Category: Biology
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Anaphase: Definition, Checkpoints, Diagram, and Examples
Continue ReadingCell Cycle, Cell Cycle Phases, and Anaphase
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.
History of Cell Cycle
The history of mitosis dates back to 18th and 19th century where the aid from microscope and visibility of cell division under the microscope supported the discovery of the cell division.
The discovery was earlier than the DNA discovery was a breakthrough in the scientific community as it answered the most intriguing question of Humans “How do we grow? Develop? Reproduce? What is the driving factor for the growth? How do we resemble our parents?” etc.,
Before the discovery of cell division there were many theories on how the cells are related to the overall development of an organism’s lifecycle.
One of which was Rudalph Virchow’s theory of cell: “Omnis cellula e cellula” which states that a cell originates from a pre-existing cell.
Walther Flemming discovered and published a detailed book on cell division in 1882 after discovering cell division in 1879.
He named it “Mitosis” after the Greek word Mito – “Wrapping thread” owing to the thread like appearances of the chromosomes.
He conducted a detailed study and deduced staining techniques to understand cell cycle and named the stages of each division as Prophase, metaphase, Anaphase and Telophase.
The cell division was identified in Salamander’s embryo. Flemming supported Virchow’s cell theory with precision and stated that “Omnis nucleus e nucleo” which states that a nucleus origin from a pre – existing nucleus and highlighting the chromosomal segregation and laid foundation for the theory of inheritance where the chromosomes play an important role carrying the genetic information from the parent to the offspring.
Cell Cycle Phases
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.
Mitotic 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.
Prophase: The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division. RNA synthesis is inhibited.
Metaphase: Nuclear membrane is eliminated completely chromosomes are completely condensed. The cytoskeleton – spindle fibers attach to the kinetochores. The chromosomes are aligned in the equatorial plate.
Anaphase: Chromosomal split forms daughter chromatids; travels to the opposite poles. The chromosomes are V – Shaped as they are dragged to the opposite sites.
Telophase: Microtubules disappear and chromosomes decondense to chromatin mass. Nuclear envelope starts to form. The disintegrated organelles form again.
Types of Anaphase
Anaphase has 2 phases Anaphase A and Anaphase B were the segregation and spindle fiber withdrawal happen respectively.
Chromosomes separates to chromatids by breaking the wounding of COHESIN which holds the chromosome intact in the metaphase; is Anaphase A.
I. Anaphase A
Anaphase A involves the cell cycle regulator proteins of the M phase.
The anaphase elucidation is poorly studied for plants rather than animals; but the cell cycle and the mechanism is conserved over evolution of the living organism.
It can give a basic picture of the mechanism which is similar to all eukaryotes were the difference is the proteins and other components involved in them governed by the external cues and time.
The mechanism is a cascade of proteolysis, degradation and activation different protein molecules.
The first step is the proteolysis of cohesins mediated by APC/C along with Cdc ubiquitinates CYCB – CDKB complex also degrades the Securin.
Securin directly inhibits Separase in chromosomes cleaves Cohesin rings at Scc1 segregate the chromatids.
The separation induces respective poleward movement of the chromatids.
I. Anaphase B
Anaphase B is the self-detachment of microtubules from the KMT complex due to loss of MT protein components.
Dynein and Kinesin are the 2 proteins; these are motor proteins which pulls the KMT to opposite poles especially Dynein.
The removal of microtubules is mediated by depolymerization of the proteins components leads to separation of each tubulin component from the minus driven ends and Dynein at the control of the fibers near plasma membrane.
Removal of KMT complex is simultaneous with chromatid retraction to the poles.
Motor protein Kinesin undergoes a sliding elongation forming interpolar MT at the plus end.
The main function of the interpolar MTs are to separate the poles far apart from the chromosomal segregation.
Anaphase Citations
- Detection of Ultrafine Anaphase Bridges. Methods Mol Biol . 2018;1672:495-508.
- Anaphase promoting complex or cyclosome? Cell Cycle . 2005 Nov;4(11):1585-92.
- Evidence for anaphase pulling forces during C. elegans meiosis. J Cell Biol . 2020 Dec 7;219(12):e202005179.
- Anaphase in vitro. Proc Natl Acad Sci U S A . 1996 Oct 29;93(22):12076-7.
- Engaging Anaphase Catastrophe Mechanisms to Eradicate Aneuploid Cancers. Mol Cancer Ther . 2018 Apr;17(4):724-731.
- Force-generating mechanisms of anaphase in human cells. J Cell Sci . 2019 Sep 16;132(18):jcs231985.
- Anaphase: a fortune-teller of genomic instability. Curr Opin Cell Biol . 2018 Jun;52:112-119.
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Prophase: Definition, Checkpoints, Diagram, and Examples
Continue ReadingCell Cycle, Cell Cycle Phases, and Prophase
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.
History of Cell Cycle
The history of mitosis dates back to 18th and 19th century where the aid from microscope and visibility of cell division under the microscope supported the discovery of the cell division.
The discovery was earlier than the DNA discovery was a breakthrough in the scientific community as it answered the most intriguing question of Humans “How do we grow? Develop? Reproduce? What is the driving factor for the growth? How do we resemble our parents?” etc.,
Before the discovery of cell division there were many theories on how the cells are related to the overall development of an organism’s lifecycle.
One of which was Rudalph Virchow’s theory of cell: “Omnis cellula e cellula” which states that a cell originates from a pre-existing cell.
Walther Flemming discovered and published a detailed book on cell division in 1882 after discovering cell division in 1879.
He named it “Mitosis” after the Greek word Mito – “Wrapping thread” owing to the thread like appearances of the chromosomes.
He conducted a detailed study and deduced staining techniques to understand cell cycle and named the stages of each division as Prophase, metaphase, Anaphase and Telophase.
The cell division was identified in Salamander’s embryo. Flemming supported Virchow’s cell theory with precision and stated that “Omnis nucleus e nucleo” which states that a nucleus origin from a pre – existing nucleus and highlighting the chromosomal segregation and laid foundation for the theory of inheritance where the chromosomes play an important role carrying the genetic information from the parent to the offspring.
Cell Cycle Phases
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.
Mitotic 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.
Prophase: The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division. RNA synthesis is inhibited.
Metaphase: Nuclear membrane is eliminated completely chromosomes are completely condensed. The cytoskeleton – spindle fibers attach to the kinetochores. The chromosomes are aligned in the equatorial plate.
Anaphase: Chromosomal split forms daughter chromatids; travels to the opposite poles. The chromosomes are V – Shaped as they are dragged to the opposite sites.
Telophase: Microtubules disappear and chromosomes decondense to chromatin mass. Nuclear envelope starts to form. The disintegrated organelles form again.
Prophase Regulators
Onset of chromosomal condensation and differentiation is marked by the prophase requires constant expression of CYCB – CDKB complex which drives the cell division and are regulated.
The cell division is a very specific period for a developing cell which requires most of the contribution of the cell proteins and RNA to be concerned on producing two daughter cells.
Therefore, regular cell functions does not take place in the cell and whole cytoplasm will be occupied by chromosome and spindle fibers in open mitosis or the division occupies the whole nucleus with intact nuclear membrane in closed type prevalent in Yeast and lower eukaryotes.
Most of cell organelle are disintegrated at the beginning of the cycle and new spindle fibers are developed to aid in the chromosomal rearrangement genetic material segregation.
CDKB – CYCB are responsible to maintain this state throughout the cycle and other CYC – CDK complexes are phosphorylated and remains inactive; especially CDKA – CYCD whose expression might initiate growth in the mitotic cell.
Prophase Induced Cell Modification
Prophase is predominant by cellular preparation for the division includes the chromosomal condensation and disintegration of the cell organelles mainly nuclear membrane.
Prophase generally involves number of phosphorylation which are induced by unavailability of free genetic material to express the suppressing role.
These are regulated and mediated by CDK – CYC complexes of the mitotic phase where the complex induces the breakdown of nuclear membrane by phosphorylating the membrane components to breakdown and further phosphorylation of other proteins of the nucleus and the cytoplasm induce disintegration of the organelles and development of new spindle fibers.
As the cell organelles disintegrates; the mitotic spindle made of microtubules and microfilaments develops along with the chromosomal condensation by inducing the action of 2 proteins CONDENSINS and COHESIONS which coils the chromatids to chromosomes with centromeres.
Prophase Induced Nuclear Modification
I. Chromosomal Condensation
Compaction of chromosome for the ease of cellular division is aided by Condensins and Cohesions an exact mechanism on how they work is not yet achieved as perceiving chromosome is well limited by the size, integrity and complexity of it.
But theories remains that the five – subunit protein forms a ring by ATP hydrolysis and combines the chromatin and coils together.
Cohesins are similar proteins which are present from the S Phase where it binds the Replicated DNA to the sister DNA and ensures the chromatid attachment to each other.
Cohesins and Condensins belong to Structural Maintenance Protein – SMC maintains the chromosome structure and prevent them from tangle.
II. Other Events
Cell must completely prepare for the cell division to support it.
This is done by Nucleolus disintegration: condensation of the chromosomes restricts the site for gene expression which is essential for the cell coordination and synchronization.
When the region is coiled in the chromosome regular functions cease to exist thereby releasing inhibition from the nuclear membrane for CDK’s to phosphorylate the nuclear inner lamina which disintegrates into small protein speckles similar to that of ribosomes.
The condensation restricts the transcription of the cell maintenance; this may lead to the disintegration of nucleolus and nuclear envelope.
The disintegrated envelope is further displaced by Microtubules with an influx of Ca and Protein Kinase C.
The spindle formed were present form the S phase which are also eliminated and new Microtubules are formed.
Primary Constriction in the same chromosome results in the formation of a chromosomal element – centromere; plays an important role in connecting spindle fibers for the chromosomal segregation.
Prophase Induced Cytoplasmic Modification
Cell division takes place during a favorable condition where it has sufficient nutrients to promote the development of the cell cycle.
Main changes in the cell cytoplasm are: the reduced protein synthesis by a mechanism which is to be deduced.
Micro tubulars and Microfilaments from interphase disintegrate and new ones are formed, additionally; the region of cytokinesis is marked in the cell cortex by the thick actin bands – Preprophase Bands.
“Tog” domain proteins initiate the microtubular formation by catalyzing the polymerization of tubulin.
The mitotic restructure is essential to govern the cell at structural level. In mitosis; the cytoplasm remains immotile due to absence of the microtubules and microfilaments of the cell causes notable changes to support the division
1. New microtubules are synthesized which will support the cell cycle of the daughter cells.
2. The rigid cell becomes round and this promotes the even distribution of the cytoplasm and other structures to ensure the integrity of daughter cells.
3. The microtubules are soluble in the cell reduces the viscosity for ease in movement as the cytoplasm as the stream gets stationary.
All these factors ensure a proper and equal division of the cytoplasm and other organelles to the daughter cells.
Prophase Citations
- Coordinating the events of the meiotic prophase. Trends Cell Biol . 2005 Dec;15(12):674-81.
- Checkpoint mechanisms: the puppet masters of meiotic prophase. Trends Cell Biol . 2011 Jul;21(7):393-400.
- Double-strand break repair on sex chromosomes: challenges during male meiotic prophase. Cell Cycle . 2015;14(4):516-25.
- The meiotic checkpoint network: step-by-step through meiotic prophase. Cold Spring Harb Perspect Biol . 2014 Oct 1;6(10):a016675.
- The role of chromatin modifications in progression through mouse meiotic prophase. J Genet Genomics . 2014 Mar 20;41(3):97-106.
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Interphase: Definition, Checkpoints, and Examples
Continue ReadingCell Cycle and Interphase
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.
Interphase Types
Interphase of the cell cycle is non dividing phase where the cells undergo major synthesis and other integrating functions transducing signals and coordinate with other cells to produce an overall reaction to the stimulus.
Biosynthetic activity is more prevalent in this phase; absence of cell division marks a resting period; varies for every differentiated cells.
Certain cells might have a short resting period and certain cells such as neurons loses the power of differentiation and remains constant.
I. Gap1 Phase
The first gap phase starts just after the 2 daughter cells formed by mitosis is long and are species specific.
Intermediate to Mitosis and the synthesis phase the cell increases in size by synthesizing proteins and RNA for Synthesis Phase.
In a cell cycle, G1 phase is regulated by the external and internal factors.
External limiting factor is the availability of the nutrients in general eukaryotic organism and additional hormonal induction in plants.
Most of the organism restrict their cycle at G1 or enter G0, few cells on proper hormonal induction will reenter the cell cycle from G0 phase; specifically, plants have a specialized region called the meristem which continuously divide to produce cell growth.
The G1 phase determines the fate of the cell whether to enter cell cycle or to retain in G1 phase.
The cell must grow to an appropriate size and must synthesize proteins and RNA to enter S phase, and this is regulated by Cyclins and CDK’s.
II. Synthesis Phase
The threshold point – restriction point in mammals and START in other organisms; when crossed the cells enter the synthesis phase.
The G1 phase prepared the cell and its components to synthesis phase to get committed to cell division.
The cell division is arrested when external aids are less or unavailable.
The DNA replication doubles the genetic material, but the chromosome is not condensed and replicated material remains as chromatin.
Synthesis of genetic material is significant in this phase where cells are committed to divide and produce 2 daughter cells in most of the metazoans.
The S phase does not induce the increase in chromosomal numbers but doubles the genetic material.
Endoreduplication a landmark in plant endosperm is the different process of the metazoans permits DNA replication without mitosis results in ploidy. In animals, Drosophila’s salivary glands and mammal’s hepatocytes exhibit endoreplication.
Gap2 Phase
Synthesis phase and Mitosis phase is separated by G2 Phase; marked by absence of synthetic function.
This phase is a preparatory phase where the cell again enters the mitotic cycle for division.
Decondensed chromatin starts a preliminary condensation marks the start of mitotic phase.
Condensation inhibits the RNA synthesis gradually as the synthesis sites are being compactly wound for replication.
G2 Phase has minimal RNA synthesis which reduces or absent in Mitotic phase.
Interphase Checkpoints and Regulation
The cell enters cell cycle to divide and produce daughter cells or it participates in regular metabolic function is determined during the interphase.
The process becomes irreversible when the cells traverse across G1 phase and enters S phase.
Determination and commitment to cell division is regulated by protein kinases CDK – CYC complex which are conserved in plants but varies across other kingdoms; are heteromeric proteins which are predominant in eukaryotic cells determines the fate of cell based on the internal and external cues.
Each transition from stages is regulated and governed by CYC – CDK complex; involves an intricate network of mechanism involves positive and negative regulation of many numbers of components to maintain a homeostasis.
Stages of cell cycle also provides checkpoint to make sure the proper functioning of the cell cycle.
G1/S Cell Cycle Checkpoints
G1 phase is the lap phase where the cell obtains essential growth and necessary elements; increase in size by biomolecular synthesis; to proceed the cell cycle or to enter G0 to restrict them by functioning regular metabolic activities.
In most of the eukaryotes the increase in CDKA – CYCD in G1 phase makes the cell cross the restriction point and irreversibly committed to cell cycle; involves an activation pathway.
The Cyclin D concentration responds to external cues such as hormone induction, availabilities of nutrients; promote CDKA-CYCD complex thereby initiating the G1 activity; the concentration of the CYCD-CDKA complex increase the phosphorylation of Rb (Retinoblastoma) proteins.
The Rb proteins are the inhibitor of transcription factors transcribing the proteins and RNA essential for the synthesis phase.
In the absence of the CYC-CDK complex the pathway is inhibited by Rb protein.
The transcription is also inhibited when a cell synthesis the essential quantity of proteins required for S Phase or when the CYC-CDK complex over express in the process.
The transition pathway is conserved in plants. Generally; plants have a single Rb homologue and 6 types of E2F transcription factors.
E2F is further divided into 2 groups: CANONICAL and ATYPICAL. E2Fa, E2Fb, E2Fc require a dimeric substance such as DP to bind to the DNA are canonical and monomeric factors E2Fd, E2Fe, E2Ff are Atypical.
Each E2F factors has a definitive function which are not yet determined correctly; but from many scientific experiments in Arabidopsis the functions are deduced as:
E2Fa – Transcriptional
E2Fb – Transcriptional
E2Fc – Down Regulation of Transcription (Repressors)
E2Fd – unidentified
E2Fe – Prevent endocycles delaying cell elongation
E2Ff – Cell expansion Also, E2Fa and E2Fb are more essential in S Phase to balance proliferation and endoreduplication in cell.
E2F transcription leads to initiation and increase in the DNA replication by setting up a replication origin where ORC – origin replication complex binds to produce the replicating fork for DNA synthesis.
The regulating checkpoint in the process is the expression of E2Fc transcriptional factors which are induced by the genetic expression mediated by E2Fa/b; inhibit the over expression of DNA Replication.
G2/M Cell Cycle Checkpoints
The transition of the particular phase involves many genes and transcriptional factors in cycle progression.
The transcriptional factors are of 5 types: MYB3R1, MYB3R2, MYB3R3, MYB3R4 and MYB3R5.
The G2/M transition involves proteins which are also present in M phase induces cytokinesis.
The functions of transcriptional Factors are:
MYB3R2 – circadian clock
MYB3R1, MYB3R4 – activates the gene expression
MYB3R3, MYB3R5 – repress the gene expression
MYB3R3 and 5 regulates the function of gene expression of G2/M phase when it over express.
Along with this; APC/C – Anaphase promoting complex/Cyclosomes targets the proteolysis which regulates the G2/M transition and supports Mitosis phase of cell proliferation.
Interphase Citations
- Functional organisation of the genome during interphase. Curr Opin Genet Dev . 2007 Oct;17(5):451-5.
- Interphase cytogenetics and its role in molecular diagnostics of solid tumors. Am J Pathol . 1998 Feb;152(2):325-7.
- Interphase cytogenetics: at the interface of genetics and morphology. Anal Cell Pathol . 1999;19(1):3-6.
- Chromosome topology in mammalian interphase nuclei. Exp Cell Res . 1991 Feb;192(2):325-32.
- Two-step interphase microtubule disassembly aids spindle morphogenesis. BMC Biol . 2018 Jan 23;16(1):14.
- Interphase microtubules: chief casualties in the war on cancer? Drug Discov Today . 2014 Jul;19(7):824-9.
- Interphase Cell Cycle Staging using Deep Learning. Annu Int Conf IEEE Eng Med Biol Soc . 2020 Jul;2020:1432-1435.
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Cell Cycle: Definition, Description, Stages, and Checkpoints
Continue ReadingCell Cycle Introduction
Beings of earth are programmed to be born, grow, reproduce, and die. Multicellular eukaryotes develop from a unit cell to an array of trillion celled creature by a simple mechanism of cell division.
The capacity to divide is the measure of growth of an organism and the capacity to grow differs for every species are limited by internal and external factors.
Proliferation of cell can be infinite which might have resulted in an overwhelming cornucopia of cells.
The cell proliferation and growth are finite and regulated by an organism by spatial and temporal means according to its functional ability.
The increase in cell corresponds to the growth an organism exhibits; regulated or stopped after a particular period.
Plants tends to show a regulated unlimited growth; developing over lifetime a distinctive character of plants to support the sedentary life; but the growth is governed by internal and external cues of development.
Specialized regions such as meristem plays a primary role in plant cell division and growth.
Growth is regulated at cellular level by the cell cycle and growth regulators plays a significant role in inducing growth and supporting the structural composition of growth.
Periodic stages of cell cycle involve the major function of a single cell over a lifetime following a repetitive phase of development and function.
What is Cell Cycle
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.
Cell Cycle History
Discovery of microscope was a breakthrough in understanding the physiology at cellular level.
Robert Hooke was first to observe cork cells; from there microscopic observation revealed the world of microbiota.
The cell was discovered and components, functions and the significance were found. In late 19th century; it was deducted that new cell develops from the pre – existing cell by the process of cell division.
Cell division led to elucidation of cell cycle processes which was determined to be the principal process regulating growth and development of the cell and the organism.
But the DNA replication restricted to S phase of the cell cycle was not discovered until 1950 by Alma Howard and Stephane Pele.
The conservative nature of the cell cycle was identified in 1980’s; were scientists found out that the molecular processes of cell cycle is similar in all eukaryotic organisms; from experimenting various research on organisms.
Cell cycle regulators and genes “START” was discovered by Leland Hartwell.
Paul Nurse discovered CDK which runs the cell cycle based on the genetic expression.
Timothy Hunt discovered cyclin activating subunit of CDK’s. Leland Hartwell, Paul Nurse and Timothy Hunt shared Nobel Prize for Medicine in 2001.
Characteristic of Cell Cycle
o The sequential progress of cell growth and cell division to form new daughter cells are termed as cell cycle.
o The sequences are divided into 4 divergent phases falls in 2 majors they are: Interphase and Mitotic Phase.
o The stages of cell cycle is common for eukaryotic organisms.
o The cell cycle through evolution is conservative because of the similarities of the phases in all organisms.
o The cycle is well regulated by genes and protein kinases.
o Protein kinase action is same in all eukaryotic species signifies the conserved evolution and a proof that all organisms arise from a common ancestor who diverse from single celled unicellular organism to complex human beings.
o The stages of cycle are governed by time at which cells enter the cell cycle.
o Cell cycle is asynchronous in a living organism.
o The cell division is regulated and controlled by the protein kinases to maintain the structural integrity and functionality of the organism.
o As the organism grows few regions remains constant with no cell growth or maintained to a threshold level where cell death is balanced by cell division.
o In few parts the cells the growth might be rapid or enter the cycle first and other cells may enter later result in asynchronous cell division.
o Length of cell cycle varies from species to species. Few organisms have rapid replication system compared to other organism.
o For Example: Yeast completes its cell cycle in 90 minutes (about 1 and a half hours) whereas in human cell cycle lasts for 24 hours and in Drosophila it is about 8 minutes.
o Phase of cell cycle is sequential in continuously dividing cells were one phase follows the other in a constant order becomes inevitable for a proper functioning of the cell cycle are regulated by the controls of the cycle.
o In multicellular organisms, higher level of cell differentiation reduces the capacity of a cell to proceed the cycle.
o A post – mitotic differentiated cell restricts it at G1 phase or enter G0 phase a quiescent phase.
o Stem cells are an ideal example for cell cycle where it keeps on proliferating to maintain the integrity of the organism replacing the dead cells by new ones.
o Most of the cells completes the cycle whereas few other remains in the synthetic phase their lifetime without entering the dividing phase.
o Proceeding the phases of cell cycle is determined by the synthesis phase where a cell cycle sets up an upper threshold level which must be crossed to enter the division phase.
Cell Cycle Phases
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.
I. Interphase
Interphase of the cell cycle is non dividing phase where the cells undergo major synthesis and other integrating functions transducing signals and coordinate with other cells to produce an overall reaction to the stimulus.
Biosynthetic activity is more prevalent in this phase; absence of cell division marks a resting period; varies for every differentiated cells.
Certain cells might have a short resting period and certain cells such as neurons loses the power of differentiation and remains constant.
II. Gap1 Phase
The first gap phase starts just after the 2 daughter cells formed by mitosis is long and are species specific.
Intermediate to Mitosis and the synthesis phase the cell increases in size by synthesizing proteins and RNA for Synthesis Phase.
In a cell cycle, G1 phase is regulated by the external and internal factors.
External limiting factor is the availability of the nutrients in general eukaryotic organism and additional hormonal induction in plants.
Most of the organism restrict their cycle at G1 or enter G0, few cells on proper hormonal induction will reenter the cell cycle from G0 phase; specifically, plants have a specialized region called the meristem which continuously divide to produce cell growth.
The G1 phase determines the fate of the cell whether to enter cell cycle or to retain in G1 phase.
The cell must grow to an appropriate size and must synthesize proteins and RNA to enter S phase, and this is regulated by Cyclins and CDK’s.
III. Synthesis Phase
The threshold point – restriction point in mammals and START in other organisms; when crossed the cells enter the synthesis phase.
The G1 phase prepared the cell and its components to synthesis phase to get committed to cell division.
The cell division is arrested when external aids are less or unavailable.
The DNA replication doubles the genetic material, but the chromosome is not condensed and replicated material remains as chromatin.
Synthesis of genetic material is significant in this phase where cells are committed to divide and produce 2 daughter cells in most of the metazoans.
The S phase does not induce the increase in chromosomal numbers but doubles the genetic material.
Endoreduplication a landmark in plant endosperm is the different process of the metazoans permits DNA replication without mitosis results in ploidy.
In animals, Drosophila’s salivary glands and mammal’s hepatocytes exhibit endoreplication.
IV. Gap2 Phase
Synthesis phase and Mitosis phase is separated by G2 Phase; marked by absence of synthetic function.
This phase is a preparatory phase where the cell again enters the mitotic cycle for division.
Decondensed chromatin starts a preliminary condensation marks the start of mitotic phase.
Condensation inhibits the RNA synthesis gradually as the synthesis sites are being compactly wound for replication.
G2 Phase has minimal RNA synthesis which reduces or absent in Mitotic phase.
V. Mitotic 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.
Prophase: The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division. RNA synthesis is inhibited.
Metaphase: Nuclear membrane is eliminated completely chromosomes are completely condensed. The cytoskeleton – spindle fibers attach to the kinetochores. The chromosomes are aligned in the equatorial plate.
Anaphase: Chromosomal split forms daughter chromatids; travels to the opposite poles. The chromosomes are V – Shaped as they are dragged to the opposite sites.
Telophase: Microtubules disappear and chromosomes decondense to chromatin mass. Nuclear envelope starts to form. The disintegrated organelles form again.
These phases mark the Karyokinesis were the nucleus and other cell parts are newly formed.
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.
Cell Cycle Regulation
The cell cycle is regulated by heterodimeric protein which was first discovered in sea urchin; the cyclic appearance named the compound CYCLIN; is the regulatory subunit activating Cyclin dependent Kinases – Catalytic subunit.
The involvement of CDK’s promotes an intricate network of mechanism triggering positive and negative response creates a loop controlling the cell cycle.
The protein kinases phosphorylate other repressors of the cell cycle traversing cell over distinct phases of development.
The cell cycle and the basic protein kinases were conserved throughout the eukaryotic cells, but the CDK types of serine – threonine special class are not conserved; varying from quantity to Plant types
The Animals has a distinctive CDK types; CDK1 – CDK7 are present in animals and CDK A – CDK E in plants.
Cyclins also falls under 5 types A, B, C, D, and H. The cyclin is transcript from most conserved part of the human genome preserving its integrity over their period.
CDK and Cyclin combines to regulate the cell cycle at each phase; characterized by specific combination of Cyclin and CDK promoting the cell to reproduce for the growth of an organism.
Distinct phases have different cyclin – CDK complex varying in animals and plants.
Monomeric CDK does not participate in regulation of cell cycle; CDK require cyclin subunit to be activated by CDK activating kinases.
These heteromeric protein can be inhibited by inhibiting proteins – Kip-related proteins by inhibitory phosphorylation.
The CDK’s and cyclins are dependent on the rate of turnover, transcription, and translational control.
Cell cycle Regulation in Plants
The CDK – CYC (cyclin) complex regulates the cell cycle. Each phase expresses specific complex for the cells to be accustomed to proceeding through cell cycle. CDK – A;1 is expressed in all phases but are activated by D type cyclins in G1 Phase to traverse the restriction point.
Transcription of CDK A – CYC D accumulates and facilitates the preparation of cell to transverse G1/S phase to commit to cell cycle.
The synthesis of CDKA – CYCD complex when increase in quantity reaches a restriction point and when it exceeds the point the cell commits to cell division.
When the point is not crossed the cell remains in G1 phase and eventually enters G0 phase indicating maturation and takes part in regular cellular activities.
In S phase CDKA – CYCA is expressed during the synthesis of DNA duplicate.
Traversing from S phase the CYCD starts expressing with the entry of G2 phase along with CYCA2 – CDKA later this followed by the expression of CDKB – CYCB which on reaching a threshold limit indicates the cells are ready for the cell division.
Again, at the end of G2 phase the positive feedback of CDKB-CYCB induces the CDKA-CYC3 to be produced all along the mitosis and prevails in G1 PHASE to increase the growth of the cell.
Cell Cycle Plant Hormones
Plant growth hormones Auxins, cytokinin and brassinosteroids has a significant role in control of the cell cycle.
Auxin and Cytokinin are essential for cell proliferation and are widely expressed in the meristematic tissues of plants promotes plant growth by cell division induces the cell cycle.
These were discovered from the mutational studies inhibiting the hormones and observing cell cycle.
Cytokinin helps in G1/S transition and G2/M transition by dephosphorylating CDK’s thereby activating the cell cycle or inhibits WEE 1 kinase by down regulation.
Abscisic acid inhibits cell cycles by promoting inhibitors such as KRP1 in certain plants.
Similarly, Jasmonic Acid inhibits DNA replication when administered during G1 phase and has minimal inhibition effects in later stages.
Ethylene promotes cell death in the developing cell cycle.
Cell Cycle Citations
- Cell cycle checkpoints. Curr Opin Cell Biol . 1994 Dec;6(6):872-6.
- Cell Cycle Control: A System of Interlinking Oscillators. Methods Mol Biol . 2016;1342:3-19.
- Cell cycle development. Dev Cell . 2004 Mar;6(3):321-7.
- Linking the Cell Cycle to Cell Fate Decisions. Trends Cell Biol . 2015 Oct;25(10):592-600.
- The cell cycle: a review. Vet Pathol . 1998 Nov;35(6):461-78.
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Phenylalanine Deaminase Test: Result, Principle, Procedure, and...
Continue ReadingPhenylalanine Deaminase Test Introduction
Biochemical tests are usually performed to detect the ability of the microorganism to distinguish their enzymatic activity and to define their characteristics.
Each of the organism acts accordingly based on their surrounding enzymatic reactions and their host cell they live upon.
These biochemical tests are performed with the help of inoculating cultures and specific chemical reagents and the indicators used.
Phenylalanine deaminase test is one such test which is used to differentiate the species belonging to the group of urea-positive gram-negative bacilli on the basis of their ability to produce phenyl.
What is Phenylalanine Deaminase Test?
Phenylalanine deaminase test is commonly known as Phenyl pyruvic acid test, which is commonly called as PPA test.
Phenylalanine deaminase test is due to ability of the organism to produce deaminase.
Phenylalanine deaminase enzyme helps in removing the amino group from the amino acid phenylalanine which thus produces and phenyl pyruvic acid and ammonia by the process of oxidative deamination of phenylalanine.
Phenyl pyruvic acid reacts along with the ferric iron and produces a visible green color.
Phenylalanine agar, commonly known as phenylalanine deaminase medium, as it contains the DL- phenylalanine and the nutrients in their medium.
Phenylalanine Deaminase Test
Phenylalanine deaminase tests is usually used to determine the ability of the organisms to produce the enzyme deaminase.
Micro organisms which are capable of producing phenylalanine deaminase.
Micro organisms that are capable of producing the enzyme phenylalanine deaminase removes the amide group from the phenylalanine thus results in release of the free molecules of ammonia.
Thus, the deamination of phenylalanine results in the formation of phenylalanine pyruvic acid with the helps of the oxidative enzymes.
Hendriksen in the year 1950 demonstrated the species of proteus which helps us in converting the amino acid phenylalanine into the acid form known as phenylpyruvicacid.
But in later times, Buttiaux et al. developed a medium of culture for detecting the formation of phenyl pyruvic acid from the members of the phenylalanine namely Proteus, Providencia and other groups such as Morganella.
This medium is modified again by Ewing et al. and further by Bynae. Bynae simplified this medium by discarding the process of protease peptone in the medium.
Usually, the micro-organisms which detects the phenylamino acid by the enzyme phenylalanine deaminase is identified by adding 4 to 5 drops of ferric chloride.
Whereas addition of ferric chloride results in the production of green colored complex between the compounds of the slant test and also in the tube which indicates the positive results.
On the other hand, the absence of green colored complex determines the negative result of the test.
Phenylalanine Deaminase Test
• The main aim of the test is to detect the ability of the organism to deaminate the phenylalanine oxidatively and to convert it into phenyl pyruvic acid.
• To differentiate the certain species of gram-negative bacilli from the Enterobacteriaceae family.
Phenylalanine Deaminase Test Reagents
Materials required for preparing a medium:
Ingredients Gram/Liter DL – Phenylalanine 2.0 Yeast extract 3.0 Sodium chloride 5.0 Disodium phosphate 1.0 Agar 12.0 Distilled water 1 liter pH 7.3 Here, yeast extract plays an important role in acting as a source of carbon and nitrogen. On the other hand, meat extract and protein hydrolysates cannot be used as the natural varying agent of the phenylalanine.
Phenylalanine Deaminase Test Procedure
Initially a loop full of inoculum is collected from an 18 to 24-hour pure culture, and it is streaked against the slanted surface with the help o a fishtail motion or by using a Phenylalanine inoculate of about 1 drop from the 24-hour brain-heart infusion medium.
The prepared medium is inoculated at a temperature of about 35 degree Celsius for about 18 to 24 hours.
After completing the process of incubation, about 4 to 5 drops of 10% ferric chloride solution is applied directly upon the slant.
Then the tube containing a slant is agitated gently and if the results are positive then the development of green color can be observed within a minimum of 1 minute and a maximum of 5 minutes.
Phenylalanine Deaminase Test Result
Positive Result: In case of positive result, the development of green color can be observed along the slant after adding few drops of ferric chloride. This reaction is usually seen within 5 minutes of addition of ferric chloride.
Negative Result: Here the negative results are determined by absence of green color in the medium after 6 to 7 minutes of addition of a ferric chloride. But negative results are determined by appearance of yellow color which is due to the presence of the ferric chloride.
Phenylalanine Deaminase Test Uses
This test is usually recommended for differentiating the species of gram-negative enteric bacilli on the basis of the ability of the micro-organism to produce the phenyl pyruvic acid by the process of oxidative deamination.
Generally, the genera like Morganella, Proteus and Providencia are differentiated by following these tests to differentiate them from Enterobacteriaceae family.
Phenylalanine Deaminase Test Citations
- Rapid identification of micro-organisms from urinary tract infections by beta-glucuronidase, phenylalanine deaminase, cytochrome oxidase and indole tests on isolation media. J Med Microbiol . 1994 Dec;41(6):389-92.
- Rapid test for urease and phenylalanine deaminase production. Appl Microbiol . 1971 Mar;21(3):545.
- Combined Medium to Determine Deoxyribonuclease Activity and Phenylalanine Deamination by Enterobacteriaceae. Appl Microbiol. 1970 Feb; 19(2): 385–386.
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Potassium Hydroxide Test: Result, Principle, Procedure, and...
Continue ReadingPotassium Hydroxide Test Introduction
Many of the biochemical tests are used to detect the ability of the micro-organisms to act against certain chemicals and enzymes.
Potassium hydroxide test is used to identify the gram-negative bacteria.
Potassium hydroxide is one of the inorganic compounds which has its chemical formula KOH, and it is most commonly referred to as caustic potash.
It can be used in many chemical and biochemical tests.
Potassium hydroxide is also known as Potassium hydroxide dissolves with the peptidoglycan which is present as a thin membrane in the cell walls of the gram-negative bacteria.
But this does not affect the gram-positive cell walls.
Disintegration of these gram-negative cell walls lyses the cell thus releasing the contents from the cell including those of DNA.
The DNA makes the solution viscous and makes the solution to stick to the plastics loop when touched.
However, Gram positive bacteria does not affect the Potassium hydroxide, as it has thicker peptidoglycan layer present in the cell wall.
Hence the cells will not be lysed, so that there will no release of DNA and there is no viscosity observed.
What is Potassium Hydroxide Test?
Potassium hydroxide test is also known as KOH string test.
Potassium hydroxide test mostly relies on the differential resistance of about 3% in the potassium hydroxide, between the gram positive and the gram-negative cells.
Here a small portion of a colony is mixed up with a small volume of 3% KOH.
If the cell lyses, the cellular DNA will be liberated making the mixture viscous or stringy.
The positive string test indicates a gram-negative organism.
Hence the potassium hydroxide can also be known as String test.
Potassium hydroxide test also helps in differentiating the Gram positive and the Gram-negative organisms and this test is also helpful in complementing the gram stain and the antibiotic disc test.
Potassium Hydroxide Test Objective
The main aim of the test is to differentiate between gram negative and the gram-positive organisms.
Potassium Hydroxide Test Principle
Similar to the Gram stain reaction, the potassium hydroxide test is basically used to identify the differences in the chemical composition of the bacterial cell wall.
During the presence of this Potassium hydroxide, Gram negative cells will be lysed, this makes the Potassium hydroxide solution to dissolve easily with the thin layers of the cells, known as peptidoglycan.
However, Disintegration of Gram-negative cell wall will lyse the cells making it to release all of it contents, including DNA.
As a result, a viscid chromosomal material is released along with the contents and it is caused by the suspension of the bacteria to become thick and stringy.
The viscous and the solution sticks to the loops.
On the other hand, Gram positive bacteria are not affected by the Potassium hydroxide as it has thicker peptidoglycan layer along the cell so there are no chances of cell lyses and no contents will be spilled out of the cell so no viscosity can be detected.
Potassium Hydroxide Test Procedure
About one drop of 3% of potassium hydroxide solution is placed in a clean microscopic slide.
Few colonies of a suspect organism are emulsifying along with the drop of potassium hydroxide placed in the slide and makes it as a dense suspension.
Then the mixture is continuously mixed for about 60 seconds and they are gently pulled using a loop such that to pull it against the suspension.
Then the medium is detected for any changes.
Potassium Hydroxide Test Result
For positive results, the organisms become thick, stringy and it forms a long strand within few seconds. This is seen only in the species of the Gram-negative bacteria.
In case of negative results, the organisms leave the suspension without any changes in the absence of the stringing. These results are mostly seen in Gram positive bacteria.
Potassium Hydroxide Test Uses
Potassium hydroxide tests are generally used in laboratories where the cultures are processed in large numbers, the above test is used is also used along with the gram stain in preliminary differentiation.
This test is also useful in reacting as a complement in the Gram stain and in antibiotic tests.
Potassium Hydroxide Test Limitation
Though, this test is useful, the test cannot be used to determine the results, as the negative tests does not prove any conclusive if an organism is Gram-positive.
In old cultures which are being cultures before 48 hours, has the chances of turning the negative results into positive after 30 seconds of mixing the bacteria along with the potassium hydroxide solution, thus gives unreliable results. This type of results are most common in species like Achromonacter genera such as Brucells melitenis, Pseudomonas paucimobilis, Moraxella species, etc.
False results mostly occur due to a heavy inoculum, where the solution appears in the form of gel, without a string form. In some cases, there is also chances of forming the inoculation with the mucoid colonies. On the other hand, False results occur when there is a very little or light inoculum or if there is too much of potassium hydroxide than normal.
Potassium Hydroxide Test Citations
- Evaluation of the KOH test and the antibiotic disk test in routine clinical anaerobic bacteriology. J Clin Microbiol . 1988 Oct;26(10):2144-6.
- The Sensitivity and Specificity of Potassium Hydroxide Smear and Fungal Culture Relative to Clinical Assessment in the Evaluation of Tinea Pedis: A Pooled Analysis. Dermatol Res Pract. 2010; 2010: 764843.
- The sensitivity and specificity of potassium hydroxide smear and fungal culture relative to clinical assessment in the evaluation of tinea pedis: a pooled analysis. Dermatol Res Pract . 2010;2010:764843.
- Successive potassium hydroxide testing for improved diagnosis of tinea pedis. Cutis . 2017 Aug;100(2):110-114.
- Use of Potassium Hydroxide (KOH) Test Reduces Antifungal Medication Prescription for Suspected Monilial Diaper Dermatitis in the Neonatal Intensive Care Unit: A Quality Improvement Project. Adv Neonatal Care . 2019 Dec;19(6):E3-E10.
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Nitrate Reduction Test: Result, Principle, Procedure, and...
Continue ReadingNitrate Reduction Test Introduction
Many of the biochemical test is usually used to detect the ability and to identify the species of bacteria b differentiating them based on the characteristics and their biochemical activities.
There are many factors such as protein, carbohydrate metabolism and the enzyme production.
These biochemical tests used in many of the clinical test to detect the pathogen and its nature of the disease-causing techniques.
Nitrite is one of the ions consisting molecule that is made up of nitrogen atom which is formed along with the oxygen atoms with the help of nitrogen bonds.
Nitrite is usually known as anion. Nitrite has the capability to inhibit the growth of bacteria hence it is used n many of the biochemical tests and also in clinical test and in food processing units.
In nitrate reduction test is based on the detection of the nitrite that is present in the medium after incubating it along with the organism.
Presence of nitrite in the medium reacts with the sultanic acid and results in the formation of the colourless complex.
This complex helps us to yield a red precipitate when the nitrate reagent is added to the medium.
What is Nitrate Reduction Test?
Anaerobic metabolism usually requires an electron acceptor other than the atmospheric oxygen.
Many of the species of the Gram-negative bacteria uses the nitrate as the final acceptor of electron.
Nitrate reduction test is a test which determines the production of the enzyme known as nitrate reductase, that results in the reduction of nitrate.
Bacterial species are differentiated according to their ability to reduce nitrite or nitrogenous gases.
Nitrate Reduction Test Objective
The main aim of the test is to determine the ability of an organism to reduce the nitrate or nitrite.
To identify the different ways of reduction of bacteria by the nitrate.
Nitrate Reduction Test Principle
To perform this test, a heavy inoculum of the test organism is collected and it is incubated in the broth containing nitrate.
The organisms which have the capability to produces the enzyme, nitrate reductase reduces the presence of nitrate in the broth, thus it forms its reduced form known as nitrite.
Nitrite further reduces to nitric oxide, nitrous oxide or nitrogen.
This test is purely based on the detection of nitrite and its ability to form a red colored compound on reacting with sulfamic acid and forms a complex known as nitrite sulfamic acid.
Sulfanilic acid further reacts with the alpha-naphthylamine which results in the formation of the red precipitate known as Prontosil, this prontosil is completely soluble in water and it is called as azo dye.
It is also noted that only when nitrate is present in the medium, formation of red color can be observed.
If there is no formation of red color then it can be proceeded by adding sulfanilic acid and Alpha-naphthylamine , when nitrite is absent in the medium.
Usually, this observation is explained in two ways, Nitrate does not have reduces, if the strain is noted as nitrate-negative.
The nitrate reduces into nitrite, which is further reduced into nitrous oxide or nitrogen, which on this reduced form does not reacts with nitrite, then the strain is said to be nitrate-positive.
However, if nitrate is not detected, it is important to test if the organism has the capability to reduce nitrate beyond nitrite.
This is usually done by adding a small amount of zinc powder catalyzes in order to reduce the nitrate into nitrite.
On adding zinc, the formation of red color can be observed, which indicates that nitrate was not reduced to nitrite.
In other case, where is no change in color even addition of zinc powder, then it indicates that the organism is reduced into any of the nitrogen compounds.
Nitrate Reduction Test Reagents
Nitrate Broth:
Peptone – 5 grams per litre
Meat extract – 3 grams per litre
Potassium nitrate – 1 gram per litre
Nitrate Reduction Test Procedure
Nitrate reduction is usually determined in two step process. Initially the reduction of nitrate to nitrite is determined by adding the nitrogen reagents into the medium.
If necessary, the reduction of nitrate beyond nitrite beyond is determined by adding nitrate reagent C, which is in the form the zinc dust.
Initially the nitrate broths are inoculated into the nitrate broth into the bacterial suspension.
Incubate the tubes at the optimum temperature of about 30 to 37ºC for 24 hours.
After incubating the formation of nitrogen gas is noted before adding the reagents.
To this medium, about 6 to 8 drops of the nitrate reagent A and the same amount the nitrate reagent B are added, once it is added, color changes can be observed in a minute or some times even less than that.
On the other hand, if there is no color change, then zinc powder is added and formation of any changes can be observed within 3 minutes of adding.
Nitrate Reduction Test Result
Positive Test:
In positive tests, the development of red color can be detected after addition of the reagents A and B.
And in Zinc powder addition, the medium should not develop the red color and it results in positive results.
Negative Test:
Development of a red color when zinc powder is added.
Nitrate Reduction Test Citations
- Simple disk technique for detection of nitrate reduction by anaerobic bacteria. J Clin Microbiol . 1977 Mar;5(3):315-9.
- Nitrate reduction: new method for testing the antibiotic susceptibility of Haemophilus influenzae. Antimicrob Agents Chemother . 1978 May;13(5):791-5.
- Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system. J Microbiol Methods . 2010 May;81(2):208-10.
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Kligler Iron Agar Test: Principle, Procedure, and...
Continue ReadingKligler Iron Agar Test
Many of the biochemical tests are performed to detect the ability of the micro-organism to utilize the enzyme in the culture medium. Kligler iron test is also one of the biochemical tests, which employs a medium for the identification of the species of Enterobacteriaceae, based on the fermentation of the double sugar and the production of hydrogen sulphide.
Kligler Iron Agar Test was first determined by Kligler in the year 1918 to describe the medium for detecting the production of hydrogen sulphide and it also helps us to differentiate the species of Salmonella.
This was first modified by Bailey and Lacey by substituting the phenol red indicator instead of Andrade indicator in the medium.
The medium used in the test is commonly known as KIA. It is also recommended that determination of the Hydrogen sulphide depends on the production of the Enteric gram-negative bacilli and also in detecting the production of H2S by using some strains of Pseudomonas.
What is Kligler Iron Agar Test?
Kligler iron agar test is used for detecting the fermentation of carbohydrate in the medium.
Here the reaction of the KIA helps us to include or exclude a particular bacterial species to isolate it from the family of Enterobacteriaceae.
If the organisms cannot ferment the carbohydrate or glucose present in the medium, then alkaline Slant in alkaline butt reaction can be observed in the Klinger Iron Agar.
This reaction is sufficient for excluding and isolating the species belonging to the Enterobacteriaceae family.
Klinger Iron Agar is also helpful in identifying the species named Salmonella shigella along with the other members of the Enterobacteriaceae family.
Kligler Iron Agar Test Objective
The main aim of the test is to differentiate the organisms by demonstrating the medium with the hydrogen sulphide production and to determine the fermentation of dextrose and lactose.
Kligler Iron Agar Test Principle
The medium of Klinger iron consists of lactose and the glucose in addition to peptone, HM peptone B and the yeast extract, which helps us to enable the difference between the species of the enteric bacilli.
Generally, in this test, phenol red indicator is used as a pH indicator, which brings about a change in colour in the medium when the acid response is detected during the fermentation process of the sugars.
Fermentation of the dextrose results in the production of acid, which in turn changes the colour of the medium from red to yellow.
If there is only minimum amount of sugar present in the medium, the indicator remains red and there will not be any change in the colour.
On the other hand, when lactose is fermented, there will be large amount of acid production in the medium and it also avoids reoxidation and results in the yellow colour of the whole medium.
The combination of ferrous sulphate and the sodium thiosulphate enables us to detect the production of hydrogen Sulphide and it is evidenced by a black colour or through the formation of butt.
In some cases, it can also be determined by ring formation near or top of the butt.
The production of butts and yellow slants in the lactose fermentation is due to production of high amount of acid in the medium which induces the change in pH under the aerobic conditions.
Whereas, the tubes which does not show any change in colour indicates that there is no presence of glucose or lactose in the medium.
However, the gas production is detected by formation of the individual bibles or by splitting of the agar, due to the formation of the cracks in the butt of the medium.
Kligler Iron Agar Test Reagents
Ingredients Gram/Litre Peptone 15.0 HM peptone 3.0 Yeast extract 3.0 Protease peptone 5.0 Lactose 10.0 Dextrose 1.0 Ferrous sulphate 0.2 Sodium chloride 5.0 Sodium thiosulphate 0.3 Phenol red 0.02 Agar 15.0 Kligler Iron Agar Test Procedure
Initially, a well isolated colony from a solid culture medium is picked from the centre of the medium using an inoculating needle.
Here the medium used for identification of the colonies includes MacConkey Agar, Bismuth Sulphide Agar, or Deoxycholate Citrate Agar are used as the plating medias.
Further the stab is taken from the centre of the medium into the deep tube which has about 3 to 5mm of depth.
Then the inoculating medium is withdrawn and it is streaked against the surface of the slant.
The caps of the tubes are closed loosely before incubating so that there will be an aeration in the medium.
Then the tubes are incubated aerobically at a temperature of about 35ºC for about 18 to 48hours.
After incubation the tubes are observed for acid production which can be detected by change in colour or formation of butts.
Kligler Iron Agar Test Result
The results are interpreted by Carbohydrate fermentation, Kligler Iron Agar colour reactions and by Hydrogen sulphide production.
Carbohyderate Fermentation
Slant Reaction:
Positive reaction- Yellow colour (acid)
Negative reaction- Red colour (alkaline)
Butt reaction:
Positive result _ Yellow (acid)
Negative result – Red (alkaline)
Kligler Iron Agar Color Reactions
Formation of red slant or yellow butt – Presence of dextrose and absence of lactose.
Formation of yellow slant or yellow butt – Presence of dextrose and lactose
Formation of red slant or red butt – Absence of dextrose and presence of lactose.
Production of Hydrogen Sulfide
Positive Test: Here the positive result is determined but formation of black colour throughout the medium and a black ring at the juncture of the slant and butt. In some cases, it is also determined by formation of black precipitate in the butt.
Negative Test: Here the negative test is indicated when there is no formation of black colour.
Gas Production
Positive Test: Formation of bubbles in the medium along with cracking or displacement of the medium. In some cases, it also leads to separation of the medium.
Negative Test: There will no bubbles and displacement or separation of the medium does not take place.
Kligler Iron Agar Test Citations
- The phenylalanine test on Kligler’s iron agar for the identification of Proteus. Nature . 1959 May 30;183(4674):1537-8.
- Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens. J Clin Microbiol. 1994 Jun; 32(6): 1583–1585.
- Biochemical differentiation of the Enterobacteriaceae with the aid of lysine-iron-agar. Appl Microbiol . 1966 Mar;14(2):212-7.
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Salt Tolerance Test: Principle, Procedure, and Result
Continue ReadingSalt Tolerance Test Introduction
Many of the biochemical test are performed in order to detect the ability of the organism to grow in a particular medium and to detect the utilization of the enzymes by them.
Once such biochemical test is salt tolerance test, which helps us to detect the ability of the bacteria to grow in the presence of the variable amount of sodium chloride and which is used to characterize the variety of bacteria.
Salt Tolerance Test also accounts the organism’s ability to tolerate the various osmotic concentrations. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans, which are present among the species of Enterococcus, and are considered as salt tolerant.
What is Salt Tolerance Test?
Salt tolerance test is usually used to identify the group of enterococcal group D Streptococcus based on the ability to tolerate the salt contents.
Thus, the capability of the bacteria to grown in the variable amount of Sodium chloride containing medium, helps us to distinguish the variety of bacteria’s, including those of Viridians Streptococci.
This test is used particularly for identifying the Species of Enterococcal Group D organisms, as it has its specialized ability to grow in a medium containing of 6.5% of the Sodium chloride.
This test is often performed with Bile Esculin tests in most of the laboratories, to distinguish the species of Enterococcus from the Group D species of Streptococci and also from Streptococcus bovis and Streptococcus lactis.
Enterococci is considered as one of the Significant causes for endocarditis, which has a high degree of mortality rate.
Salt Tolerance Test and Broth
In Salt tolerance test, Brain Heart infusion broth is used most commonly, it is supplemented with 6.5% of Sodium chloride and the Bromocresol purple as the indicator to denote the pH of the medium.
The use of this indicator helps us to read the results easier.
The broth also consists of dextrose.
Where the fermentation of this dextrose results in acid production, which leads to the change in pH and the color of the medium changes from purple to yellow.
Salt Tolerance Test Objective
The main aim of the test is to determine the ability of the organism to grow in high concentrations of the salt.
It is also used to differentiate the variety of Enterococcus species from the Non- enterococcus species.
Salt Tolerance Test Principle
Generally, Salt has its own characteristic to act as a selective agent and it also has the capability to interfere with the membrane permeability and the osmotic equilibrium.
The salt tolerance in the medium is calculated as a selective and the differential one according to the capability of the organisms to produce heavy growth in the broth and on the solid agar medium within 48 hours.
The salt tolerance medium was first formulated by Hajna.
Where as the high salt concentration inhibits a range of the bacteria which allows the salt-tolerant organisms like enterococci to grow in the medium, the quadric formaulation includes the carbohydrates, that can be fermented, dextrose, color indicator, bromocresol purple.
Organisms which have the capability to grow in the slaine medium, utilize the sugar and it releases the acid as its by product during their metabolism in the medium.
Which results in the decrease in the pH, the indicator bromocresol changes its purple color into yellow color.
Enterococci are usually resistant to high concentration of the salts and it often shows growth in the medium which contains the species like Enterococcus faecalis, Enterococcus zymogenes, Enterococcus liquefactions and Enterococcus durans which are known as salt tolerant species of the Enterococcus.
Salt Tolerance Test Reagents
The medium is composed of Sodium chloride of about 6.5% which is often used in brain heart infusion broth, commonly called as BHI and it can be used instead of individual components along with the sodium chloride and the indicator.
Components Required:
Herat digest of about 10 grams
10 grams of Enzymatic digest of animal tissue
Sodium chloride
Bromocresol purple indicator (it is added Per 1000 ml)
Salt Tolerance Test Procedure
Initially, one or two colonies are inoculated from 18 to 24-hour culture into the 6.5 of the sodium chloride broth without an indicator or 6.5 % of Nacl broth with indicator.
Then the tube is further incubated at temperature of about 35 to 37ºC in an ambient air for about 48 hours.
Then the incubated medium is examined for the presence of turbidity or growth or formation any colonies without an indicator. In some cases, there will also be a color change in the media with an indicator, here the color changes from purple to yellow.
Salt Tolerance Test Result
Positive Test: Here the positive result is indicated by visible turbidity or growth in the broth, and a color change from purple to yellow with the use of indicator.
Negative Test: Here the negative test is indicated by no turbidity or absence of color change, and the color remains the same.
Salt Tolerance Test Uses
This test is often used to differentiate the species of enterococci from the non-enterococci species.
It is also used to differentiate the species of non-beta hemolytic strains of the catalase from the negative gram-positive cocci based on their ability to grow in a medium containing 6.5% of sodium chloride broth.
Where as the Aerococcus species such as the A. viridians and Aerococcus Urinae has also has the capability to grow in the medium containing 6.5% of sodium chloride, hence this salt tolerance broth has the capability to differentiate the different species of Aerococcus from the other similar organisms such as Stomatococcus and Helcoccus possessing the same characteristics.
Salt Tolerance Test Limitation
It is often insisted that the biochemical immunological, molecular or mass spectrometry test is performed on the colonies from pure culture for complete identification of the test.
Some strains of pseudococcus, Leuconostoc and Beta-hemolytic streptococcus species grows in the Salt tolerance Broth.
Infusion broth having 6.5% Sodium chloride produces slow reactions in the medium which results in making the interpretations difficult.
Usually, a light inoculum must be used while inoculating a broth. • If too heavy inoculum is used it produces turbidity and results in a false positive result.
Salt Tolerance Test Citations
- THE SALT-TOLERANCE TEST. Triangle . 1964 Oct;7:268-72.
- FURTHER STUDIES WITH THE SALT TOLERANCE TEST IN NORMAL INDIVIDUALS AND IN PATIENTS WITH ADRENAL CORTICAL HYPERFUNCTION. J Clin Invest . 1949 Sep;28(5 Pt 2):1091-3.
- Taxonomic study of a salt tolerant Streptomyces sp. strain C-2012 and the effect of salt and ectoine on lon expression level. Microbiol Res . Feb-Mar 2014;169(2-3):232-8.
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Biuret Test for Protein: Principle, Procedure, and...
Continue ReadingBiuret Test Introduction
Many of a biochemical test are used to detect the ability of the microorganism to utilize the enzymes and the proteins in the medium. Biuret test is one such test, which is used to detect the proteins in a compound.
What is Biuret Test?
Protein are one of the complex molecules which are formed by millions of amino acids.
Amino acids are known as amphoteric electrolytes as they have carboxyl and the amino groups and it acts like an acid and base, it has one positive charge and one negative charge and these ions are considered as electrically neutral and they do not migrate in the electrical field.
The two amino acids are linked together with the help of a peptide bond which results in the formation of a dipeptide and this process is often called as condensation reaction.
However, the amino acids are linked together with the help of three peptide bonds and they are known as tripeptide and as well as the chain elongates, it is also called as polypeptide.
Biuret is one of the compounds which is formed by heating urea at 180ºC, which results in condensation with two molecules of urea.
The peptide bond in the Biuret usually gives a positive result in the tests.
Biuret test is considered as one of the generally performed tests for the compounds such as proteins, which have their two or more peptide bonds.
Biuret Test Objective
The main aim of the test is to detect the protein in the given sample or a solution.
To detect the presence of the peptide pond.
Biuret Test Principle
As mentioned above, biuret test is one of the biochemical tests, which is used to detect the presence of a peptide bond in the compounds or substances given.
This test is purely based on the structure of peptide which consists of about two peptide linkages and results in producing a violet or purple color when it is treated along with the copper sulfate.
During the presence of an alkaline solution, the blue colored copper II ion, forms a complex with the peptide bonds.
These peptide bonds does not share their pair of electrons with the nitrogen and oxygen that is present in the water.
The colored coordination complexes is usually formed between the ion carbonyl oxygen and the amide nitrogen (=NH) of the peptide bond.
On formation, of this complex the solution changes its color blue to purple. When the purple color changes deeper, in the number of peptide copper complexes.
This compound containing at least two H2N-C, H2N-CH2-, H2N-CS- or similar groups they are joined together either directly or with the help of a carbon or a nitrogen atom.
One copper ion is appropriately linked to about 6 peptide bonds present in the molecule of protein which reacts and also the number of a protein molecules present in reaction system.
The Biuret reagent is a solution composed of Sodium hydroxide or potassium hydroxide along with hydrated copper II sulfate, and potassium sodium tartrate.
Sodium hydroxide and potassium hydroxide thus provides the alkaline medium and the potassium sodium tartrate is added to chelate and to stabilize the cupric ions in the solution or in order to maintain their solubility in alkaline solution.
Biuret Test Reagents
5% of egg white (albumin)
Biuret reagent
Water bath
Pipettes
Dry test tubes
Biuret Reagents:
Copper sulfate
Sodium hydroxide
Sodium potassium tartarate (Rochelle Salt)
Preparation of Biuret Reagents
This reagent is prepared by adding sodium hydroxide and copper sulfate solution, making it alkaline.
To prepare 1000 ml of a Biuret reagent, about 1.5 gram of a pentavalent copper sulphate and about 6 gram of sodium potassium tartarate and dissolve in a 500ml of distilled water.
Sodium potassium acts as a chelating agent and it also helps stabilize the copper ion.
Further 375 ml of a two molar sodium hydroxide and it is mixed both the solution in volumetric flask and make it final volume to 1000 ml by adding water.
Biuret Test Procedure
About 1ml of test solutions are taken in a dry test tubes and in another tube take 1 ml distilled water is added.
To this about 1ml of biuret reagent to all the test tubes and it is mixed well.
Then the test tube is observed for the developed for blue colors.
Then there will be change in color to the purple.
Hence, it is concluded that there is a presence of peptides and proteins and it resulted in positive tests.
It is also to be noted that in a biuret test of protein, Histidine is the only amino acid to give a positive result.
Biuret Test Result
Observation Interpretation If there is no color change and the solution remains blue Absence of proteins or peptides Here the test is detected as negative The solution changes into deep purple It determines the presence of proteins. Here the test is detected as positive. Biuret Test Uses
This test is generally used to detect the amount of protein present in the urine.
Biuret reaction with the protein is applied to determine quantitative analysis of the total protein by using the spectrophotometric analysis.
Biuret Test Citations
- The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. J Hosp Infect . 2006 Nov;64(3):288-92.
- Comparison of tests for failure of passive transfer in neonatal calf serum using total protein refractometry and the biuret method. Prev Vet Med . 2021 Apr;189:105290.
- Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens. J Am Vet Med Assoc . 2016 Apr 1;248(7):789-94.
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