Category: Biology
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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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Bile Solubility Test: Principle, Reagents, Procedure, and...
Continue ReadingBile Solubility Test
Generally Biochemical tests are used to identify the species of bacteria by differentiating the species on the basis of their biochemical activities.
The factors such as protein and fat metabolisms and the enzyme production and ability of the organism to utilize the compounds in the mediums helps us in differentiating and identifying the specific species of the bacteria.
Bile solubility test is one such biochemical test, which helps us to identify and differentiate the species of bacteria namely, Streptococcus pneumonia from another species named Alpha-hemolytic Streptococci.
Whereas the Streptococcus pneumoniae is soluble in bile, where as the other species of Alpha-hemolytic streptococci are resistant to the bile and it does not dissolve.
Here let us discuss briefly about the objectives and the preparations of this test.
What is Bile Solubility Test?
Bile solubility test is a biochemical test which is used for differentiation and confirmation of the species of Streptococcus namely S. pneumoniae from all other alpha-hemolytic Streptococci.
This bile solubility test is used as one of the vital tests for differentiating S. pneumoniae as it allows the differentiation between the two species of the Streptococcus namely S. pneumonia and the S. pseudopneumoniae, this is considered as the most challenging task.
This test is usually based on the lysis of the bacterial cells in the presence of a few bile salts like Sodium deoxycholate under specific conditions.
The organisms which lyse in the presence of these specific salts are considered as positive and the others which do not lyse in such medium is known as negative.
However, the exact mechanisms of these specific tests are not clearly understood but it has been detected that the lysis is brought about by inducing the autolytic enzymes.
This test is generally considered as an accurate test for identifying Streptococcus pneumoniae from other species of Streptococci.
Anyhow the results are difficult to interpret as it is based on the evaluation of subjective human.
Bile Solubility Test Principle
Bile solubility test us usually sed for identifying and distinguishing the species of Pneumonia from the Alpha-hemolytic streptococcus species.
This test is usually performed by using a suspension of cell on slide or in a tube depending upon the method we use, and adding the reagent directly to the colony.
The basic principle of the bile solubility test is the lysis of the pneumococcal cells when the sodium deoxycholate is applied on the colony under some of the specific conditions depending on the time and temperature where as the other species do not lyse.
As pneumococcus has an intracellular autolytic enzyme, known as amidase and causes the organisms to undergo an autolysis when the species is cultivated on an artificial membrane.
The bile salts alter the surface tension of the medium which causes the rearrangement of the cell-membrane.
Bile Solubility Test Objective
 To identify and to detect the difference between S. pneumoniae and Alpha-hemolytic streptococcus.
 To observe the capability of the organism to undergo lysis on the presence of bile salts.
Bile Solubility Test Reagents
Bile salts:
• About 10 % of bile solution is either purchased commercially or prepared in the laboratory. Bile salts is prepared usually by adding 10 grams of Sodium deoxycholate in a beaker containing 100 ml of distilled water.
• Then the prepared solution is dispensed in a small amount in the test tubes to minimize the contamination.
• The shelf life of the bile salts which was prepared in the laboratory is usually 270 days.
• It should be stores at a temperature of about 15 to 30ºC.
• If it is stored less than the defined temperature, the solution becomes thickened.
• 0.85% of sodium chloride • Broth culture medium
Reagents:
• Loops
• Slides
• Test tubes
• Pipettes
Bile Solubility Test Procedure
Bile solubility test can be done in two ways, either in the test tube method or by using Direct plate method.
1. Test Tube Method
About 0.5 ml of sterile saline or the suitable broth is dispensed into a small tube.
A heavy suspension of the organism is prepared in the saline and the suspension is then shaken by hand or by using a vortex to form the uniform suspension.
The suspension is further divided into two tubes and it is labelled as TEST and the other as CONTROL.
Then two to five drops of the reagent are dispensed into the both the tubes and they are mixed thoroughly.
The tubes are then incubated at a temperature of 35ºC, for about 3 hours. And the tubes are checked at an interval of each hour and they are examined by adding a gram stain of methyl blue wet mount for lysis of cells at an interval of about 15 minutes.
2. Direct Plate Method
About 2 drops of bile reagent is placed near a suspected colony from the 18 to 24-hour culture.
Then the drops are rolled gently over several colonies by tilting the plate gently, we must be awarded that colonies should not be dislodged against the bile reagent.
Then the plate is placed in the upper right side and it is incubated at a temperature of about 35ºC for a period of about 15 to 30 minutes or in some cases it can also be incubated till it evaporates.
Here instead of incubator, heat block can also be used.
The flattening of the colony is seen.
The colony is observed properly and it should ensure that colony simply float away.
3. Direct Slide Blood Culture Test
A drop of blood culture broth is added to the drop of a bile reagent on a glass slide and it is allowed to let dry.
Then one drop of the blood culture is added to one drop of water and it is let to dry away.
Further, the resulting suspension is gram stained and they are examined if there is presence of any cocci.
Bile Solubility Test Result
Test Tube method: Generally, bile solubility test is detected by the test tube methods a clear zone or a loss of turbidity can be observed when compared to the CONTROL tube, within 3hours. This results in the lysis of the cells and it can be observed under the microscope.
Direct slide blood culture method: The organism is considered as bile soluble if all the cocci in the smear are lysed completely and the Control smear shows an intact bacterium.
Direct plate method: Here the bile solubility is determined by observing the integrity or flattening of the colony within 30 minutes. In case of negative results, there will be no change in the integrity of the colony within half an hour.
Bile Solubility Test Citations
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Butyrate Disk Test: Principle, Procedure, Results, and...
Continue ReadingButyrate Disk Test
Many of the biochemical tests are performed to identify the characteristics of a specific micro-organism and to find about its pathogenic activities, especially during some of the specific enzymes.
These tests help us to find the nature of the pathogen and its disease-causing activity. Which helps us to define the organism and its characteristics in the hosts body especially in humans.
One such biochemical test is Butyrate disk test which helps to detect the morphology and identification, of the specific organism Moraxella Catarrhalis, commonly called as Branhamella.
What is Butyrate Disk Test?
Butyrate disk test is one of the rapid identification tests for detecting the Moraxella catarrhalis based on their ability to utilize bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate.
In recent days Moracella catarrhalis is recognized as the major pathogen which causes sinusitis, conjunctivitis, bronchitis, ottis media and pneumonia. In addition to large number of Moraxella catarrhalis strains produces an enzyme known as Beta-lactamase which are resistant to penicillin and ampicillin.
For the completion of conventional test, it requires up to 24 to 48 hours, However, the alternative tests are easy to perform, whereas for identification methods butyrate disk tests can be used.
Usually in biochemical tests, butyrate esterase is demonstrated by hydrolyzing the substrates like bromochloro-indolyl butyrate or four-methyl umbelliferyl butyrate.
The test can be performed either by using disk test or using the tube method. Both of these tests are included as the rapid identification methods, But the disk tests are considered as a most feasible one as it does not require any preparations like reagents in the test tubes.
Butyrate Disk Test Objective
• The main aim of this test is to identify and detect the species known as Moracella catarrhalis in the clinical samples, as a presumptive identification method.
• To detect the capacity of the organism to hydrolyze butyrate substrate by producing butyrate.
Butyrate Disk Test Principle
Butyrate test is considered as one of the rapid qualitative tests used for detecting the enzyme known as butyrate esterase, which further provides a judgment for identifying the organism, Moracella catarrhalis.
While performing this test, it is important to know the characteristic morphology of a specific organism which is placed on the plate of blood agar, typical gram stain and the positive oxidase tests are very much useful in identification of this species.
Both the reagents, Bromochloro-indolyl butyrate and the 4-methylumbelliferyl butyrate serves as a substrate.
During hydrolyzing the bromochloro-indolyl substrate with butyrate esterase it releases indoxyl.
Thus, indoxyl in the presence of oxygen changes its form into indigo. Indigo is one of the chromogenic compounds, which appears in the blue color or in a shade of blue-violet.
On hydrolyzing 4-methylumbelliferyl substrate it produces a fluorescent compound, which is visible only under a UV light.
However, Hydrolysis is detected by change in color or by the detecting the fluorescence from the products.
Butyrate Disk Test and Types of Micro Organism
Here, the Gram-negative species of oxidase positive diplococci that is growing on the blood agar plates in the form of white colonies which remain together when lifted using a loop or a wire is detected.
Butyrate Disk Test Reagent
Reagents:
• Disks impregnated with Bromochloro-indolyl butyrate and 4-methylumbelliferyl
Supplies:
• Bacteriologic loops
• Sterile wooden applicators
• Petri dish
• Slide
• Tube
• Distilled water
• Long wave of UV light, about 360nm.
Butyrate Disk Test Procedure
Initially the disks are removed from the vial and they are placed on the clean glass slide or in a petri dish.
Then a drop of deionized or distilled water is placed on the disk, in order to provide a moisture.
A heave inoculum, that is visible is collected from the 24 to 72-hour culture using a sterile wooden applicator stick and it is placed on the disk.
Further the inoculated disks are incubated by maintaining a room temperature of about 15 to 30ºC for about 5 minutes.
Incubation for a slightly longer period is required for yielding a false-positive result.
Butyrate Disk Test Quality Control
• The disks are discarded in case if they are not appearing in white color or without any visible colors.
• According to quality control, the organisms like Catarrhalis ATCC gives a butyrate positive result. Neisseria gonorrhea or Neisseria lactamica gives butyrate negative results.
Butyrate Disk Test Result
• A positive result is determined in the butyrate disk test when there is a change in color from blue to violet or appearance of fluorescence under UV light. This reaction is seen within 5 minutes, which indicates the hydrolysis of bromochloro-indoyl and the 4-methylumbelliferyl, by the enzyme butyrate esterase.
• Whereas the negative test is indicated by no change in the color
Butyrate Disk Test Uses
• This test helps us to identify the Moraxella catarrhalis.
• This test also helps us to differentiate Neisseria gonorrhea and Moraxella catarrhalis; where both are oxidase positive, gram negative diplococci.
Butyrate Disk Test Limitations
• Incubation should not be maintained more than 5 minutes, as it results in false positive results.
• False-negative reactions also occurs of the inoculum is smaller than usual.
• Tests are often performed in conjunction with the chromogenic substrate tests or in carbohydrate fermentations, oxidase reaction, gram stain and morphology for the complete identifation of the organism.
• Non-human species of Branhmella subgenus, Moraxella are butyrate esterase-positive. Some strains of the subgenus Moraxella five a positive weak reaction in few cases.
• IN some cases, the unrelated organisms like Staphylococcus and pseudomonads gives a positive result,
Butyrate Disk Test Citations
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CAMP Test: Principle, Procedure, Results, and Uses
Continue ReadingCAMP Test
Many of the biochemical tests are identified and followed to test the ability of the micro- organisms to survive in the particular media. One such test is known as CAMP test.
CAMP test is usually used to identify the Group B- Beta hemolytic Streptococci based on their formation of a substance known as CAMP factor, which enlarges the area of hemolysis formed by the Beta-hemolysin elaborated from Staphylococcus aureus.
CAMP Factor
CAMP factor is a diffusible, and a heat stable protein that is produced by a group B- Streptococci.
This test is considered as one of the synergistic tests between the two species of Staphylococcus namely S. aureus and S. agalactiae S. agalaciae produces the CAMP factor and S. aureus produces the sphingomyelin C, which has the capability to bind with the membranes of the red blood cells.
What is CAMP Test?
Camp test is usually done to distinguish the species of Streptococcus agalacctieae from other species of the Beta-hemolytic species of Streptococcus.
Streptococci agalactiae is one of the members of the Lance field Group B Streptococci It is also considered as one of the causative agents of mastitis in cows.
CAMP is simply an acronym of the authors of this test namely Christie, Atkinson, Munch, and Peterson. They identified this test in the year 1944.
CAMP Test Objective
 The main aim of the test is to differentiate the Species of Streptococcus agalactiae from the other Beta-hemolytic species of Streptococci
 To determine the ability of the organism of an organism to produce the camp factor
CAMP Test Principle
Streptococcus agalactiae usually synthesis a diffusible and a thermostable, extracellular protein which interacts synergistically with the Beta-hemolysin that is produced by Staphylococcus aureus, and results in the formation of a Zone which enhances the lysis of the sheep or bovine erythrocytes.
In Standard CAMP tests elaboration of two toxins relies n the growth which forms a typical arrowhead or a flame shaped clearing the the interaction point of the two organisms and it is streaked perpendicular to each other.
The rapid disks helps us in utilizing the extract of the staphylococcal Beta-hemolysin which interacts directly with the CAMP factor that already left diffused in the medium around the colony of Streptococcus agalactiae.
Thus, the hemolysis produced from the Beta-hemolytic strains of the Staphylococcus aureus is enhanced but one of the extracellular proteins which is produced by the species of Group-B- streptococcus.
The interaction between the S. aureus and the CAMP factor of the Group B streptococci results in the formation of the Synergistic hemolysis which thus results in the formation of the arrowhead done of the hemolysis.
Thus, this test is very useful for identifying the S. agalactiace and many other Gram- positive rods including Listeria monocytogenes.
CAMP Test Requirements
Media:
 Blood agar
Reagents:
 Beta-lysin reagent
 Culture of aureus
 Disks containing Beta-lysin of aureus
Supplies:
 Sterile wooden applicator
 Bacteriologic loops
 Distilled water
 Petri dish and slide
CAMP Test Procedure
The CAMP test is usually performed in two ways, either by using Standard method or by using Disk method.
1. Standard Method
Initially a streak of Beta-lysin that produces strain of S.aureus is put down at the center of the sheep blood agar plate.
It should be made clear that the streak should be only at a length of 3 to 4 cm.
The streak test organism is put across the agar plate, perpendicular to the streak at the length of about 2mm or even less than that.
Then the agar plate is let to incubate at a temperature of about 35 to 37ºC in an ambient air for about 18 to 24 hours.
After incubation the enhancement of Beta-lysin aureus strain is seen which is produced by Group B streptococci and other species of Beta-streptococci.
2. Disk Method
Here the disk is removed from the vial and it is placed on the warm Blood agar plate.
Then the micro-organism is streaked there, from the edge of the disk, for about 2 to 3mm.
Then the plate is incubated at a temperature of about 35 degree Celsius for a whole night in the carbon dioxide incubator.
CAMP Test Quality Control
Control Incubation Results Streptococcus agalactiae It is usually for about 24 to 48 hours at a temperature of about 33 to 37ºC in the air having 5% of carbon dioxide. It is considered as CAMP positive, as the formation of arrowhead hemolysis is seen at a the intersect ion of the streaks. Streptococcus pyrogens The culture is incubated for about 24 to 48 hours at a temperature of about 33 to 37ºC in an air contain 5% of carbon dioxide. Here the results are considered as CAMP negative, as Beta-hemolysis does not form a 3nchnaced arrow head. CAMP Test Result
 A position results in the standard method is concluded by the formation of the distinct arrowhead of the hemolysis at the interaction of the two streaks made from Streptococcus and the test organism.
 Whereas, the Positive reverse CAMP or phospholipase D is shown by a typical arrowhead or formation of no hemolysis at the junction o the two hemolytic organisms.
 In case, of the Disk test, a positive result is detected by distinct arc shaped done, of the complete hemolysis at the point of the junction, with the Beta lysin and the test organism.
 A lack of enhanced hemolysis near the colony is being tested as the negative result.
CAMP Test Citations
- Is a positive Christie-Atkinson-Munch-Peterson (CAMP) test sensitive enough for the identification of Streptococcus agalactiae? BMC Infect Dis . 2019 Jan 3;19(1):7.
- The CAMP test performed by using Staphylococcus aureus sphingomyelinase (beta-haemolysin) and Clostridium perfringens lecithinase. NIPH Ann . 1981 Dec;4(2):43-8.
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Cetrimide Agar Test: Principle, Procedure, Results, and...
Continue ReadingCetrimide Agar Test
Many of the biochemical tests are identified and performed in the laboratories and in experiments to differentiate a characteristic of a microbe and they are differentiated from other organisms.
One such biochemical test is cetrimide agar test. Cetrimide agar test is performed in order to identify the Pseudomonas aeruginosa and it also helps in differentiating it from other organisms.
Cetrimide Agar
Cetrimide agar is one of the selective and differential mediums which is used for isolating the Pseudomonas aeruginosa from other clinical and non-clinical specimens.
Cetrimide is considered as one of the selective agents which inhibits the detergent activity of most of the bacteria.
Cetrimide agar is also known as Pseudomonas cetrimide Agar or Pseudosol agar.
What is Cetrimide Agar Test?
Cetrimide agar test works on the principle based on the ability of the organisms to grow in the presence of cetrimide.
Cetrimide is one of the toxic quarterly ammonium detergents. This ammonium detergent is toxic to most of the bacteria except few organisms such as Pseudomonas aeruginosa.
P. aeruginosa plays active role in causing infections in the urinary tract and other infections like bacteremia, which occurs especially in humans.
This Pseudomonas aeruginosa is usually identified on their basic characteristics such as production of pyocyanin, and its blue colour its ability to soluble in water, presence of a phenazine Pigment, morphology of their colony and its characteristic grape like odour.
Pseudomonas aeruginosa is the only species of Pseudomonas or its gram-negative rod which has the capability to excrete pyocyanin, and this capability is usually detected by performing this cetrimide agar test.
Cetrimide Agar Test Objective
 Aim of this test is to identify the species, Pseudomonas aeruginosa and to differentiate it from other micro-organisms.
 To detect the ability of various organisms to grown on the cetrimide agar.
Cetrimide Agar Test Principle
As mentioned above, cetrimide is a quaternary ammonium salt, and it also acts as a cationic detergent in most of the bacterial cells.
This cationic detergent is considered as one of the toxic substances. Cetrimide reduces the surface tension of the medium at the time of point of contact which results in complex precipitants which denatured the effect of proteins that were present on the bacterial membranes.
Where as the inhibition of growth in various micro-organisms is observed in this cetrimide agar medium including Pseudomonas species except P. aeruginosa.
When cetrimide is Brought in contact with the bacteria there will be release of nitrogen and phosphorus in the bacterial cell.
In such case, the organisms like P. Aeruginosa and other species of pseudomonas cannot withstand this germicidal activity.
This media also contains gelatine peptone which provides necessary nutrients for Pseudomonas aeruginosa.
Sodium chloride helps in maintaining the osmotic equilibrium in the cetrimide medium and it also prevents the change of pH during the growth in the medium.
Micro Organisms Tested
Cetrimide agar is usually used for testing the isolated colonies of non-glucose fermentation, gram negative rods which are suggestive for Pseudomonas aeruginosa.
Cetrimide Agar Test Reagent
Cetrimide agar can be purchase commercially in the format of dehydrated powder or it can be prepared in the laboratory using the necessary chemicals and materials available.
Supplies:
 Sterile inoculating loops
 Sterilized sticks
 Uv light
Cetrimide Agar Test Procedure
Media Preparation
About 46.7grams of dehydrated powder is added in a beaker containing 1000 millilitres of distilled water. Instead of dehydrated powder, media prepared in lab can also be used.
The prepared medium is now heated till it boils, such that the medium dissolves completely.
The boiled medium is then transferred into the tubes and it is sterilized in an autoclave at a temperature of 121ºC or at 15 lbs pressure for about 15 minutes.
After autoclaving the tubes are taken out and placed in a slanted position and it is allowed to cool till the temperature reaches about 40 to 45ºC.
The slanted position is maintained until it develops a butt in a depth of 1.5 to 2.0 cm.
Cetrimide Agar Test
A well isolated colony is collected from an 18 to 24-hour culture using sterile inoculating loops or needles.
The cetrimide agar tubes are then inoculated by streaking the surface of the slants along the medium, in the direction of froth and back and it is also ensured for proper inoculation with the inoculating stick.
The caps of the tubes should not be closed tightly so that the medium gets an adequate aeration.
Further the tubes are incubated aerobically at a temperature of about 35 to 37ºC for about 7 days.
The test tubes are checked daily, and they must be kept at least for 7 days without discarding in order to get the proper and appropriate results.
Cetrimide Agar Test Result
Positive Result: A positive result is usually determined by observing the growth in the medium, and sometimes along with a growth there will also be a change in colour from yellow green to dark blue green. Positive results are usually detected in P. aeruginosa.
Negative Result:Â Negative result is usually determined when there is no change in colour or no growth is seen. Negative results are usually detected in Escherichia coli.
Cetrimide Agar Test Citations
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Catalase Test: Principle, Procedure, Results, and Uses
Continue ReadingCatalase Test
Many of the biochemical tests are identified and performed in the laboratories and in experiments to differentiate a characteristic of a microbe and they are differentiated from other organisms. One such biochemical test is catalase test.
Catalase test is mostly used to detect the production of catalase enzymes in the aerobic organisms.
What is Catalase Test?
Catalase enzyme is one of the common enzymes which is found in all living beings which survive in oxygen and it also catalyses the decomposition of hydrogen peroxide, releasing water and oxygen.
Catalase is considered as one of the important enzymes in the pathogen organisms as it helps in protecting the organism from oxidative damages from the reactive oxygen species.
Catalase enzyme also results in neutralising the bactericidal effects of the hydrogen peroxide, and its concentration in bacteria has been correlated depending upon the pathogenicity of the organism.
Catalase test is being used from olden days in order to differentiate the positive catalase organisms from the negative organisms like streptococci.
Catalase positive organisms includes Staphylococci. This test is very much useful in identifying the characterises of most of the bacteria.
In aerobic conditions only 3% H2O2 is used, whereas in case of anaerobic conditions 15% of H2O2 is used.
Catalase Test Purpose
 The main aim of this test is to detect the ability of the organism to produce the enzyme catalase.
 To differentiate the catalase positive organisms from the catalase negative organisms.
Catalase Test Principle
The metabolic activity of the aerobic and the facultative anaerobic micro-organisms has the capability to produce toxic by-products like Superoxide radical and hydrogen peroxide.
Where these products are toxic to the organisms and they may also result in lysis of the cell if the cell is not broken.
Considering the pathogenic organisms, different mechanisms are used in order to break the products into non-toxic substances.
The bacteria which is capable of synthesising the enzyme catalase, hydrolyses the hydrogen water into gaseous oxygen and water that results in the liberation of gas bubbles.
Thus, the production of this enzyme, catalase protects the organism from suffering those lethal effects of the hydrogen peroxide which is accumulated at the end of the aerobic metabolism.
The presence of this enzyme can be detected by adding hydrogen peroxide in the bacterial inoculum, which thus results in the liberation of rapid air bubbles.
If the enzyme is absent then there will be no liberation of such air bubbles.
Micro Organisms Tested
The organisms that can be used for catalyse test includes the colonies of young bacteria, which are left in a culture of 18 hour or less than that, and mostly agar media is preferred here.
In case of anaerobes the colonies should be exposed to the air for about 30 minutes prior to the test.
Catalase Test Reagents
• Hydrogen peroxide Reagent
• But the reagent of hydrogen peroxide various for each organism, where as it is 30% for Neisseria, 15% for anaerobes, 3% for other bacteria.Â
• Glass slide
• Sterile glass sticks
• Sterilised Platinum loops or wires
Catalase Test Procedure
The procedure for catalase test includes more than one method. The methods used are slide or drop catalase test, tube method, heat-stable catalase test, Semiquantative catalase test, Capillary tube method and cover -slip method.
Here the Heat-stable catalase test is mostly used for differentiating the species of Mycobacterium, the semi-quantative catalase method is mostly used for identifying the species, Mycobacterium tuberculosis.
From all these methods, the test which are most commonly performed in clinical bacteriology includes slide or drop catalase method, as this method requires only a small number of organism and it also works relatively as an uncomplicated technique.
1. Slide Method
To perform this slide method, first the microscopic slide is placed inside a petri dish. But it is also not necessary that the slide must be placed inside a petri dish as it is used only to limit the catalase aerosols, that carries viable bacterial cells.
Then a small amount of organism is collected from a well isolated colony from an 18 to 24-hour culture using the inoculating loops and it is placed on the microscopic slide.
No agar should be picked up along with the colony when the culture is taken from the blood agar.
About one drop of 3% of hydrogen peroxide is added upon the organism that is placed on a microscopic slide, using a Pasteur pipette or a dropper.
Then we can observe the formation bubbles which is formed against a dark background.
2. Tube Method
In tube method about 4 to 5 drops of 3% of hydrogen peroxide is added into a test tube.
Then the small amount of organism is picked up using an inoculation loop from an 18 to 24-hour colony and it is placed into the test tube.
Then the test tube is placed against the dark background and we can observe the bubbles that are formed immediately.
Catalase Test Quality Control
The organisms listed below are tested foe positive and negative results.
 If the result is positive then it denotes the presence of Staphylococcus aureus.
 If the results are negative then it denotes the presence of Streptococcus pyogenes.
Catalase Test Result
 The positive test is usually determined by appearance of immediate bubbles
 Whereas if only one or two bubbles are appeared, then it indicates the weak reaction
 The negative test is indicated by no bubbles are only few bubbles.
Catalase Test Citations
- Semiquantitative catalase test as an aid in identification of oxidative and nonsaccharolytic gram-negative bacteria. J Clin Microbiol . 1979 Oct;10(4):525-8.
- The catalase test–a cautionary tale. Med Lab Technol . 1972 Apr;29(2):203-4.
- Capillary tube catalase test. Appl Microbiol . 1973 Oct;26(4):631-2.
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Citrate Utilization Test: Results, Purpose, and Principle
Continue ReadingWhat is Citrate Utilization Test?
Many biochemical tests are studied and experimented in the laboratories. These tests help us to find the nature of the micro-organisms and their characteristics against certain enzymes and indicators used. One such test is citrate Utilization test.
Citrate tests are usually performed to determine the ability of the bacteria to utilize sodium citrate as the source for carbon and inorganic ammonium hydrogen phosphate as the source for nitrogen.
This citrate utilization test is possible only for the organisms which are capable of fermenting the citrate. This processes usually takes place with the help of an enzyme citrase.
Characteristic of Citrate Utilization Test
Citrate utilization test is often performed along with IMViC test. IMViC test is an abbreviated form of Indole, Methyl Red, Vogues-Proskauer and citrate test.
This test helps in differentiating the organisms on the basis of their ability to use citrate as a source of energy. This citrate is test mostly performed with other IMViC tests which helps us to differentiate Gram-negative bacilli of the Enterobacteriaceae family from other species.
It is considered as one of the important tests as it has a capability to detect the species-level identification in the family of Enterobacteriaceae.
However, it also has exception with the few species of the Enterobacteriaceae family. Some species such as Salmonella, Edwardsiella, Serratia Providencia, Citrobacter, Enterobacter, etc. gives a positive reaction in a citrate utilization test, and other species like Escherichia, Morganella, Shigella and Yersinia gives a negative reaction during this test. Proteus is considered as citrate variable.
Citrate utilization test is most commonly called as Simmon’s citrate test; as it helps in utilizing Simmon’s citrate agar that contains a major source of energy in the form of carbon.
This medium also comprises of ammonium hydrogen phosphate as the sole source of nitrogen.
The organisms which gives positive result during this test has the capability to ferment citrate during the presence of an enzyme citrate.
Citrate Utilization Test Principle
Citrate agar is used to test the ability of an organism whether the particular has citrate as the source of energy.
The agar medium contains citrate as the source of carbon and the other inorganic ammonium salts as the source of nitrogen.
Thus, the growth of organism in the medium is usually indicates by the amount of citrate that is utilized, as citrate is one of the intermediate compounds in the process of Krebs’s cycle.
The enzyme citrase breaks the citrate into oxoacetate and acetate.
Oxoacetate is again broken down into pyruvate and carbon dioxide. Hence the carbon dioxide released, induces the metabolism of ammonium salts, and results in the formation of ammonia or sodium carbonate, which increases the alkalinity in the medium.
On adding an indicator bromothymol blue, there will be a shift in pH and the color of the medium changes from green to blue, when the pH is above 7.6.
Thus, the growth of the organism is detected by change in colour of the medium, as a result Citrate metabolism determines a positive citrate test.
Citrate Utilization Test Requirements
Media: For citrate utilization test, simmon’s citrate is generally used as the medium to test the ability of the organism to utilize citrate as a sole source of energy.
Simmon’s citrate agar is now a days sold commercially in the form of dehydrated powder by commercial vendors.
At the same time, it can also be prepared in the laboratory using the necessary ingredients.
The ingredients and their composition are listed below;
Ingredients Gram/liter Ammonium dihydrogen phosphate 1.0 Magnesium sulphate 0.2 Dipotassium phosphate 1.0 Sodium citrate 2.0 Sodium chloride 5.0 Bromothymol blue 0.08 Bacteriological agar 15.00 Supplies:
 Inoculating loops or needles
 Incubator
Citrate Utilization Test Procedure
About 24.28 grams of dehydrated powder or the media prepared in laboratory are added in a beaker containing 1000 milliliters of pure distilled water.
Then the solution is heated until it boils, so that the medium dissolves completely.
Further the dissolved medium is shifted into the tubes and the tubes are sterilized in an autoclaved at a temperature of 121ºC for about 15 minutes.
After completion of the autoclaving process, the tubes are taken out and they are cooled at temperature of about 40 to 45ºC in a slanted position and this position is maintained till obtaining the butts in a depth of about 1.5 to 2.0cm
Utilization Test
An isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating loop.
Then the citrate agar tubes are inoculated by streaking the surface of the slant, in the back and froth direction using the inoculating sticks.
The caps of the tubes are usually left loosened in order to ensure adequate aeration and they are incubated aerobically at a temperature of 35 to 37ºC for about 4 days.
This test is examined each day.
It must be kept for a minimum to 4 days to finalize the results.
The change in color is observed if present.
Citrate Utilization Test Quality Control
Aseptic simmon citrate agar is maintained in a forest green color which is slightly opaque at the gel slants.
The agar is inspected for the evidence of freezing, any contamination, dehydration, cracks or formation of bubbles prior to use.
If any tubes are seen as blue perform usage then it must be discarded.
Positive control of the tests includes Klebsiella pneumoniae, and the negative control include Escherichia coli.
Citrate Utilization Test Results
Positive Result: Positive test is usually determined by growth in the medium along with the color change. Here the color changes from green to deep blue along the direction of the slant.
Negative Results: The test is concluded as negative if there is no growth or change in color in the medium, such that the color remains the same, green.
Citrate Utilization Test Citations
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