NEET MDS Lessons
General Microbiology
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.
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.
CHEMICAL AGENTS
Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also.
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.
Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine.
Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs.
Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin.
When used in sufficiently high concentration it destroys the bacteria and their spores.
Classification of chemical sterilizing agents
Chemical disinfectant
Interfere with membrane functions
• Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids
• Phenols : Phenol, cresol, Hexylresorcinol
• Organic solvent : Chloroform, Alcohol
Denatures proteins
• Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid
Destroy functional groups of proteins
• Heavy metals : Copper, silver , Mercury
• Oxidizing agents: Iodine, chlorine, Hydrogen peroxide
• Dyes : Acridine orange, Acriflavine
• Alkylating agents : Formaldehyde, Ethylene oxide
Applications and in-use dilution of chemical disinfectants
Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%
Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %
Silver nitrate : Antiseptic (eyes and burns) Dilution Used 1 %
Phenolic compound : Antiseptic skin washes Dilution Used .5 -5 %
Iodine : Disinfects inanimate object, Skin antiseptic Dilution used 2%
Chlorine compounds : Water treatment Disinfect inanimate objects , Dillution used 5 %
Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %
Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %
Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.
Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.
Evaluation of Disinfectants
Two methods which are widely employed are:
Phenol coefficient test, Kelsey -Sykes test
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
Immunofluorescence
This is precipitation or complement fixation tests. The technique can detect proteins at concentrations of around 1 µg protein per ml body fluid. Major disadvantage with this technique is frequent occurrence of nonspecific fluorescence in the tissues and other material.
The fluorescent dyes commonly used are fluorescein isothocyanate (FITC). These dyes exhibit fluorescence by absorbing UV light between 290 and 495 nm and emitting longer wavelength coloured light of 525 nm which gives shining appearance (fluorescence) to protein labelled with dye. Blue green (apple green) fluorescence is seen with FITC and orange red with rhodamine.
Enzyme Immunoassays
These are commonly called as enzyme linked immunosorbent assays or EL1SA. It is a simple and versatile technique which is as sensitive as radioimmunoassays. It is now the
technique for the detection of antigens, antibodies, hormones, toxins and viruses.
Identification of organisms by immunofluorescence
Type of agent Examples
Bacterial Neisseria gonorrhoeae, H. influenzae ,Strept pyogenes, Treponema pallidum
Viral Herpesvirus, Rabiesvirus, Epstein-Barr virus
Mycotic Candida albicans
Enzymatic activity results in a colour change which can be assessed visibly or quantified in a simple spectrophotometer.
Enzymes:
Serum lysozyme:
Provides innate & nonspecific immunity
Lysozyme is a hydrolytic enzyme capable of digesting bacterial cell walls containing peptidoglycan
• In the process of cell death, lysosomal NZs fxn mainly to aulolyse necrotic cells (NOT “mediate cell degradation”)
• Attacks bacterial cells by breaking the bond between NAG and NAM.
• Peptidoglycan – the rigid component of cell walls in most bacteria – not found in archaebacteria or eukaryotic cells
• Lysozyme is found in serum, tears, saliva, egg whites & phagocytic cells protecting the host nonspecifically from microorganisms
Superoxide dismutase: catalyzes the destruction of O2 free radicals protecting O2-metabolizing cells against harmful effects
Catalase:
- catalyzes the decomposition of H2O2 into H2O & O2
- Aerobic bacteria and facultative anaerobic w/ catalase are able to resist the effects of H2O2
- Anaerobic bacteria w/o catalase are sensitive to H2O2 (Peroxide), like Strep
- Anaerobic bacteria (obligate anaerobes) lack superoxide dismutase or catalase
- Staph makes catalase, where Strep does not have enough staff to make it
Coagulase
- Converts Fibronogen to fibrin
• Coagulase test is the prime criterion for classifying a bug as Staph aureus – from other Staph species
• Coagulase is important to the pathogenicity of S. aureus because it helps to establish the typical abscess lesion
• Coagulase also coats the surface w/ fibrin upon contact w/ blood, making it harder to phagocytize
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
NORMAL MICROBIAL FLORA
A. Properties. Normal microbial flora describes the population of microorganisms that usually reside in the body. The microbiological flora can be defined as either
1) Resident flora - A relatively fixed population that will repopulate if disturbed,
2) Transient flora - that are derived from the local environment. These microbes usually reside in the body without invasion and can
even prevent infection by more pathogenic organisms, a phenomenon known as bacterial interference.
The flora have commensal functions such as vitamin K synthesis. However, they may cause invasive disease in immunocompromised hosts or if displaced from their normal area.
B. Location. Microbial flora differ in composition depending on their anatomical locations and microenvironments. The distribution of normal microbial flora.