NEET MDS Lessons
General Microbiology
PHAGOCYTOSIS AND INTRACELLULAR KILLING
A. Phagocytic cells
1. Neutrophiles/Polymorphonuclear cells
PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells.
The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.
2. Monocytes/Macrophages
Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker.
B. Response of phagocytes to infection
Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an infection. SOS signals include N-formyl-methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue.
Some of the SOS signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium.
Vasodilators produced at the site of infection cause the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing” between the endothelial cells in a process called diapedesis.
Once in the tissue spaces some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in increased phagocytosis and intracellular killing of the invading organisms.
C. Initiation of Phagocytosis
Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:
1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).
2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst.
3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.
4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.
D. Phagocytosis
The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.
E. Respiratory burst and intracellular killing
During phagocytosis there is an increase in glucose and oxygen consumption which is referred to as the respiratory burst. The consequence of the respiratory burst is that a number of oxygen-containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.
1. Oxygen-dependent myeloperoxidase-independent intracellular killing
During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).
2. Oxygen-dependent myeloperoxidase-dependent intracellular killing
As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).
3. Detoxification reactions
PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase.
4. Oxygen-independent intracellular killing
In addition to the oxygen-dependent mechanisms of killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent killing pathways are able to kill bacteria. However, since the oxygen-dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections.
Types of microscopy used in bacteriology
Light microscopy
Phase contrast microscopy
Fluorescence microscopy
Darkfield microscopy
Transmission electron microscopy
Scanning electron microscopy
Fluorescent microscopy in which ultraviolet rays are used to examine cells after treatment with fluorescent days.
Phase contrast microscope enhances the refractive index differences of the cell components. This microscopy can be used to reveal details of the internal structures as well as capsules, endospores and motility
Electron microscope The resolving power is more than 200 times that of light microscope.
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
Complement Fixation Test (CFT)
This test is based upon two properties of the complement viz:
a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.
Test system
It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.
Indicator system
To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.
BACTERIAL GROWTH
The conversion of a parental cell into two daughters constitutes the bacterial life cycle and the time taken to complete cell cycle is known as generation_time. This is around 15 minutes in vegetative bacteria except mycobacteria.
Bacterial Growth Curve
In the presence of fresh growth medium a bacterium shows following four phases;
The Lag phase -> The Log phase -> The Stationary phase -> The Decline phase
The Lag Phase : short duration , bacteria adapt themselves to new environment
The Log Phase (Exponential Phase) : Regular growth of bacteria occurs The morphology of bacteria is best developed in this phase and organisms manifest typical biochemical characters.
- Most of the cidal Abx work best in this phase
• i.e. Ampicillin
- Best phase for staining bacterial cultures
Chemostat and turbidostat are examples of technique by which this phase can be prolonged.
Stationary Phase : balanced growth and cell division cannot be sustained. The total cell Count remains static till lysis supervenes, but the viable cell count quickly declines.
Decline Phase: death phase. Dyeing bacteria exceed the dividing bacterias.
ANTIGEN-ANTIBODY REACTIONS
I. NATURE OF ANTIGEN-ANTIBODY REACTIONS
A. Lock and Key Concept
The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a lock (i.e. the antibody).
B. Non-covalent Bonds
The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds.
C. Reversibility
Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.
II. AFFINITY AND AVIDITY
A. Affinity
Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody .
B. Avidity
Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.
III. SPECIFICITY AND CROSS REACTIVITY
A. Specificity
Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions.
B. Cross reactivity
Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen.