Saturday, 31 October 2020

How Meiosis Occurs in Germ Cells and Human Life Cycle

 Every living creature on earth begins as a single cell. That cell undergoes mitosis, dividing over and over again, with certain cells becoming specialized, until eventually. cell division begins with an existing cell. Instead, that first cell came about because of meiosis and fertilization. Let’s take a look at the details of meiosis,and how it differs from mitosis.

  First, understand that meiosis is another type of cell division, but it doesn’t produce two identical cells like mitosis does. Human cells have 46 Chromosomes, which are two of each of 23 types. Each pair of chromosomes of a particular type are called homologous, meaning they carry genes controlling the same characteristics. After mitosis occurs in somatic cells, which are most of the cells in your body, you end up with two new cells with identical copies of those 46 chromosomes. These are called diploid cells, represented by 2n, meaning they contain two sets of chromosomes.

 With meiosis, when one cell divides, the product is four haploid cells. These have 23 chromosomes each, or just a single set, represented by n, and these haploid cells are reproductive cells called gametes. These are the cells that transmit genetic information from one generation of an organism to the next. In humans, these gametes take the form of sperm and egg cells.

  As we said, these are haploid cells with 23 chromosomes, and when fertilization takes place, the sperm and egg merge, and a new cell is formed, complete with the full 46 chromosomes, which are a combination of genetic material from the mother and father. So while mitosis, a kind of asexual reproduction, just produces identical copies of an original cell, meiosis and subsequent fertilization comprise sexual reproduction, which produces new cells with a novel set of genes. This is how we get variation from generation to generation.

  All humans have a combination of the genetic material from their parents, so no child will be 100% identical to either one of them.

Meiosis is similar to mitosis in a lot of ways, we will first note that the main difference between these processes is that meiosis consists of two cell divisions rather than one. These are called meiosis one and meiosis two. In meiosis one, homologous chromosomes, one maternal and one paternal, duplicate and are separated during cell division to produce haploid cells with duplicated chromosomes. Then in meiosis two, these haploid cells divide again to produce four haploid daughter cells, each with just a single set of chromosomes.

Meiosis (I) :
  Meiosis one consists of : prophase (I),
metaphase (I),
anaphase (I), and
telophase (I)
followed by cytokinesis. In prophase one, each chromosome, already duplicated, exchanges information with the homologous
recombination. which is a process called crossing over.

What is crossing over ?
  This begins when certain proteins break apart the DNA of two non-sister chromatids at exactly the same location. Then during synapsis, a complex of proteins called the synaptonemal complex holds the homologous together and each broken end of DNA is joined to the corresponding section of DNA from the other chromatid. in this process the two chromosomes have essentially swapped a tiny section of DNA with one another.

The areas on the chromosome where crossing over occurs are called chiasmata, and there will be at least one instance of this per chromosome, resulting in what we call recombinant chromosomes. This is just a fancy way of saying that they carry genes from what were originally two completely different chromosomes. In this case, they carry genes from both the maternal and paternal chromosome.

Then the nucleus comes apart just like in mitosis,


  In prophase (I) the mitotic spindle forms, attaching to kinetochores at each centromere.
  In metaphase (I), chromosomes line up at the metaphase plate, and in random fashion. The maternal and paternal chromosomes for each homologous pair can be in either order.
  In anaphase (I), the homologous  separate and are pulled towards the poles by the spindle. Notice that both chromatids of each chromosome are pulled to one side or the other together, rather than being pulled apart at the centromere,l ike in mitosis.
  In telophase (I) the nuclear membrane reforms, cytokinesis occurs, and we get two haploid daughter cells.

Difference between Mitosis and Meiosis (I)

  So meiosis one differs from mitosis in that the sister chromatids of an individual chromosome are not getting pulled apart, it is a pair of homologous chromosomes that are getting pulled away from each other after having
exchanged some genetic material. This is why the daughter cells are haploid,because each of them has only half the genome, with only one chromosome from each pair of homologous.

Meiosis (II)
  In meiosis (II), again we have prophase (II),
metaphase (II),
anaphase (II),
telophase (II) and cytokinesis.
This part looks just like mitosis.

  In prophase (II), the spindle apparatus forms.
  In metaphase (II), the chromosomes align at the metaphase plate, but unlike mitosis, the sister chromatids are not all genetically identical, because of the crossing over that occurred in prophase one. Again, the spindle attaches to kinetochore that
   In anaphase (II), the sister chromatids are pulled apart towards the poles.
Then telophase (II) and cytokinesis occur,where nuclei form, and cells are left with four haploid cells, each with 23 unduplicated chromosomes.

Each of these four daughter cells is different from the parent cell, and they are all different from each other. In fact, due to all the different possibilities present for the assortment and distribution of the chromosomes, each haploid daughter cell, or gamete, represents one unique outcome out of millions of possible outcomes. This is the secret to biological variation that sexual reproduction offers, which gives rise to the wide variety of phenotypes in living organisms.

So the human life cycle begins with haploid cells, in this case a sperm and an egg. These specialized cells are products of meiosis, and contain just one set of 23 chromosomes each. When these fuse during fertilization, the product is a single diploid cell with both sets of chromosomes, one from each of the parents. From here, it is mitosis that leads to the development of a human being, which will then exhibit characteristics from both parents.

Thursday, 29 October 2020

Mitosis: How One Cell Become Two

  Mitosis is a part of cell cycle, which mean the cells copy the genome and grow in preparation for cell division. The actual process of cell division is called mitosis, is happening all over your body right now, and it’s quite complex. Mitosis is divided into five phases. There’s the prophase, prometaphase, metaphase,anaphase, and telophase. At the completion of telophase, there is also cytokinesis. Once all this is finished, we end up with two identical cells, each with all the genetic information pertaining to that organism.
  Before mitosis begins, when the cell is still in the G2 phase of the cell cycle, we have two copies of all the chromosomes sitting in the nucleus, but they are loose and strewn about. In addition, the centrosome of the cell, which as we recall, contains two centrioles, has duplicated, so there are two pairs of centrioles

  Then, as mitosis begins, during the Prophase, the chromatin becomes tightly coiled, and forms the shape we are familiar with for chromosomes,with sister chromatids linked by a centromere. It is also in the prophase that something called the mitotic spindle begins to form. This is made up of the two centrosomes and a number of microtubules that begin to form between them. Each centrosome also has a radial array of microtubules surrounding it called an aster. As the cytoskeleton disassembles, the microtubules between the centrosomes grow and grow, which pushes them apart. 

  
  Then, in the prometaphase, the nucleus breaks apart and the growing microtubules cover the area where the nucleus used to be, so that they can attach to special proteins called kinetochores, which have assembled on the chromosomes at their centromeres. Things are starting to get organized as a kind of tug of war plays out. 

  
 Then in the Metaphase, the centrosomes have settled at the poles of the cell with the asters attaching to the plasma membrane, and all of the chromosomes have been arranged nicely along a plane in the middle of the cell. This imaginary plane is called the metaphase plate. At this stage, there is a checkpoint to ensure that each pair of sister chromatids is firmly attached to opposite ends of the mitotic spindle. Once all the kinetochores are attached to the spindle and everything is lined up nicely, a regulatory protein complex becomes activated,allowing the cell to pass through the M checkpoint, which means we are ready for the anaphase. 

  In Anaphase, the shortest of all the phases,the enzyme separase cleaves the cohesins that keep the sister chromatids together, and the kinetochores attached to the two sister chromatids pull the chromatids apart on each chromosome,thus generating the two separate sets of the genome. These chromosomes are then pulled by motorproteins that attach to the kinetochores, which reel them in by their centromeres to opposite ends of the cell, with the microtubules they are attached to coming apart as they go. The cell also elongates during this phase,until the two sets of chromosomes are far apart. 

  Then in the Telophase, two new nuclei form,rebuilt from the fragments of the original nucleus that came apart in the prometaphase. The chromosomes loosen up a little, the microtubules finish coming apart, and mitosis is complete, with two genetically identical nuclei. 

  To finish things up, cytokinesis will occur,which is where the cytoplasm, which has already begun dividing the cell into two smaller ones,will continue until the cells are distinct and separate. This starts with a cleavage furrow at the metaphase plate, caused by actin microfilaments that pull the cell inwards like a drawstring,which eventually pinches the cell in two.

   Your body is constantly producing new cells by mitosis, to make new skin, heal a wound, or when you grow rapidly in childhood. Every single somatic cell in your body was produced by mitosis, except the very first one. This first cell is an egg cell that has been fertilized by a sperm cell, and these reproductive cells, or gametes, are produced by a different process, it's called meosis

Thursday, 22 October 2020

The Composition and Function of Blood

 Of all the substances within the human body,blood is one of the more familiar ones, as we have all had an injury that involves bleeding. For a long time it was not well-understood exactly what blood is, or what it does, we just knew that if you lose enough of it, you die. But we now have an intimate understanding of this fluid, as well as the circulatory system whose function it is to continuously pump blood around the body. We will get to this system in a moment, first let’s examine blood itself, what it’s made of, and why it is so critical for human life.

First let’s mention that blood is technically considered a connective tissue, and as such it is the only fluid tissue in the body, full of fibrous proteins. It is comprised of formed elements, which are blood cells, suspended in a fluid called plasma.


If we place blood in a centrifuge, it will separate into its components. The densest section is comprised of erythrocytes,or red blood cells, and the least dense section will be the yellowish plasma. They are separated by the buffy coat, a white layer containing platelets as well as leukocytes, otherwise known as white blood cells.

As a whole, blood is responsible for distributing various substances around the body, most notably oxygen, which we can’t survive very long without. But it also carries nutrients absorbed from the digestive tract, and hormones secreted by endocrine organs. Blood also delivers waste products to the organs that will dispose of them, like the carbon dioxide that we exhale. Beyond this, blood serves to regulate pH in various tissues, maintain body temperature, and prevent infection.

Let’s discuss each component of blood
Beginning with plasma. This is a sticky fluid made mostly of water,but also containing a variety of proteins, nutrients, ions, gases and hormones. The most abundant plasma protein is called albumin, which contributes significantly to plasma’s osmotic pressure, and this is followed by a variety of globulins, which bind to certain molecules for transport. Moving to the formed elements, these are erythrocytes,or red blood cells, leukocytes, or white blood cells, and platelets.

Red blood cells and platelets are interesting in that they don’t possess all the typical organelles and they don’t divide, they are replaced by stem cells in the bone marrow. Red blood cells are very numerous in the bloodstream,and they are shaped like flattened discs with depressed centers. There is no nucleus, not much of anything in side other than lots of hemoglobin. This is the protein that allows for the transport of oxygen throughout the bloodstream, which is picked up in the lungs and then released for tissue cells throughout the body. There are other proteins as well that have structural or protective functions, but hemoglobin will be the focus here.

structure of hemoglobin,

it is made of a protein called globin, consisting of four polypeptides, two identical alpha chains and two identical beta chains, each of which is bound to a heme group with iron at the center. The iron in this heme is able to bind to an oxygen molecule in reversible fashion, so that it can bind and then release when necessary,so each hemoglobin can bind four oxygen molecules, and there are around 250 million hemoglobins per red blood cell, so one red blood cell can transport one billion oxygen molecules.

Blood cells are produced through a process called hematopoiesis, and this occurs in the bone marrow, which is a soft network of connective tissue found on certain blood capillaries, and which contains hematopoietic stem cells. For erythrocytes, this is more specifically called erythropoiesis, and billions of new red blood cells are made every day to maintain a nearly constant number, given that red blood cells function properly for only about three months(120 days), only to be destroyed by macrophages, which are a phagocytic type of white blood cell.

let’s look at leukocytes, or whiteblood cells
which unlike the far more abundant red blood cells, are complete cells with nuclei and organelles. These are part of the immune system, that these help us defend against pathogens and other harmful things. These use the circulatory system to get around the body, but they can also slip out into other connective tissues to do their work. There are two types of white blood cells,granulocytes and a granulocytes, which differ in the presence or absence of granules. The three types of granulocytes are neutrophils,which kill bacteria, eosinophils, which kill parasitic worms, and basophils, which contain histamine that attracts other white blood cells to a site of inflammation.
A granulocytes, on the other hand, can be lymphocytes,which fight viruses and tumors, and also give rise to plasma cells, which produce antibodies, or they can be monocytes, which become macrophages that can eat up intruders. Leukocytes are produced by leukopoiesis, which is stimulated by certain chemical messengers.


Lastly we get to the platelets. These are fragments of large cells called megakaryocytes. These fragments are essential during blood clotting, which happens when blood vessels are damaged, as platelets can plug up any holes or tears to seal things off. They flow through the bloodstream in an inactive state unless needed, dying every ten days or so and constantly regenerated. Megakaryocytes form due to repeated mitotic cycles that do not perform cytokinesis, so the result is one huge cell with a multi lobed nucleus. This then presses against a sinusoid, and its extensions burst to release the platelets. These platelets are important during hemostasis,which is the process by which the body will stop bleeding through vascular spasm, platelet plug formation, and then coagulation, or blood clotting.
   This essentially means that where there is damage to a vessel, smooth muscle will contract, platelets will plug the tear, and a protein called fibrin will form a mesh to patch everything up. Once the vessel has healed, the clot is removed through a process called fibrinolysis, so that there is no blockage in the vessel. So those are the components of blood.

We can also briefly mention the different blood types that humans can exhibit. These are A, B, AB, and O. These have to do with glycoproteins and glycolipids found in the plasma membranes of red blood cells. A and B refer to two different agglutinogens that can be found in these membranes, so blood group A has one of them, B has the other,AB has both, and O has neither. This is important for blood transfusions,because if someone’s body only recognizes A and gets blood with B, the new blood cells will be recognized by antibodies as foreign and destroyed, which can be fatal, so someone with AB blood can receive any blood, since both A and B will be recognized, hence they are universal recipients, and someone with O blood can give blood to anyone, since there are no markers to be recognized, hence they are universal donors.


There are also Rh blood groups which refer to agglutinogens called Rh factors, and for these a person is either positive or negative. This is reported along with the ABO blood group by tacking on positive or negative to the end, giving us groups like O positive,A negative, and so on. And with that, we are familiar enough with the structure and function of blood that we can begin to examine the circulatory system as a whole.

Sunday, 18 October 2020

Protein Polymerisation, Structure and Stability


 Protein is a monomers of amino acids. So we know about amino acids, and these are the monomers that will form proteins, which are also known as polypeptides. Proteins are polymers of amino acids, and they are the most diverse type of biomolecule in your body. 

  Different kinds of proteins include enzymes that catalyze chemical reactions, receptors that control signaling in your body, hemoglobin, which carries oxygen throughout the bloodstream, muscle and organ tissue, which gives your body structure and mobility, and so many other things. 

So how do amino acids polymerize?

 This happens when amino acids form peptide bonds with one another, such as the peptide bond between two amino acids. Peptide bond formation is an example of a dehydration reaction because the two hydrogens and the oxygen are lost, and two hydrogens plus one oxygen equals a water molecule, so as a water molecule is lost these two amino acids come together to form a peptide bond, which results in an amide

 An amide is a functional group with a nitrogen atom next to a carbonyl and this is the functional group that will connect each amino acid during polymerization.

 If two amino acids combine we get a dipeptide. If between three and ten come together, we would call that an oligo peptide, since oligo means just a few. And if more than ten come together we will call that a polypeptide, since poly means many and proteins are large polypeptides of around three hundred to a thousand amino acids that are folded in such a way that they have some biological activity. When we look at any peptide, we must notice that there is an N-terminus, meaning the side of the chain that ends with the amino group, and a C-terminus, the side that ends with the carboxyl group. 


 Proteins are very large compared to simple molecules. They contain hundreds of amino acid residues and they have very specific shapes from which their function is derived

Structure of Protein :
 A polypeptide chain can fold up to form specific shape (conformation). This conformation is a 3D arrangement of atoms and is determined by the sequence of amino acids, Four levels of structures are observed in proteins. They are 
1. Primary structure, 
2. Secondary structure, 
3. Tertiary structure and 
4. Quaternary structure
1. Primary structure 
    A linear sequence of amino acids, joined by peptide bond represents primary structure. Hence, the proteins with primary structure are under influence of only one type of chemical bond i.e. covalent bond.

2. Secondary structure 
    The protein molecules, under influence of two types of chemical sources form secondary structure. These are the forces of covalent bonding (represented by peptide bond) and force of hydrogen bonding. Partially charged secondary groups of amino acids are responsible for the formation of hydrogen bonds. 
 The secondary structure represents regular folding of regions of poly peptide chains. The two most common types of protein folds are α-helix and β-sheet structures 

α-helix
 These structures are found in fibrous, linear or rod shaped proteins, where the peptide chain shows a regular helical conformation. The structure is formed by hydrogen bonding between carbonyl oxygen of each peptide bond and amino group of fourth amino acid away. The hydrogen bonds run nearly parallel to the axis of helix. The side chains of amino acids are positioned along the outside of the cylindrical helix. e.g. myoglobin. 

β-sheet
  In this case, hydrogen bonds are formed between the peptide bonds in different peptide chains are in different regions of the same peptide chain. These bonds form sheet or plate like structures. The polypeptide chains, oriented in these structures may be parallel or anti parallel. Multiple ?- pleated sheets provide strength and rigidity to structural proteins. 

3. Tertiary structure 
    Protein molecules, under influence of additional chemical forces, other than that of covalent boniding and hydrogen bonding show tertiary structure. Proteins with tertiary structure have a three dimensional structure. Proteins with tertiary structure exhibit folding of their polypeptide chain in aqueous medium (water), where hydrophobic non polar groups of the amino acids are buried interior and hydrophilic polar group remain on the surface. The chemical forces involved in such folding of protein molecule include electrostatic forces, hydrogen bonding and disulfide bonds. e.g. globular protein of myoglobin in water.

4. Quaternary structure
    Proteins having more than one polypeptide chains exhibit fourth level of protein structure, called quaternary structure. These proteins have two or more polypeptide subunits joined by covalent links (disulfide bonding) or non-covalent forces such as ionic forces, hydrogen bonding or hydrophobic interactions.


Protein stability :
  The native three dimensional conformation of proteins maintained by influence of different types of covalent and non-covalent bonds. They include electrostatic forces, hydrogen bonding, hydrophobic forces, disulfide covalent bonds.

Electrostatic forces 
  These include interactions between two ionic groups of opposite charge such as that between positive charge of ammonium ion of lysine and negative charge of carboxyl ion of aspartic acid. 

Hydrogen bonding
  These bonds involve electrostatic attractions between weakly acidic donor group and acceptor group, that has alone pair of electrons.

Hydrophobic forces
  Non polar groups are hydrophobic and attempt to remain away from water or to minimize their contact with water. These forces are hydrophobic forces. 

Disulphite bonds
  These are covalent bonds that form between cysteine residues and are formed under oxidizing conditions.these bonds are mainly use to making high heat stable proteins

Tuesday, 13 October 2020

Structure of Nucleic acids DNA & RNA

Nucleic acids DNA and RNA structure


  Nucleic acids are polymers of nucleotides.They were first reported from nucleus of pus cells by Friederich Miescher in 1868 as nuclein.

Basic chemistry of nucleic :
acids Nucleotides are the building blocks of nucleic acids, which are linked by phosphodiester bonds.  Nucleotides are made of nitrogen bases, pentose sugar and phosphoric acid,


a). Nitrogen bases
   The bases of nucleic acids are ring structures of carbon that contain nitrogen atom within.  There are two types of bases found in nucleic acids. 
    l).  Purines
   ll).  Pyrimidine

Purine :
  Purines are fused ring structures having one six member and another five member ring.  Depending on the type and position of side chain attached, they are of two different types;  adenine and guanine. 

Pyrimidines :
Pyrimidines consist of a single six member ring structure.  Three types of pyrimidines are found: uracil, cytosine and thymine.  They differ in the type and position of side chains attached to the ring or these, DNA contains thymine and cytosine, while RNA contains uracil cytosine. there is a difference between thymine and uracil is 2' carbon methyl group which present on thymine insted of hydrogen in uracil.


b). Pentose Sugar


The pentose sugar found in nucleic acid can be either ribose or deoxyribose. Ribose is associated with RNA while deaxyribose is found in DNA.
Phosphoric acid Nucleotides contain a phosphate group attached to pentose.


c). Nucleoside : When a nitrogen base is attached to a pentose sugar, the compound is called nucleoside. The nitrogen base attaches to the pentose sugar at its first carbon by N- glycosidic bond.

There are eight types of nucleosides depending on the type of nitrogen base and pentose sugar associated. Deoxynucleosides: Deoxyadenosine, Deoxyguanosine, Deoxycytidine, Deoxythyimidine Ribonucleosides: Adenosine, Guanosine, Cytidine, Thmidine


d). Nucleotide : When a phosphate group is bound to a nucleoside by phospoester bond, the complex is called nucleotide. The phosphate group is bound to nucleoside at fifth carbon of pentose sugar.

Based on the type of sugar and nitrogen base involved, there are eight types of nucleotides, which could be classifled into two major categories: Deoxyribonucleotides and Ribonucleotides.
  Depending on the number of phosphate groups present, the nucleotides can be designated as nucleoside monophosphate (NMP), nucleoside diphosphate (NDP). nucleoside triphosphate (NTP). The nucleotides containing deoxyribose sugar are designated dNTP, dNDP, and dNMP. In nucleotides, the last two phosphate groups are linked by high energy phosphoester bonds.

Polynucleotide :
The nucleotides can be held together by phosphodiester linkage to form a polymer.The bond forms between the -OH group present on the 3rd carbon of pentose of one nucleotide and PO4, group present on fifth carbon of sugar of the other nucleotide. Hundreds to millions of nucleotides can be polymerized in a nucleic acid. Each polymer possesses two ends: 3' - OH end and 5' - PO4, end having a free OH group and phosphate group respectively.


e). Types of nucleic acids:
  There are two types of nucleic acids: DNA and RNA. DNA exists as the genetic material in all living organisms, except in certain viruses. RNA plays vital role in protein synthesis, by carrying information from DNA to cytoplasm. Also, in certain viruses,


DNA

Deoxyribonucleic acid is a polymer of deoxyribonucleotides. It is the genetic material, found in all living organisms, except certain viruses. It carries the blue print of the characters shown by organisms.
  The DNA possesses a characteristic base sequence that forms the basis of genetic information and diversity. The sequence of bases, responsible for the expression of a particular character is known as gene.

Chemistry of DNA :
  DNA contains four types of deoxyribonucleotides having adenine, cytosine, guanine and thymine. Studies of Chargaff on DNA chemistry have shown that 

a).  All DNA possess purine and
     pyrimidine in equal proportion  
     (1 : 1 ratio)
b). All DNA have A=T and G=C. c). DNA form different sources 
      have a characteristic AT/GC  
      ratio and AT GC.

Structure of DNA :
Watson and Crick (1953) worked out a three dimensional structure of DNA based on X-ray diffraction photographs. They proposed a double stranded helical structure of DNA.

Salient features of Watson and Crick model of DNA double helix

1).  The DNA molecule consists of two strands of polynucleotides held together by hydrogen bonds between them. 2).  The two strands run anti parallel at a distance of 20A. One strand runs from 5 → 3' direction, while the other strand runs from 3'→ 5' direction.
3).  The two strands are helically coiled around an imaginary axis, forming right handed helix.
4).  Each turn of the double helix is 34 A° long and accommodates ten base pairs. The distance between two successive base pairs in the DNA is 3.4 A°.
5).  The bases are arranged almost perpendicular to the central axis of the double helix. In the structure, the hydrophilic sugar phosphate groups form outer back bone. The hydrophobic nitrogen bases remain inside the structure.
6).  The double helix shows presence of two grooves: a major or deep groove and a minor or shallow groove.
7).  In the double helix, pairing takes place between adenine and thymine as well as guanine and cytosine.

Adenine pair with thymine by forming two hydrogen bonds between  them. Guanine and cytosine pair by forming three hydrogen bonds between them.

Acceptability of Watson and Cricks structure of DNA

1). Pairing is possible between purine and pyrimidine bases only to allow and accommodate hydrogen bonds between them. Two purines, being larger molecules cannot be accommodated in the double helix having diameter of 20 A°. Two pyrimidines, being smaller molecules, also cannot form hydrogen bonds, leaving a too large space between them. This explains, why in DNA purine - pyrimidine ratio is 1, according to Charggaff's rule.

2.) Structural complimentarily exists between adenine and thymine as well as guanine and cytosine allowing hydrogen bonds between them and pair. This also explains, why in DNA, A=T and G=C, according to Chargaff's rule.

3). The structure docs not put any restrictions on the sequence of bases in DNA. Therefore, in DNA it is not cssential to have AT = GC. Its ratio can be different in different DNA, as observed by Chargaff.
Further, this also explains, how a sequence of four bases can decide for diversity in characters.

4). The structure can also allow understanding how DNA replicates. Crick proposed that at the time of replication, the two strands of DNA could separate, and act as template, allowing complementary strand to be synthesized. As a result, two daughter molecules of DNA could be formed having identical sequence, as found in mother DNA molecule. This pattern of replication is called semi conservative replication.

RNA

  RNA is a polymer of ribonucleotides held together by phosphodiester bonds. The nucleotides of RNA contain adenine, guanine, cytosine and uracil.
A large variety of RNA forms are found in cell, having molecular weight ranging from just 25000 to several millions. Most RNAS are single stranded. But many times, folded RNA molecules having secondary and tertiary structures are also observed.

Types of RNA :
  There are threc major types of RNA.

1. Transfer RNA or tRNA
2. Messenger RNA or mRNA
3. Ribosomal RNA or rRNA

tRNA
These are the smallest types of RNA having 70 to 80 bases in the structure. They are named so because they transfer amino acid from cytoplasm to ribosome for protein synthesis. They comprise about 10- 20% of total RNAs in cell. There are at least 20 types tRNAs in the cell, one each for a specific amino acid. tRNAS are unique in composition as they contain abnormal or modified bases such as pseudo uridine, inosine and methyl nucleosides.
   

 tRNA have a secondary structure, and are clover leaf in shape having


1). Aminoacyl arm at which amino acid can bind.
2). D loop arm and loop, that contain abnormal base, dihydroxy uridine and possess binding site for amino acid activating enzyme that allows tRNA to carry amino acid.
3). Anticodon arm and loop, that contain anticodon, which has complementarily with codon and allow proper codon anticodon pairing between tRNA and mRNA during protein synthesis. 

4). T loop arm that contain abhormal base, pseudouracil in the sequence TΨC. It allows proper positioning of tRNA on ribosome during protein synthesis. 

5). Optional or pseudo arm having variable length with C. 3 to 21 bases.

mRNA
  The RNA that carries message for the type of protein to be synthesized is called mRNA or messenger RNA. The length of mRNA depends on the size of protein it codes for.
  mRNA possess a codon sequence, which decides for sequence of amino acids to be polymerized during protein synthesis. Codon is a sequence of three bases, triplet, specific for cach amino acid.

In addition, mRNA also possesses signal codons.
1. Initiation codon which provide signal for initiation for protein synthesis and
2. termination codon, which provide signal for termination of protein synthesis.
  Initiation codon is always first AUG codon on 5' end of mRNA. Termination codon is a nonsense codon, that does not code for any amino acid. They are UAA, UAG and UGA.

rRNA
  rRNAs associated with structure of ribosome are called ribosomal RNA or rRNA. They are structural RNAs, associated with ribosomal proteins. Different species of rRNA are found in cell as shown below in table 

Sunday, 11 October 2020

Culture Media - Definition, Principle, Preparation and Types and Microbiology

Culture media can be defined as the environment from which the organisms satisfy entire nutritional requirements for growth. They are used for the cultivation of microorganisms. 

Principles involved in construction of culture media -

Construction of a cultural medium requires consideration of following criteria.

1). The medium should consist of a balanced mixture of all the required nutrients at a concentration that permits good growth. This requires a thorough knowledge of nutritional requirements and habitat of the organism to be cultivated.

2). The medium should not possess nutrients in great excess. This is due to inhibitory effect of many nutrients at a higher concentration. e.g. fatty acids. certain metal lons etc.
  High concentration may also create unfavorable osmotic conditions for the organisms to be cultivated.

3). Due to the growth and metabolic activities of the organisms, unfavorable conditions gradually develop into the medium. This may be reflected as
a). increase in concentration of toxic metabolites,
b). depletion of availability of dissolved oxygen,
c). change in pH etc.
This results into the inhibition of growth. Hence, more supply of nutrients in excess is meaningless.

4). The microorganisms differ widely in their nutritional requirements. Hence, the medium should consist of those ingredients only. which can be attacked and utilized by the organisms to be cultivated.


Ingredients used for the preparation of bacteriological media -

A bacteriological medium should contain
1). Suitable sources of carbon, nitrogen, energy and electron donor. 
2). Mineral sources
3). Growth factors, if required
4). Water

Ingredients used as sources of carbon -

Various carbohydrate and non carbohydrate compounds are used as the sources of carbon in the medium. They include:  
a. Carbohydrates like
1. Monosaccharides such as glucose. fructose and others.
2. Disaccharides such as lactose, sucrose, maltose etc.
3. Polysaccharides such as starch, cellulose and others.

b. Noncarbohydrates like
1. Protein and protein hydrolysates
2. organic acids
3. hydrocarbons

Ingredients used as sources of nitrogen -

A wide variety of nitrogen compounds, both organic and Inorganic, can be used as sources of nitrogen in the medium construction.
Organic sources of nitrogen include proteins and protein hydrolysates such as peptone, peptides, amino acids, meat and meat extract, urea etc.
  Inorganic sources of nitrogen include nitrate, nitrite. ammonium salts, liquor ammonia etc.

Ingredients used as sources of energy and electron donor -

  Energy source is another important ingredient as nutrient, which is used for driving cellular metabolism. A wide variety of substances are used as sources of energy. Chemotrophs obtain energy from chemical which include both organic
and inorganic.
The substances incorporated into medium as sources of energy are as under.

a). Usually organic substances are used as sources for energy by chemoorganotrophs. Usually these organisms use same organic matter as source of both carbon and energy.

b). Usually inorganic substances are used as sources of energy by chemolithotrophs. These substances include reduced sulfur and iron compounds, ammonia or molecular hydrogen.

Organisms normally use the same chemical as electron donor, which is used as energy source. Upon oxidation of these compounds, they generate reducing power, required for biosynthesis.

Mineral sources -

Minerals are usually supplied as corresponding salts into the medium. Often, the Ingredients used as sources of carbon, nitrogen and energy as well as water used to prepare medium may contain some of the minerals needed by organisms.

Growth factors -

Growth factors may be added to the medium in pure form or materials containing them. Their addition supports growth of fastidious organisms. It also allows luxuriant growth of other organisms. Generally, substrates like meat and beef extract, yeast extract are used as source of growth factors.

Water -

All media ingredients are dissolved in water. The water used may be distilled water or tap water. Tap water is used, where media are used for commercial (fermentation) purposes.

Buffers -

  Apart from all these nutritional requirements, presence of suitable buffer is necessary in the medium. Buffer is the system that resists any change in pH of the medium. It is a mixture of weak acid and its conjugate base. Buffers have maximum buffering capacity at a pH. where concentration of the acid equals to Its conjugate base.

  When bacteria grow in the medium their metabolic activity affects the pH of the medium. This in turn affects the growth. Buffers, when incorporated into the medium, help in maintenance of optimum pH required for the growth. A variety of buffer systems can be incorporated in media. A few examples are listed below.
-COOH / -COO¯ e.g. acetate buffer
H2PO4¯/ HPO4²¯ e.g. phosphate buffer
NH3+ / -NH4 e.g. proteins

Of these, phosphate buffers are widely used In bacteriological media because they have Optimum buffering capacity at pH 6-8.
Proteins and peptones, used in the preparation of media as source of carbon, nitrogen and energy can also act as buffers.

Solidifying agents -

  Certain media are convenient to use if they are solid. The bacteriological media, therefore, can be solidified by dissolving a suitable solidifying agent into the medium. The solidifying agents used are agar, gelatin and silica gel.
- Agar is the most commonly used solidifying agent.
- Gelatin was the first solidifying agent employed in construction of solidified media. However, Its use became limited due to following reasons,
1). Property of gelatin to melt at temperature above 35°C.
2). Easy degradation by many microorganisms.
- Silica gel is inorganic substance and it is widely used for preparation of solidified media meant for cultivation of chemoautotrophs.


Some important ingredients used in construction of bacteriological media -

Following are the most widely used media ingredients, which are used for construction of media.

• Bacteriological peptone

  Peptone is the product obtained from hydrolysis of native proteins, such as gelatin, casein etc.
They are hydrolysed by acids or enzymes.
These digested proteins are easily attacked by most microorganisms.
They can serve as source of organic nitrogen. In addition, it can also satisfy the need of carbon, energy as well as growth factors for the organisms.

• Beef extract

It is the extract of beef and obtained as paste, when concentrated.
It contains water soluble substances of the animal tissues, organic nitrogenous compounds, some carbohydrates, vitamins and growth factors. It is supplied to the medium mainly as the source ofr growth factors.

• Yeast extract

  It is the extract obtained from yeast and available as powder. It is rich source of vitamins and used as source of growth factors in the medium.

• Agar

Agar is the most widely used solidifying agent in preparation of bacteriological media. It is obtained from cell walls of marine algae. It is hetero polysaccharide in nature. Following characteristics of agar make it most suitable as the solidifying agent.
1. Ability to melt at 92°C - 93°C and gets solidified at 42°C.
2. Resistance to degradation by microorganisms.
3. Non interfering with the growth of microorganisms.


Types of culture media :

The culture media can be classified into different types on the basis of various criteria:
A). Solid, solidified, semisolid and liquid media.
B). Natural, synthetic and complex media.
C). Routine and specialized media.

A. Solid, solidified semi solid and liquid media -

Based on physical status, the media can be classified as solid, solidified, semisolid or liquid media.

Solid media

  Solid media are prepared by mixing solid ingredients. Water is used to provide adequate moisture to the organisms to be cultivated.
  These media are commonly used in industry for the fermentative production of enzymes as well as preparation of inoculum.
e.g. use of moistened rice bran, dried bread powder, grass soaked in buffer etc.

Liquid or broth media

  A liquid medium, without any solidifying agent is called broth medium.
  They are prepared by dissolving nutrients into water. They are used mainly for
1. Cultivation of microorganisms.
2. Primary cultivation of microorganisms from natural sources.
3. Study of various biochemical and physiological characters of organisms etc.

Solidified media

  Solidified media are prepared by dissolving suitable solidifying agent Into the liquid media. Depending on the type of solidifying agent used, these media are commonly called as agar media, gelatin media or silica gel media. These media are commonly used for
1. Isolation and cultivation of microorganisms.
2. Preservation of microorganisms.
3. Enumeration of microorganisms by viable count. bloassay etc.

Semi solid media

  Semi solid midea are prepared by dissolving suitable solidifying agent into the liquid media. But, in this case, the concentration of solidifying agent is much less. AS a result, the gel strength of medium remains low. These media are widely used for study of motility of organisms.


B. Natural, synthetic and complex media

Based on the composition, the bacteriological media can be classifled as natural, synthetic and complex media,

Natural media

  These media are prepared from natural ingredients and extract, which can provide natural environment to the organisms aimed for isolation and cultivation.
  These media were first employed by Winogradsky to isolate organisms from soil. The type of natural extracts used depends on the type of organisms to be Isolated.
e.g. Soil extract for isolation of soil organisms. Potato extract for isolation of plant pathogens etc.
  These media are usually preferred for primary isolation of organisms from their natural habitats.

Synthetic media

  These media are commonly known as chemically defined media. These are the media where the exact chemical nature and composition of all the ingredients used in the preparation of media is known. These media are prepared by adding
- Known concentration of sugar or other source of carbon and energy, whose chemical composition is known.
- Nitrogen source, usually as ammonium or nitrate salt or Liquor ammonia.
- Salt solutions to provide mineral nutrients requirements.
- Amino acids and growth factors, If required, in known concentration.

These media are widely used to study
1. influence of various chemicals on the growth
2. bioassay etc.

Complex media

  The media, where, the exact chemical composition of all the Ingredients of the medium is not known, are called complex media.
  These media are widely used for growth and cultivation of microorganisms. These media are relatively easy to prepare, comparatively cheaper than synthetic media and allow better growth of organisms. Media containing peptone, beef extract etc. fall in this category.


C. Routine and specialized media 

  Based on the nature of use. the media can be classifled into two broad categories: routine and specialized.

Routine media

  The media, which are routinely used for cultivation of micro organisms are called routine media. They are designed in a manner that they permit growth of most of the types of organisms. Nutrient agar and nutrient broth media fall in this category because they are used widely in the laboratory.

Preparation of n-agar & n-broth
Composition of nutrient agar and nutrient broth

Specialized media

  The media which are used for specialized purposes fall in this category, Following are the diferent categories of such specialized types of media.

Enriched media -

  Certain mieroorganisms possess complex nutritional requirements. They grow poorly on the routine laboratory media. They need supply of specific growth factors. Therefore, special media are constructed which are supplemented with highly nutritious substances such as blood, serum, egg. yeast extract etc.
  Such media are nutritionally enriched. Hence, they are called enriched media. They favor luxuriant growth of microorganisma, Including fastidious organisms.
e.g. Blood agar medium, Chocolate agar medium, Glucose yeast extract agar medium, Egg media etc.

Enrichment media -

  These are the media which favor growth of destred type of organisms, without affecting normal growth of other organisms. These media are constructed by using those
growth of desired group of organisms.
e.g.
a) Incorporation of aromatie hydrocarbon in the medium as the sole source of carbon will favor growth of hydrocarbon degrading organisms.
b). Adjusting pH of the medium at a very low value, 2 to 3 will favor growth of acidophiles.

Usually, these media are used as liquid media and are used for improving probability of Isolation of desired type of organisms from natural populations.

Selective media -

  These are the media which allow growth of only selective group of microorganisms and Inhibit growth of other undesired types of organisms. These media are prepared by adding speciflc inhibitors into the medium, which inhibit particular class of organisms.
e.g.
a). Crystal violet or bile salt may be used to construct selective media for isolation of gram- negative bacteria. These agents are able to inhibit growth of gram-positive bacteria.
b). Streptomycin is used in preparation of rose bengal agar medium for inhibiting growth of bacteria and increase probability of isolation of fungi.

Selective media are normally used as agar media and are widely used for the isolation of desired types of microorganisms from a mixed population.

Differential media -

  The media, which allow differentiation of two groups of organtsms on the basis of their growth and cultural characteristics are called differential media.
  These media are prepared by, Incorporating suitable reagents or chemicals or Indicators Into the medium, which helps in differentiation of different groups of organisms.
e.g. Mac Conkey's agar medium. It is incorporated with pH Indicator dye Neutral red to allow differentiation of lactose fermentor and non fermentor colonies.

Assay media -

  The media which are used for the purpose of bioassay of antibiotics or vitamins and growth factors are called assay media. These media have a specifically prescribed composition.
  These media do not contain any substance that bind with substance to be assayed and interfere with it's estimation. Further these media do not contain even a trace of a substance to be assayed.

Enumeration media -

  Media, which are used for enumeration of bacteria from the specimen are called enumeration media. These media allow most types of bacteria to grow. Usually these media are used as agar media.

Maintenance media -

  Special media are used for the maintenance of culture. These media are designed in a manner that the properties of organisms are not affected and organisms grow slowly such that the intervals between two subculturing can be longer.

   Usually these media are solid. The use of solid /agar media allow diffusion of toxic metabolites away from the growing culture and protect them from their toxic effect.


Some differential and selective media :

A). Mac-Conckey agar -

   Mac-Conkey agar is a selective as well as a differential medium, used for selective Isolation of Enterobacteriaceae and related enteric gram-negative rods.
  The medium contains bile salt and/or crystal violet, which inhibit the growth of gram-positive bacteria and some fastidious gram- negative bacteria. Thus, the medium is a selective medium.
 
The medium also incorporates lactose as a sole fermentable carbohydrate and neutral red as pH indicator dye. The organisms, capable of fermenting lactose produce acid.
  Hence their colonies become acidic, which appear pink because of the conversion of neutral red (Neutral red is pink under acidic conditions).
  Colonies of lactose non-fermenting bacteria appear colorless. Thus, the medam ls also differential, allowing differentiation of bacteria as lactose fermentor and non-fermentor.

B). Eosin Methylene Blue (EMB) agar -

  EMB agar is also a selective and differential medium. useful for differentiation of Enterobacterlaceae and related coliforms. The medium incorporates aniline dyes; eosin and methylene blue, which inhibit gram-positive and fastidious gram-negative bacetria and hence it acts as selective medium.

  The medium also incorporates lactose as a sole fermentable carbohydrate and neutral red as pH Indicator dye. Organisms that ferment lactose and produce large amount of acid, allow precipitation of aniline dyes on their colonies.

Typical coliforms like E. coli produce colonies with greenish blue metallic sheen. Atypical coliforms like Enterobacter produce pink colonies but not metallic sheen.
  Enterobacter can convert acid in to a neutral compound, 2.3 butanediol and hence they do not produce sufficlent acidity to allow precipitation of anfline dyes. Pink colonies are due to presence of neutral red in medium.
  Lactose non fermentor organisms produce colorless colonies. Thus, the medilum also acts as a differential one.

C). Deozycholate citrate agar (DCA) -

  DCA agar is also selective and differential medium, used for isolation of members of Enterobacteriaceae.The medium contains
- About three times the concentration of bile salts than Mac-Conkeys agar medium. This permits selective isolation of Salmonella from mixed cultures contaminated with coliforms and other gram-positive bacteria.
- Sodium and ferrie citrate that retard growth of coliforms.
- Lactose and neutral red. Their presence in the medium helps in differentiation of lactose fermentors from lactose non-fermenting bacteria.

D). Endo agar -

  The medium is a differential one, used for differentiation of lactose fermentors and non-fermentors. Incorporation of sodium sulfite and basic fuchsin inhibits growth of gram- positive bacteria.
  Lactose fermentors produce pink colonies due to release of basic fuchsin from neutralized sodium sulfite-basic fuchsin complex. Acetaldehyde. produced as one of the intermediates during fermentative metabolism is trapped by sodium sulfite allowing basic fuchsin to be released from the complex, that impart pink color to the colonies.

E).Salmonella - Shigella (SS) agar -

  SS agar medium is highly selective for Isolation of Salmonella and Shigella from clinical specimens. Presence of high concentration of bile salts and sodium citrate Inhibits gram- positive and many gram-negative bacteria, Including coliforms.
   Presence of lactose and neutral red allows differentlation of organisms as lactose fermentor and non- fermentors.
  Similarly. presence of sodium thiosulfate and ferric citrate allows the detection of bacteria that produce H₂S. Organisms that use sodium thiosulfate and reduce it to produce H₂S develop black colonies due to formation of iron sulfide.