NEET MDS Lessons
General Microbiology
Immunoglobulin (Ig)
Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field
FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.
2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.
STRUCTURE OF IMMUNOGLOBULINS
The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.
A. Heavy and Light Chains
All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)
B. Disulfide bonds
1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.
2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.
C. Variable (V) and Constant (C) Regions
When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids) and CL (110 amino acids)
2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.
E. Domains
Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides
Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations.
IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin fragments produced by proteolytic digestion –
A. Fab
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.
Antigen binding – These fragments are called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL.
B. Fc
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.
Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment .
Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
Precipitation Reaction
This reaction takes place only when antigen is in soluble form. Such an antigen when
comes in contact with specific antibody in a suitable medium results into formation of an insoluble complex which precipitates. This precipitate usually settles down at the bottom of the tube. If it fails to sediment and remains suspended as floccules the reaction is known as flocculation. Precipitation also requires optimal concentration of NaCl, suitable temperature and appropriate pH.
Zone Phenomenon
Precipitation occurs most rapidly and abundantly when antigen and antibody are in optimal proportions or equivalent ratio. This is also known as zone of equivalence. When antibody is in great excess, lot of antibody remains uncombined. This is called zone of antibody excess or prozone. Similarly a zone of antigen excess occurs in which all antibody has combined with antigen and additional uncombined antigen is present.
Applications of Precipitation Reactions
Both qualitative determination as well as quantitative estimation of antigen and antibody can be performed with precipitation tests. Detection of antigens has been found to be more sensitive.
Agglutination
In agglutination reaction the antigen is a part of the surface of some particulate material such as erythrocyte, bacterium or an inorganic particle e.g. polystyrene latex which has been coated with antigen. Antibody added to a suspension of such particles combines with the surface antigen and links them together to form clearly visible aggregate which is called as agglutination.
Application of precipitation reactions
Precipitation reaction Example
Ring test Typing of streptococci, Typing of pneumococci
Slide test (flocculation) VDRL test
Tube test (flocculation) Kahn test
Immunodiffusion Eleks test
Immunoelectrophoresis Detection Of HBsAg, Cryptococcal antigen in CSF
Test for Antigen - Antibody Reactions
Antigens are those substance that stimulates the production of antibodies which, when enter into the body it reacts specifically in a manner that are clearly visible.
Some antigens may not induce antibody production, but instead creates immunological tolerance.
An antigen introduced into the body produces only specific antibodies and will react with only those specific antigens.
These antibodies appear in the serum and tissue fluids. All antibodies are considered as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE.
Antigen- antibody reactions are known as serological reactions and are used as serological diagnostic tests for the identification of infectious diseases.
The reaction occurs mainly in three stages;
1. The initial interaction between the antigen and antibody, which produces no visible effects. It is a reversible and rapid reaction.
2. The secondary stage leads to the demonstration proceedings, such as precipitation, agglutination, etc.
3. The tertiary reaction follows the neutralization or destruction of injurious antigens. These results in clinical allergy and other immunological diseases.
There are certain characteristics for antigen-antibody reactions;
1. Reaction is specific.
2. The whole molecules participate in the reaction, and not just a part of it.
3. No denaturation of antigen or antibody occurs during the reaction.
4. The combination usually occurs at the surface.
5. The combination is firm, but reversible
6. Agglutinins formed after agglutination usually are formed by both antigen and antibody together.
7. They can combine in varying proportions.
Measurement of antigen and antibody are made in terms of mass or as units or titre.
Serological reactions include;
1. Precipitation reaction
a soluble antigen combining with the specific antibody in the presence of electrolytes at a suitable temperature and pH forming insoluble precipitins. Commonly used tests are ring test, slide test, tube test, immunodiffusion, etc.
Radial Immunodiffusion
In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed .
This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.
Immunoelectrophoresis
In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs .
This tests is used for the qualitative analysis of complex mixtures of antigens
This test can also be used to evaluate purity of isolated serum proteins.
Countercurrent electrophoresis
In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line.
2. Agglutination reaction
when a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. When the antigen is an erythrocyte the term hemagglutination is used.
Applications of agglutination tests
i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
Passive hemagglutination
The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen . This is called passive hemagglutination.
The test is performed just like the agglutination test.
Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.
Coomb's Test (Antiglobulin Test)
DIRECT ANTIGLOBULIN TEST (DAT)
The DAT is used to detect IgG or C3 bound to the surface of the red cell. In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.
A positive DAT can occur without hemolysis
Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT.
INDIRECT ANTIGLOBULIN TEST (IAT)
The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination. If the antibody screen is positive, additional testing is required to determine the specificity of the antibody.
The IAT is used to detect red cell antibodies in patient serum. Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.
3. Complement fixation test (CFT)
the ability of antigen antibody complexes to fix complement is made use in this test. Complement is something which takes part in any immunological reaction and absorbed during the combining of antigen with its specific antibody.
The best example of CFT is the Wassermann reaction done for the detection of Syphilis.
4. Neutralization test
different types of these are available. Virus neutralization, toxin neutralization, etc. are some of its kind.
5. Opsonization
this makes use of the determination of opsonic index, which is the ratio of the phagocytic activity of patient’s blood to the phagocytic activity of the normal patient’s for a given bacterium.
6. Immunfluorescence
the method of labeling the antibodies with fluorescent dyes and using them for the detection of antigens in tissues.
7. Radioimmunoassay (RIA)
is a competitive binding radioisotopes and enzymes are used as labels to conjugate with antigens or antibodies.
8. Enzyme Immuno Assay (EIA)
the assays based on the measurement of enzyme labeled antigen or antibody. The most common example is ELISA used to detect HIV.
9. Immunoelectroblot
it uses the sensitivity of Enzyme immunoassay with a greater specificity. Example is Western blot done for the serodiagnosis of HIV infection.
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.
CROSS INFECTION AND STERLIZATION IN DENTISTRY
Cross infection is defined as the transmission of infectious agents amongst patients and staff with in hospital environment.
Routes of Infection
Two routes are important : transdermal and respiratory.
In transdermal route microorganisms enter the tissues of the recipient by means of injection through intact skin or mucosa (usually due to an accident involving a sharp instrument) or via defects in the skin e.g. recent cuts and abrasions.
Microorganisms causing cross infection in dentistry
Transmitted through skin
Bacteria : Treponema pallidum, Staphylococcus aureus
Viruses :Hepatitis virus, HIV ,Herpes simplex virus, Mumps, Measles , Epstein-Barr virus
Fungi: Dermatomycoses, Candidiasis,
Transmitted through aerosols
Bordetella pertussis, Myco.tuberculosis, Streptococcus pyogenes, Influenza virus
Rhinovirus, Rubella
PHYSICAL AGENTS
Heat occupies the most important place as a physical agent.
Moist Heat : This is heating in the presence of water and can be employed in the following ways:
Temperature below 100°C: This includes holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and in its flash modification where in objects are subjected to a temperature of 71.1°C for 15 seconds. This method does not destroy spores.
Temperatures Around 100°C : Tyndallization is an example of this methodology in which steaming of the object is done for 30 minutes on each of three consecutive days. Spores which survive the heating process would germinate before the next thermal exposure and would then be killed.
Temperatures Above 100°C : Dry saturated steam acts as an excellent agent for sterilization. Autoclaves have been designed on the principles of moist heat.
Time-temperature relationship in heat sterilization
Moist heat (autoclaving)
121°C 15 minutes
126°C 10 minutes
134 C 3 minutes
Dry heat
>160°C >120 minutes
>170°C >60minutes
>180°C >30 minutes
Mechanism of microbial inactivation
The autoclaving is in use for the sterilization of many ophthalmic and parentral products. surgical dressings, rubber gloves, bacteriological media as well a of lab and hospital reusable goods.
Dry Heat: Less efficient, bacterial spores are most resistant. Spores may require a temperature of 140° C for three hours to get killed.
Dry heat sterilization is usually carried out by flaming as is done in microbiology laboratory to sterilize the inoculating loop and in hot air ovens in which a number of time-temperature combinations can be used. It is essential that hot air should circulate between the objects to be sterilized. Microbial inactivation by dry heat is primarily an oxidation process.
Dry heat is employed for sterilization of glassware glass syringes, oils and oily injections as well as metal instruments. -
Indicators of Sterilization:
These determine the efficacy of heat sterilization and can be in the form of spores of Bacillus stearothermophilus (killed at 121C in 12 minutes) or in the form of chemical indicators, autoclave tapes and thermocouples.
Ionizing Radiations
Ionizing radiations include X-rays, gamma rays and beta rays, and these induce defects in the microbial DNA synthesis is inhibited resulting in cell death. Spores are more resistant to ionizing radiations than nonsporulating bacteria.
The ionizing radiations are used for the sterilization of single use disposable medical items.
Mechanism of microbial inactivation by moist heat
Bacterial spores
• Denaturation of spore_epzymes
• Impairment of germination
• Damage to cell membrane
• Increased sensitivity to inhibitory agents
• Structural damage
• Damage to chromosome
Nonsporulating bacteria
• Damage to cytoplasmic membrane
• Breakdown of RNA
• Coagulation of proteins
• Damage to bacterial chromosome
Ultraviolet Radiations :
wave length 240-280 nm have been found to be most efficient in sterilizing. Bacterial spores are more resistant to U.V. rays than the vegetative forms. Even viruses are sometimes more resistant than vegetative bacteria.
Mechanism of Action :
Exposure to UV rays results in the formation of purine and pyrimidine diamers between adjacent molecules in the same strand of DNA. This results into noncoding lesions in DNA and bacterial death.
Used to disinfect drinking water, obtaining pyrogen free water, air disinfection (especially in safety laboratories, hospitals, operation theatres) and in places where dangerous microorganisms are being handled.
Filteration
Type of Filters
Various types of filters that are available are /
Unglazed ceramic filter (Chamberland and Doulton filters)
Asbestos filters (Seitz, Carlson and Sterimat filters)
Sintered glass filters
Membrane filters
Membrane filters are widely used now a days. Made up of cellulose ester and are most suitable for preparing_sterile solutions. The range of pore size in which these are available is 0.05-12 µm whereas the required pore size for sterlization is in range of 0.2-0.22 p.m.