrolysed area to look clear.
This test is commonly used to determine the ability of an organism to produce the extracellular proteolytic enzymes, gelatinases, which hydrolyses the gelatin, a component of the vertebrate connective tissue.
This reaction usually occurs in a series of two steps, in first reaction process, Gelatinases hydrolyses gelatin into polypeptides and further the polypeptides are converted into amino acids.
The amino acids are taken up by the cells and they are used for their own metabolic purposes. Generally, the presence of gelatinases can be detected using a nutrient gelatin medium.
When an organism produces gelatinase, the enzymes liquifies the growth medium by liquifying the gelatin that is present in the medium.
Gelatin Hydrolysis Test and Microorganism
Gelatin hydrolysis test is generally used to detect the micro-organisms Gram negative rods require gelatine for the identification of specific fluorescent pseudomonas. Such as Pseudomonas putida (negative) from pseudomonas fluorescens (positive). Whereas the gram-positive rods are needed for identifying the pathogens in species level.
Gelatin Hydrolysis Test Materials
Nutrient Gelatin media is generally used for the purpose of demonstrating the hydrolysis of gelatin by adding mercuric chloride or by using liquification of gelatin.
Ingredients Gram/litre Tryptose 20.0 Gelatin 10.0 Magnesium sulphate 0.01 Beef extract 3.0 Agar 15.0 Reagents:Â
• Mercuric chloride
Supplies:
• Inoculating needle
• Incubator
• Pipettes
Gelatin Hydrolysis Test Procedure
1. Preparation of the media
First, the media is prepared. 128 grams of dehydrated medium mixed with 1000 millilitres of warm distilled water in a beaker.
The prepared solution is heated with agitation to bring about the boiling point and the media is let to dissolve completely.
Then the prepared medium is dispensed into series of test tubes and it is autoclaved at 15lbs pressure for about 15 minutes. If agar plate method is used, the medium is autoclaved in the beaker.
The tubes are cooled after autoclaving to about 49 to 50 degree Celsius by keeping it in an upright position.
2. Gelatin Hydrolysis
Gelatin hydrolysis can be identified by nutrient gelatin slab method or by agar plate flooding method by using mercuric chloride.
a. Stab Method of Gelatin Hydrolysis Test
Gelatin medium in a tube is inoculated with 4 to 5 drops of a 24-hour broth medium.The inoculated tubes are then incubated at a temperature of about 37 ºC in the air for 24 to 48 hours, If the organisms have the capability to grow at 25ºC then the incubation should be done at 25ºC. After the first incubation, the tubes are placed at 4ºC for one 24 hours.
b. Plate Method of Gelatin Hydrolysis Test
In plate method, heavy inoculum of a culture is taken using an inoculating loop and it is inoculating using nutrient gelatin medium. And the plates are kept in an incubator, by setting a temperature of 37ºC for about 24 to 48 hours.
Gelatin Hydrolysis Test Result
1. Tube Method: In positive result, either partial or total liquefication of the gelatin can be observed in the tubes. In negative result, complete solidification of gelatin is seen at a temperature of 4ºC.
2. Plate Method: In positive result, clear zone around the colonies is noted after adding mercuric chloride. If there is no clear zone after adding mercuric chloride, it indicates the negative result.
Gelatin Hydrolysis Test Uses
 Gelatin hydrolysis test is usually used to test the capability of an organism to produce gelatinase.
 This test also helps in identification of Serratia, Pseudomonas, Flavobacterium and clostridium.
 Gelatin hydrolysis test also helps in distinguishing the gelatinase positive staphylococcus aureus from the gelatinase negative non-pathogenic S. epidermidis.
 Using this test; genera of bacteria such as Serratia and proteus are differentiated from other members of Enterobacteriaceae family
Gelatin Hydrolysis Test Citations
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Hippurate Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingHippurate Hydrolysis Test
Hippurate hydrolysis test is one of the biochemical tests, which is used to differentiate the micro-organisms on the basis of their ability to hydrolyze Hippurate into benzoic acid and glycine by the action of the enzyme hippuricase, present in the bacteria.
Hippuricase is one of the constitutive enzymes which helps in hydrolyzing the Hippurate and helps in producing amino acid, glycine.
Glycine can be detected by the oxidizing the Ninhydrin reagent, that results in the production of a deep purple color.
Hippurate hydrolysis test is used in the identification of Gardnerella vaginalis, campylobacter jejuni, Listeria monocytogenes and group B streptococci; to detect the ability of the organism to hydrolyze Hippurate.
What is Hippurate Hydrolysis Test?
In olden days this test was performed using a ferric chloride indicator to identify the benzoic acid, but those traditional methods would take a longer time to resolve.
But now a days with the modern techniques, this test has been modified and it is used as a rapid test by detecting the glycine by adding ninhydrin as an indicator.
This test can also be used to distinguish the Group B streptococci from other groups A, C, F and G which cannot hydrolyze sodium Hippurate.
Other group of viridians like Group D can be also detected by using Hippurate sodium hydrolyze.
This test is considered as one of the class of test which differentiates the bovine-β-hemolytic Group B streptococci from human β-hemolytic Group B species of streptococcus.
Hippurate Hydrolysis Test Objective
Hippurate hydrolysis test is generally used to detect the production of the enzyme hippuricase; for identifying the presumptive of different microorganisms. Its main aim is to differentiate bacteria based on their ability to produce hydrolyzed Hippurate.
Hippurate Hydrolysis Test Principle
Hippurate test is based on the ability of the organism to hydrolyze sodium Hippurate into glycine and benzoic acid with the action of an enzyme hippuricase.
This test is primarily used in identifying the campylobacter jejuni, Gardnerella vaginalis, Listeria monocytogenes and streptococci’s agalactiae.
This ability of the few bacterial species to hydrolyze the Hippurate was classically tested using a ferric chloride indicator which helps in detecting the benzoic acid.
However, now a days a 2-hour rapid method is opposed to 48-hour tractional method, for detecting the Hippurate hydrolysis.
The rapid is done by using ninhydrin as an indicator, which on reaction with protein or amino acids detects glycine.
As glycine is deaminated by oxidizing action of ninhydrin, which results in the reduction of ninhydrin and the substance resembles in purple color.
The test medium used here should contain only Hippurate as a source of protein as ninhydrin acts with the free amino acids that is present.
Thus, rapid Hippurate hydrolysis test helps in detecting the by- product of the benzoic acid which is seen as a sensitive and classical method.
Hippurate → Glycine + Benzoic acid
Glycine + Ninhydrin → Purple colored complex
The two important reagents used in this test are Hippurate solution and Ninhydrin.
1. Hippurate Solution
Hippurate reagent can be found commercially in the form of dehydrated tubes or in the form of disks or tablets. This can also be prepared in the laboratory in the form of 1% of Hippurate solution, for preparing in the laboratory, one gram of sodium Hippurate is added into 100 ml of distilled water.
2. Ninhydrin
Ninhydrin can also to purchased commercially. But mostly it is prepared in the laboratories while performing the diagnosis, for laboratory preparation, of the ninhydrin, 50ml of 1-butanol is added into a dark colored glass bottle. Then 3.5 grams of ninhydrin is added to the bottle and it is mixed well.
Hippurate Hydrolysis Test Materials
 Sterilised wooden stick and inoculating loops
 Incubator
 Test tubes
 Distilled water
Hippurate Hydrolysis Test Procedure
1. Preparation of Hippurate Solution
If dehydrated Hippurate is used, 0.2ml of distilled water is added at a pH of 6.8 to 7.2 is added with the test reagent. Then the two drops of distilled water are added to an empty tube for disk or tablets If Hippurate solution is prepared in the laboratory, 0.4ml of the reagent is added to the tube for each test.
2. Hippurate Hydrolysis Test
A heavy suspension is prepared in a tube from an 18 to 24-hour culture. The colony is then picked up carefully where the agar which contains protein should not be taken. The tube is then incubated for 30 minutes, at 35ºC to 37ºC.
Then the tube is observed for every for 10 minutes intervals till the deep blue colour; The change in colour appears usually appears within 15 minutes, after the addition of ninhydrin solution.
Hippurate Hydrolysis Test Result
 A positive Hippurate hydrolysis test results in the appearance deep blue which almost looks like a crystal violet within 30 minutes.
 A negative Hippurate hydrolysis test, results in the appearance of a faint blue colour or there will be no colour change.
Control Organism in Hippurate Hydrolysis Test
 Streptococcus agalactiae: it indicates Hippurate positive
 Streptococcus pyogenes: It indicates Hippurate negative
Hippurate Hydrolysis Test Citations
- Rapid, colorimetric test for the determination of hippurate hydrolysis by group B Streptococcus. J Clin Microbiol . 1976 Jan;3(1):49-50.Â
- A rapid hippurate hydrolysis test for the presumptive identification of group B streptococci. Pathology . 1983 Jul;15(3):251-2.
- Evaluation of the rapid hippurate hydrolysis test with enterococcal group D streptococci. J Clin Microbiol . 1977 Mar;5(3):290-2.
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