Neutralization Test

These are basically of two types:

•    Toxin neutralization
•    Virus neutralization

In toxin neutralization homologous anti-bodies prevent the biological effect of toxin as observed in vivo in experimental animals (e.g. detection of toxin of Clostridia and Corynebacterium diphthenae) or by in vitro method (e.g. Nagler’s method).

In virus neutralization test various methods are available by which identity of virus can be established as well as antibody against a virus can be estimated.

THE PLASMIDS

The extrachromosomal genetic elements, called as plasmids are autonomously replicating , cyclic ,double stranded DNA molecules which are distinct from the cellular chromosome 

Classification

Plasmids can be broadly classified as conjugative and nonconjugative. 

Conjugative plasmids are large and self-transmissible i.e. they have an apparatus through which they can mediate their own transfer to another cell after coming in contact with the same. Example:  RF and certain bacteriocinogen plasmids.

Nonconjugative plasmids are small in size and can be mobilised for transfer into another cell only through the help of a conjugative plasmid. To this group belong some ‘r’ determinants and few bacteriocinogenic plasmids. Plasmids can also be transferred without cell contact by the process of transfection.

Properties of plasmids

Double stranded DNA , Autonomously replicate in host cell, Plasmd specific, Free DNA is transferred b transfection

Significance of Plasmids :The spread of resistance to antibiotics is one such well known example. These also play an important  role in the geochemical  cycle by spreading genes for the degradation of complex organic compounds.
 

GENETIC VARIATION

Two methods are known for genetic variation in bacteria: mutation and gene transfer.

Mutation : Any change in the sequence of bases of DNA, irrespective of detectable changes in the cell phenotype. Mutations may be spontaneous or induced by various agents which are known as mutagens. 

Spontaneous Mutations: Arise from enzymatic imperfections during DNA replications or with transient insertions of transposable elements.

Induced Mutations: Mutation by physical and chemical mutagens.

Physical mutagens  ultraviolet rays and high-energy ionizing radiations. The primary effect of UV rays on DNA is the production of pyrmidine dimers whereas ionizing radiations cause single_stranded breaks the DNA molecules.

Chemical mutagens :Affecting nucleotide sequence

(i) Agents which cause error in base pairing (e.g. nitrous acid and alkylating agents).
(ii) Agents which cause errors in DNA replication (e.g. acridine dyes such as acridine orange and profiavine).
(iii) Base analogs which are incorporated into DNA and cause replication errors (e.g. 5-bromouracil)

Gene Transfer

Transformation: Uptake of naked DNA

Transduction    : Infection by a nonlethal bacteriophage

Conjugation    : Mating between cells in contact

Protoplast fusion

Transformation: Gene transfer by soluble DNA is called as transformation. it requires that DNA be absorbed by the cell, gain entrance to the cytoplasm and undergo recombination with the host genome. 

Artificial Transformation(transfection) :Some of the bacteria (such as Escherichia coli) resist transformation until they are subjected to some special treatment such as CaCl2 to make the bacterium more permeable to DNA. Such modified cells can also take up intact double stranded DNA extracted from viruses or in the shape of plasmids. Though the process is same as transformation, it is 9 as transfection because it results in infection by an abnormal route

Transduction :The type of gene transfer in which the DNA of one bacterial cell is introduced into another bacterial cell by viral infection is known as transduction. This introduces only a small fragment of DNA. Because the DNA is protected from damage by the surrounding phage coat, transduction is an easier to perform and more reproducible process than transduction. ,

Two types of transduction are known.

- Generalized transduction When a bacteriophage picks up fragments of host DNA at random and can transfer any genes

-  Specialised transduction: phage DNA that has been integrated into the host chromosome is excised along with a few adjacent genes, which the phage can then transfer.

After entry into the host cell, the phage DNA gets incorporated into the host chromosome in such a way that the two genomes are linearly contiguous (lysogeny). The phage genome in this stage is known as prophage, The host cell acquires a significant new property as a consequence of lysogeny because it becomes immune to infection by homologous phage. This is hence called as lysogenic conversion and endow toxigenicity to Corynebacterium diphtheriae

Abortive Transduction :phage DNA fails to integrated into the host chromosome, the process is called as abortive transduction The phage DNA does not replicate and along with binary fission Of the host it goes into one of the daughter cells.

Conjugation :This is defined as the transfer of DNA directly from on bacterial. .cell to another by a mechanism that requires cell-to-cell contact. 

The capacity to donate DNA depends upon the possession of the fertility (F) factor. The F pili  also retard male-male union. Concomitant with effective male-female pair formation, the circular DNA bearing the F factor is converted to a linear form that is transferred to the female cell in a sequential manner. DNA replication occurs in the male cell and the newly synthesized, semiconserved DNA molecule remains in the male. This ensures postmating characters of the male.

Conjugation in Different Bacteria: Unusual form of plasmid transfer, called phase mediated conjugation has  been reported to occur with some strains of Staphylococcus aureus.

Protoplast Fusion: Also called as genetic transfusion. Under osmotically buffered Conditions protoplast fusion takes place by joining of cell membrane and generation of cytoplasmic bridges through which genetic material can be exchanged.

Transposons: Transposons  Tn  are  DNA sequences which are incapable of autonomous existence and which transpose blocks of genetic material back and forth between cell Chromosome and smaller replicons such as plasmids. insertion sequences (IS ) are another similar group of nucleotides which can move from one chromosome to another

Genetic material. IS and  Tn are collectively also known as transposable elements or Jumping genes. These are now recognised to play an important role in bringing about vanous types of mutations.

STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES

A.  IgG

1. Structure

 All IgG’s are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.

2. Properties

IgG is the most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.

a) IgG is the major Ig in serum – 75% of serum Ig is IgG

b) IgG is the major Ig in extra vascular spaces

c) Placental transfer – IgG is the only class of Ig that crosses the placenta. Transfer is mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.

d) Fixes complement – Not all subclasses fix equally well; IgG4 does not fix complement

e) Binding to cells – Macrophages, monocytes and neutrophils and some lymphocytes have Fc receptors for the Fc region of IgG.  A consequence of binding to the Fc receptors on such cells  is that the cells can now internalize the antigen better. The antibody prepares the antigen for killing by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. (Coating of the surface of pathogen by antibody is called opsonization).IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.

IgM

1. Structure
 IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.

2. Properties

a) IgM is the third most common serum Ig.

b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.

c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.

d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.

e) IgM binds to some cells via Fc receptors.

f) B cell surface Ig 

Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane . Cell surface IgM functions as a receptor for antigen on B cells.

IgA

1. Structure

Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure 10. When IgA exits as a dimer, a J chain is associated with it.

When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions . The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.

2. Properties

a) IgA is the 2nd most common serum Ig.

b) IgA is the major class of Ig in secretions – tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.

c) Normally IgA does not fix complement, unless aggregated.

d) IgA can binding to some cells – PMN’s and some lymphocytes.

IgD

1. Structure

 IgD exists only as a monomer.

2. Properties

a) IgD is found in low levels in serum; its role in serum  is uncertain.

b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen.

c) IgD does not bind complement.

E. IgE

1. Structure

IgE exists as a monomer and has an extra domain in the constant region.

2. Properties

a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.

b) Involved in allergic reactions – As a consequence of its binding to basophils and mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.

c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.

d) IgE does not fix complement.

Method of Sterilization for common items

Autoclaving :  Animal cages, Sugar tubes, Lab. Coats, Cotton , Filters, Instruments Culture media, Rubber, Gloves , Stopper, Tubing, Slides,  Syringe and Wax needles , Test tubes, Enamel metal trays ,Wire baskets, Wood, Tongue depressor, Applicator, Endodontic instruments, Orthodontic pliers , Orthodontic kits, Saliva ejector, Handpieces Cavitron heads, Steel burs, Steel tumbler, Hand instruments    

Hot air oven

Beakers, Flasks, Petri dish, Slides, Syringes, Test tubes, Glycerine, Needles ,Oil, Paper Saliva ejector, Matrix Band

Ethylene oxide

Fabric, Bedding, Blanket, Clothing, Matteresses, Pillows, Disposable instruments , Instruments, Blades, Knives, Scalpels, Scissors ,Talcum powder, Books, Cups, plates , Plastics., Flask, Petridish, Tubes, Tubing, Rubber , catheters, Drains, Gloves ,Special items - Bronchoscope, Cystoscope, Heart lung machine

Glutaraldehyde

Orthodontic kits, Orthodontic pliers , Steel burrs, 3 in 1 syringe tips ,Cystoscope ,Endoscope

Filtration

Antibiotics, Serum, Vaccines