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MICROBIOLOGY QUICK REVISION NOTES GENERAL MICROBIOLOGY Historical Introduction Antony van Leeuwenhoek Father of microscopy / Father of Microbiology a. Observe bacteria using single-lens microscope....

MICROBIOLOGY QUICK REVISION NOTES GENERAL MICROBIOLOGY Historical Introduction Antony van Leeuwenhoek Father of microscopy / Father of Microbiology a. Observe bacteria using single-lens microscope. Q b. Named - small organisms as ‘Little animalcules’ Paul Ehrlich Father of Chemotherapy a. Acid-fast nature of tubercle bacillus. b. Side chain theory of antibody production. c. Chemotherapy- Discovered salvarsan (‘magic bullet’) - first effective medicinal treatment for syphilis. Louis Pasteur Father of Modern Microbiology a. Principles of fermentation b. Sterilization techniques. c. Pasteurization of milk. Q d. Coined the term ‘Vaccine’ e. Vaccine development against - Anthrax, Cholera Robert Koch and Rabies. f. Disproved theory of spontaneous generation Father of Bacteriology and postulated ‘germ theory of disease’ a. Staining techniques using aniline dye. b. Isolation of pure strains of bacteria. c. Discovered the anthrax bacillus, tubercle bacillus and the cholera vibrios d. Koch’s postulates e. Koch’s phenomenon Joseph Lister Father of Antiseptic surgery a. Antiseptic techniques (diluted carbolic acid) in Q surgery 204 Cerebellum Quick Revision Notes Koch’s postulates: Father of Virology  WM Stanley Father of Mycology  Raymond Sabouraud Father of Immunology  Edward Jenner Other Important Contributors Scientist Contributions Hans Christian Developed - ‘Gram stain’ Gram Founder of electron Ernst Ruska microscope Alexander Fleming Discovered penicillin Described - viral culture Goodpasture technique in chick embryo Described - mobile genetic Barbara McClintock elements in bacteria - transposons Walter Gilbert and DNA sequencing Frederick Sanger Discovered PCR and was Karry B Mullis awarded noble prize Bacteria named after the discoverers Common name Scientific name Kleb-Loeffler Corynebacterium diphtheriae bacillus Preisz Nocard Corynebacterium bacillus pseudotuberculosis Koch Week Haemophilus aegyptius bacillus Pfeiffer’s Haemophilus influenzae bacillus Whitmore Burkholderia pseudomallei bacillus Battey bacillus Mycobacterium intracellulare Johne’s bacillus Mycobacterium paratuberculosis Micro-organism not meeting the Eaton’s agent Mycoplasma pneumoniae criteria of Koch’s Postulates Gaffky Eberth Salmonella Typhi bacillus No LPG MICROSCOPES M. Leprae Types of Microscopes T. Pallidum N. Gonorrheae Father of Bacteriology  Robert Koch  205 Microbiology 2. Dark Field Microscope Object appears bright against a dark background Q by use of a special dark field condenser Principle Central opaque area  blocks light from entering the objective lens Peripheral annular hollow area  allows the light to pass through and focus on the specimen obliquely 1. Bright-Field or Light Microscope Reflected light by specimen enters the Forms a dark image against a brighter objective lens background. Unreflected light does not enter the objective. Principle So the specimen is bright; and the background Rays from the light source pass through the iris and appears dark. fall on the specimen  Applications Light rays are gathered by the objective and a Identify thin bacteria like spirochetes magnified image is formed 206 Cerebellum Quick Revision Notes 3. Phase Contrast Microscope Applications Used to visualize the living cells by creating difference q. Microbes coated with fluorescent dye in contrast between the cells and water. b. Immunofluorescence 5. Electron Microscope Invented by Ernst Ruska in 1931. Resolution power  0.2 - 0.5 nm Source of illumination - Accelerated electrons Applications Virus detection STAINING TECHNIQUES The light rays go through  condenser  specimen (e.g. bacteria)  phase ring  objective lens  ocular lens. Common Staining Techniques Applications a. Microbial motility b. Shape of living cells 4. Fluorescent microscope UV light is used Fluorescent dyes are used Auramine, Rhodamine, Lissamine, Acrihne orange 1. Simple stains Same colour to all the bacteria in a smear. Eg. Methylene blue or Basic fuchsin 2. Negative staining Q Background is stained, while the structure to be demonstrated is not stained Eg. Indian ink and Nigrosin dyes  207 Microbiology Gram StainQ Spirochetes Acid-fast Stain (Ziehl - Neelsen Stain) Q Capsulated Bacteria 3. Impregnation methods Bacterial cells and structures that are too thin to be seen under the light microscope So they are thickened by impregnation of silver on the surface to make them visible e.g. demonstration of bacterial flagella and Q spirochaetes 4. Differential stains They impart different colours to different bacteria or bacterial structures. The most commonly employed differential stains are  a. Gram stain b. Acid-fast stain c. Albert stain Acid - Fast Organism 208 Cerebellum Quick Revision Notes No Longer Separate Booking for RIM Sim When nucleic acid synthesis is prevented by starvation, the phosphate accumulate in the Card cytoplasm in the form of granules Nocardia Also known as  Legionella Volutin granules (first described in spirillum Smegma bacilli volutans) Bacterial spores Babes Ernst granules (described by Babes Ernst) Rhodococcus Polar bodies (Seen at poles of bacteria) Isospora Metachromatic granules (reddish violet) Mycobacterium Volutin Metachromatic Granules Spermatic head Bacterias → Stained with → Cryptococcus cyst, Cyclospora MSC Garden in Agra PLAN Mycobacterium Ponder’s stain Modification in percentage of sulfuric Spirillum volutants Loeffler's methylene acid: Q Corynebacterium blue (M.C.) i. 5% H2SO4  Mycobacterium leprae diphtheiiae Albert’s stain ii. 0.5 H2S04  Nocardia Gardernella vaginalis Neisser’s stain iii. 0.25-0.5% H2S04  Oocysts of cryptosporidium Aglobacterium and isospora and bacterial spores. tumefaciens Bipolar Staining (Safetypin Appearance)Q Some bacteria display a safety pin appearance due to the accumulation of dye at the poles of the cells  bipolar staining Alberts Stain Differentiates bacteria having metachromatic Can Produce Very High Yield granules from other bacteria that do not Can - Campylobacter granulomatis have them. Produce - Pseudomonas mallei/psedomalloi Corynebacterium diphtheriae  Green coloured Very - Vibrio parahemolyticus bacilli with bluish black metachromatic granules High - Hemophilus ducreyi These bacilli are arranged in Chinese letter or Yield - Yersinea pestis cuneiform arrangement STRUCTURE OF BACTERIAL CELL Polymetaphosphate (volutin) granules Q  209 Microbiology Cell Wall Peptidoglycan  linear polymer of alternating monosaccharide subunits: 3. a. N-acetylglucosamine (NAG) 4. b. N-acetylmuramic acid (NAM) Cross linked via tetrapeptide side chains and pentaglycine bridges (only in Gram positive) Tetrapeptide side chain from NAM molecule - composed of  L-alanine - D-glutamine - L-lysine - D-alanine Gram Negative Bacteria Bacteria with Defective Cell Wall Mycoplasma Naturally Occurring bacteria Q without cell walls. Protoplasts  Derived from Gram positive bacteria Gram Postive Bacteria –– Produced artificially by lysozyme in a hypertonic medium. –– These are unstable Spheroplasts  Derived from Gram negative bacteria –– Produced in presence of penicillin. –– These are unstable –– They differ from the protoplast in that some cell wall material is retained L-forms  Kleineberger-Nobel, named them L-forms after the Lister Institute. –– Develop either spontaneously or in the Gram Negative Bacteria presence of penicillin –– L-forms are more stable than protoplasts and spheroplast Capsulated Bacteria Yes Very Killer Bacterias Have Pretty Nice Capsule Yersinia V. parahemolyticus Kleibsella Bordetella, Bacillus anthrax H. influenza Pneumococcus N. meningococci Cl. perfringes and butyricum Gram positive bacteria 210 Cerebellum Quick Revision Notes Demonstration of Capsule Peritrichous Negative staining M'Faydean capsule stain Motile with polar flagella: by India ink, nigrosin Very Protective Solution HCL Vibrio Pseudomonas Spirochetes H.pylori Campylobacter Legionella Motile with peritrichous flagella: Quellung reaction Cute Baby SLEP Clostridia all except cl.perfringens & cl.tetany Flagella Bacillus except b.antrax Thread like structures composed of a protein Salmonella except salmonella gallenarum – pullorum (flagellin) Listeria monocytogen Organ of locomotion  Confer motility to the E.Coli bacteria Proteus Types of Motility Q Darting  V. cholera Tumbling  Listeria at 20-25°C Stately  Clostridia Cork screw  T. pallidum Lashing  Borrelia Gliding  Mycoplasma Swarming  P. mirabilis, P. vulgaris, CI. tetani, Bacillus cereus Flagella types Swarming growth Rapid (2–10 μm/s) and coordinated translocation of a bacterial population across solid or semi- solid surfaces Swarming growth is due to motility of bacteria. Polar  211 Microbiology Remember BACTERIAL GROWTH → Gram positive bac with swarming growth → Clostridium tetani and bacillus cereus → Gram negative bac with swarming growth → Proteus mirabilis and Proteus vulgaris Fimbriae or Pili Pili - made up of protein called pilin Antigenic; but, the antibodies against pilin antigens are not protective. Functions  Not related to motility a. Help in bacterial adhesion b. Special type of pili (called sex pilus) - helps in conjugation. Lag phase Maximum size of cell Cells are smaller and stain Log phase uniformly Stationary Sporulation phase Exotoxin production Phase of decline Formation of involution forms Bacterial Spore On basis of their oxygen requirements Spores are highly resistant resting stage formed bacteria are classified as: in unfavourable environmental conditions Obligate aerobes: Grow only in presence of oxygen Obligate anaerobes: Grow only in absence of oxygen. Facultative anaerobes: Aerobes that can also grow anaerobically Facultative aerobes: Anaerobes that can also grow aerobically Spore Producing Bacteria Microaerophilic bacteria: Can grow in the presence of 5-10% of oxygen BSC Chemistry Aerotolerant anaerobe: Can tolerate oxygen B. anthrax and subtilis for some time, but do not use it Sporosarcina Clostridia Obligate anaerobes Coxiella Bumetti Can’t Breathe Air Clostridia Bacteroides Actinomyces 212 Cerebellum Quick Revision Notes Microaerophilic bacterias Hell Boy in Micro Camp Helicobacter pylori Borrelia burgdorferi Campylobacter Aerobic Bacteria B3 KLMN2 P3V Bacillus anthrax Bordetella pertusis Brucella Kleibsella Listeria monocytogenes Mycobacteria Nocardia Neiserria Pseudomonas Proteus Pasteurella group except Y. pseudo-TB, Y. enterocolitica Vibrio cholera 2. Enrichment Media Temperature In mixed cultures, substances which have a  stimulating effect on the bacteria to be Psychrophiles: Grow below 20°C, e.g., grown or an inhibitory effect on those to be saprophytes. suppressed incorporated in the liquid medium. Mesophiles: Grow between 25°C and 40°C, e.g., most of the pathogenic bacteria Thermophiles: Grow above of 55°C - 80°C, e.g., Bacillus stearothermophilus. CULTURE MEDIA Q Q Types of Media 3. Selective Media 1. Enriched Media Q In mixed cultures, substances which have a When basal medium is added with some  stimulating effect on the bacteria to be nutrients such as blood, serum or egg, it is grown or an inhibitory effect on those to be called enriched medium. suppressed incorporated in the Solid medium They are used to grow bacteria which are more exacting in their nutritional needs. Q 4. Indicator Media  213 Microbiology Media contain an indicator which changes colour Consists of  when a bacterium grows in them. Nutrient broth Pieces of fat-free minced cooked meat of ox heart Layer of sterile liquid paraffin over it. 5. Differential media A medium which has substances incorporated in it, enabling it to bring out differing characteristics of bacteria and thus helping Principle to distinguish between them, is called a 1. Unsaturated fatty acids present in meat utilise differential medium. oxygen for autooxidation, this reaction is catalysed by haematin in the meat. 2. Glutathione and cysteine (both are reducing agents) present in meat also utilize oxygen. 3. Sulphydryl compounds (present in cysteine) also contribute for a reduced oxidation- reduction (OR) potential. 6. Transport Media These are used in the case of delicate organisms (e.g. gonococci) which may not survive the time taken for transit or may be overgrown by nonpathogenic bacteria Bacterias Selective media Bacillus cereus MYPA medium Organism Transport media Bordetella Bordet Gengou medium Neisseria Amies medium Q Serum dextrose agar, Stuart’s medium Trypticase soy agar, Vibrio cholerae VR (Venkatraman- Brucella Potato infusion agar, Ramakrishnan) medium Castaneda method Autoclaved sea water Campy BAP Q Cary Blair medium Campylobacter jejuni Skirrow’s or Butler’s Shigella, Salmonella Buffered glycerol saline media Cary Blair medium H. Pylori Skirrow’s media Chlamydia HeLa cells Anaerobic Media Robertson’s cooked meat Clostridia (anaerobes) a. Robertson Cooked meat broth (CMB) broth b. Thioglycollate broth Corynebacterium Tellurite blood agar diphtheriae Tinsdale media 1. Robertson cooked meat (RCM) broth 214 Cerebellum Quick Revision Notes Semisynthetic media such as 2 types  Leptospira EMJH, Stuart’s, Korthoff s 1. Intrinsic media 2. Acquired Listeria Mueller-Hinton agar Mutational and Transferable Drug Legionella, Nocardia BCYE medium Resistance Neisseria Thayer-Martin medium Mutational drug Transferable drug gonorrhoeae Chocolate agar resistance resistance Pseudomonas NYC medium Resistance to one drug at Multiple drug resistance Deoxycholate Citrate agar a time at the same time Shigella (DCA) Low-degree resistance High-degree resistance Spirochaetes Smith Noguchi’s medium Resistance can be Cannot be overcome by Ludlum’s, polymixin, salt, overcome by combination Staphylococcus drug combinations mannitol of drugs Trypanosomes, Novy, McNeal, Nicole medium Virulence of resistance Virulence not decreased Leishmania (NNN) mutants may be lowered Pasturella/Yersinia Ghee broth Resistance is transferable Resistance is not Vibrio TCBS to other organisms. transferable to other Spread by: Horizontal Wilson-Blair organisms but spread to Salmonella spread (conjugation, or Triple sugar iron off- springs rarely by transduction/ E coli 0157, H-7 Sorbitol, Mac Conkey media by vertical spread only transformation) Mycobacterium TB LJ media Fungi Sabouraud’s dextrose agar Proteus CNA agar media ANTIMICROBIAL AGENTS Agents that kill or inhibit the growth of microorganisms Classification  Mechanism of action a. Inhibit cell wall synthesis b. Cause leakage from cell membranes c. Inhibit protein synthesis d. Interference with nucleic acid synthesis ANTIMICROBIAL RESISTANCE Development of resistance to an antimicrobial agent by a microorganism.  215 Microbiology Bacterial Toxins Replicate independently Transferred from one cell to another Important tool in genetic engineering Episomes  Present in nucleus as integrated with chromosomal DNA EXOTOXINS ENDOTOXINS 1. Protein (polypeptides) 1. Lipopolysaccharide 2. Heat labile 2. Heat stable Gene Transfer 3. Actively secreted by 3. Integral part of cell wall; Various methods of gene transfer are as follows: living cells into medium. released on disruption of a. Transformation (uptake of naked DNA) bacterial cell. b. Transduction (through bacteriophage) 4. Highly antigenic; 4. Weakly antigenic; c. Lysogenic conversion stimulates formation of antitoxin is not formed antitoxin which neutralizes but antibodies against d. Conjugation (plasmid-mediated) toxin. polysaccharide are raised. Transformation 5. Converted into toxoid by 5. Cannot be toxoided. formaldehyde 6. Enzymic in action. 6. No enzymic action. 7. Specific pharmacological 7. Non-specific action of all effect for each exotoxin. endotoxins. 8. Very high potency. 8. Low potency. 9. Highly specific for 9. Non-specific in action. particular tissue e.g., tetanus toxin for CNS. 10. Produced by Gram- 10. Produced by Gram- negative bacteria. positive bacteria Types Of Infectious Disease Transformation is the transfer of genetic information through free or naked DNA. Griffith Experiment Hereditary material (genes) from the dead bacteria had entered the live cells and changed BACTERIAL GENETICS them genetically so that their progeny were encapsulated and therefore virulent. Extrachromosomal Genetic Element Plasmids  Circular DNA in cytoplasm 216 Cerebellum Quick Revision Notes Transduction Transmission of a portion of DNA from one bacterium to another by a bacteriophage (Virus) Lysogenic Conversion / Temperate cycle F plasmid is also called as ‘sex factor’ or ‘fertility’ (F) factor. Phage DNA remains integrated with the bacterial chromosome as the prophage, which Donors (F+ or male cells) those bacteria multiplies synchronously with bacterial DNA. that contain F plasmid Example  Diphtheria bacillus produces toxin Recipients (F- or female cells) those only when lysogenisation with the phage beta bacteria lacking F plasmid occurs. Elimination of the phage from a toxigenic strain renders it nontoxigenic Remember In transduction  Phage acts only as a vehicle carrying bacterial genes In lysogenic conversion  Phage DNA itself is the new genetic element Conjugation Transfer of genetic material from one bacterium (donor or male) to another (recipient or female) by mating or contact 2. Colicogenic (Col) Factor 1. F Plasmid / F factor /Sex factor/ fertility factor Some strains of coliform bacteria produce colicins which are antibiotic like substances 2. Colicogenic (Col) factor lethal to other enterobacteria. 3. R factor 3. R factor Q 1. F Plasmid / F factor R factor = Resistance transfer factor (RTF) + r determinants RTF  responsible for conjugational transfer  217 Microbiology r determinant  carries resistance for several drugs. An R factor can have several r determinants and resistance to many drugs can be transferred simultaneously TYPES OF ANTIGEN Complete Ag  Immunogenic as well as antigenic i.e., they can induce Ab formation as well as can Blotting Techniques react with Ab Incomplete Ag/ Haptans  Not immunogenic but antigenic i.e., they can NOT induce Ab formation but can react with Ab Molecular weight < 100 KD They are not immunogenic but they became immunogenic when linked to large carrier proteins (protein in nature) Polymerase Chain Reaction (PCR) Immunogenic  induce Ab formation as well as Polymerase chain reaction (PCR) was first can react with Ab developed in 1983 at Cetus Corporation, USA Antigenic can NOT induce Ab formation but In 1993, Kary Mullis was awarded the Nobel can react with Ab prize for this work It is a DNA amplification system that produces a large amount of DNA in vitro from small amounts of starting material. AdjuvantQ A substance, different from antigen but when mixed with an antigen  increases immunogenicity of that antigen Examples  Alum (Aluminium potassium sulphate), Aluminium human vaccines. IMMUNOLOGY Bordetella pertussis acts adjuvant for diphtheria and tetanus toxoid in triple vaccine (DPT vaccine) Epitope and Paratope 218 Cerebellum Quick Revision Notes Epitope / Antigenic determinants  immunologically Directly bridge non-specifically between major active regions of an antigen that actually binds to histocompatibility complex (MHC)-II of APCs specific receptors on lymphocytes or antibodies. and T cells. Smallest unit of antigenicity Paratope  Combining area of the antibody, corresponding to the epitope. Specific antigen determinants on paratope are called Idiotopes Epitope and paratope  determine specificity of immunological reaction. Bacterial superantigen Staphylococcal toxin → Toxic shock syndrome toxin-1 (TSST-1); Exfoliative toxin; Enterotoxins Streptococcal toxin → Streptococcal pyrogenic exotoxin (SPE)-A and C Mycoplasma arthritidis mitogen-I Yersinia enterocolitica Yersinia pseudotuberculosis Viral superantigen Epstein-Barr virus associated superantigen Cytomegalovirus associated superantigen Rabies nucleocapsid HIV encoded superantigen (nef- negative regulatory factor) Fungal superantigen Malassezia furfur Determinants of Antigenicity 1. Foreignness 2. Molecular size  Polysacchrides > Lipids > nucleic acid 4. Physical form  Particulate Ag > soluble Ag 5. Degradability  Antigens  phagocytosed  more immunogenic 6. Route  SC > IV 7. Genetic 8. Species specificity 9. Auto specificity (except lens protein and sperm) 10. Heterogenetic / heterophile specificity Superantigen ANTIBODY Potent activators of T-lymphocytes without 2 Heavy chains  2H relation to their epitope specificity. 2 Light chains chains  2L Unique feature of superantigens  they activate T cells directly without being processed by VL = Variable domain of light chain. antigen presenting cells (APCs). CL = Constant domain of light chain. Receptor for superantigens  Variable β region VH = Variable domain of heavy chain. of T cell receptor (vβ of TCR)  219 Microbiology CH = Constant domain of heavy chain. Property IgG IgA IgM IgD IgE S-S = Disulphide bond Molecular weight 150,000 160,000 900,000 180,000 190,000 Sedimentation Immunoglobulin class H. Chain  L. Chain  coefficient(S) 7 7 19 7 8 50,000 25,000 Heavy chain Gamma Alpha Mu Delta Epsilon IgG γ (gamma) K/λ Light Chain K or L K or L K or L K or L K or L IgA α (alpha) K/λ Serum concentration 12 2 1.2 0.03 0.00004 (mg/ml) IgM μ (mu) K/λ Placental transport + - - - - IgD δ (delta) K/λ Half life 23 days 6-8 days 5 days 3 days 2-3 days IgE ε (epsilon) K/λ Intravascular 45 42 80 75 50 Distribution (%) Constant region = Carboxy terminus (C terminus) = Fc Present in milk + + - - - contains only heavy chain Heat stability (56oC) + + + + - Determines biological properties Abnormal Ig Variable region = Amino terminus (A terminus) = Fab = Antigen binding region Bence Jones Protein (BJP) Contains both Heavy and Light chains Monoclonal Ig consist of light chain Determines immunological specificity Found typically in multiple myeloma. Identified in urine by its characteristic Valency of antibody property of Coagulation when heated to 50 C but redissolving at 70 C. COMPLEMENT SYSTEM A system of some non-specific proteins which occur in normal serum Activated characteristically by Ag – Ab interaction and subsequently mediate a number of biologically significant consequences 9 are complement factors 3 Pathways 220 Cerebellum Quick Revision Notes Classical Alternative Lectin Features pathway pathway pathway Carbohydrate residue of Activator Ag - Ab bacterial cell Mechanism Endotoxin (initiator) complex wall (mannose Marrack proposed the lattice hypothesis binding Multivalent antigens combine with Bivalent protein) antibodies First complement C1 C3b C4 activated C3 C4b2a C3bBb C4b2a convertase C5 C4b2a3b C3bBb3b C4b2a3b convertase Complement All C1-C9: C1, C4, C2- C1- Normal level in Low Normal Others- Low serum Others- Low Immunity Acquired Innate Innate AG-AB REACTIONS Zone phenomenon Q 1. Precipitation Prozone = Ab excess = Absent 2. Aglutination Zone of equivalence = Proper proportion = Peak 3. Nutralisation Post zone = Ag excess = Absent 4. Complement fixation test 5. Opsonisation 6. Immunofluorescence (IF) 7. Radioimmunoassay (RIA) 8. Enzyme Linked Immunosorbent Assay (EIA/ ELISA) 9. Chemiluminescence Assay (CLIA) 10. Immunochromatography 11. Immunoblotting Precipitation and Aglutination  221 Microbiology Examples a. Wassermann reaction (Syphillis) b. Treponema pallidum immobilization test (Syphillis) c. Sabin-Feldman dye test (Toxoplasma) Complement Fixation Test Only an Ag - Ab complex will “consume” complement if it is present, whereas free antigens or antibodies do not. Enzyme Linked Immunosorbent Assay Used to test antibodies for a particular antigen (ELISA) / Enzyme Immunoassays (EIA) Antigen may be soluble or particulate Used for detection of Ag or Ab. Source of complement is guinea pig serum An enzyme is used which acts on substrate to Free complement lyse Sheep RBC produce a color in a positive test. It was once very popular, now is almost obsolete 222 Cerebellum Quick Revision Notes Radioimmunoassay (RIA) Detect antigens up to picogram (10-12g) Q quantities Principle RIA is based on competition for a fixed amounts of specific antibody between a known radio labelled antigen and unknown unlabelled (test) antigen Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ag* Immunofluorescence Immunofluorescence is a technique in which is used to detect an antibody-labeled with a fluorescent dye (fluorescein or rhodamine) the presence of an antigen or antibody by the fluorescence emitted by the bound antibody Similar to ELISA, but Fluorescent dye is used instead of enzyme for labelling of antibody. The fluorescent dyes commonly used are 1. Fluorescein isothiocynate  blue-green Under standard conditions the amount of 2. Rhodamine  orange-red fluorescence labelled antigen bound to the antibody will decrease as the amount of unlabelled antigen in the sample increases. Unlabelled Ag concentration is inversely related to radioactivity of labelled Ag Example 4 Ag* + 4 Ab  4 Ag*Ab 4 Ag + 4 Ag* + 4 Ab  2 AgAb + 2 Ag*Ab + 2 Ag* + 2 Ag 12 Ag + 4 Ag* + 4 Ab  3 AgAb + 1 Ag* Ab + 3 Ag* + 9 Ag Uses Immunochromatography Direct  Detection of rabies virus antigens in brain smears. Immunochromatography also called as lateral Q flow immunoassay. Indirect  Detecting syphilis Ab It can detect both antigens and antibodies.  223 Microbiology It has advantage of being a one step test. Coomb and Gel classification It can be completed within 30 minute. 1. Type I (Anaphylactic) Q It is a strip-based test. 2. Type II (Cytotoxic) Specimen applied to sample pad 3. Type III (Immune complex)  4. Type IV (Delayed or cell mediated). Flows laterally by capillary action Type I: Allergy / Anaphylactic /  Atopic Reaction React with conjugate pad Commonly referred to as Allergy  2 forms  Bind to conjugate if antigen or antibody is present in the specimen and forms antigen antibody complex Acute, potentially fatal, systemic form Anaphylaxis  Complex then flow laterally to reach capture line Recurrent non-fatal localised form  Atopy  Examples Here it is captured by antigen or second antibody present in the capture line All Allergies + PCTS (proximal convulated tubules)  P  PK rxn Presence of colour line is a positive test C  Casoni test T  Theoblad smith phenomena S  Schultz Dale phenomenon Type II: Antibody-mediated (Cytotoxic) Reaction Mediated by antibodies directed toward antigen on cell surfaces or extracellular matrix. Examples My Myasthenia gravis Blood Blood transfusion reactions Goodpasture syndrome and Graves’ Group disease Is Insulin resistant diabetes, ITP R Rheumatic fever H Hyperacute graft rejection Positive Pemphigus vulgaris Type V (Stimulatory Type) Reaction It is a modification of Type II hypersensitivity HYPERSENSITIVITY REACTIONS reaction. Immune response is generally a protective Antibodies interact with antigens on cell process but it may sometimes be injurious to surface that leads to cell proliferation and the host. differentiation instead of inhibition or killing. Hypersensitivity is defined as an exaggerated Antigen-antibody reaction enhances the activity state of normal immune response which results of affected cell. in adverse effects on the body. Example Grave’s disease, Myasthenia gravis 224 Cerebellum Quick Revision Notes Examples John gave TU LIP and Pop Corn to Hashi John mote reaction Tuberculin reaction LePromin reaction Pernicious anemia Contrast dermatitis Hashimoto thyroditis AUTOIMMUNE DISEASES Normally, the immune system does not attack the self. Autoimmune diseases  group of disorders in which the immune system does attack self- cells Normally immune system does not react to its own antigens due to a protective mechanism called tolerance. Any breach in tolerance mechanisms predispose to several autoimmune diseases. Immunological Tolerance State in which an individual is incapable of Type III: Immune Complex Mediated developing an immune response against his own (Arthus) Reaction tissue antigens. Arthus reaction (localised) due to relative antibody excess Serum sickness (generalised) due to relative antigen excess. Examples S Serum sickness, Schick test and SLE H Henoch-Schonlein Purpura A Arthus reaction R Reactive arthritis, Raji assay P Polyarteritis nodosa (PAN) and Post Streptococcal glomerulonephritis (PSGN) Type IV: Delayed Hypersensitivity Reaction Type IV or delayed hypersensitivity reaction is tissue injury by T cell-mediated immune response without formation of antibodies (contrary to type I, II and III) It is a slow and prolonged response.  225 Microbiology Anergy: Sequestration of self-antigen: Defined as unresponsiveness to antigenic Certain self-antigens can evade immune stimulus. recognition by sequestration in immunologically privileged sites The self-reactive T cells interact with the APCs presenting the self-antigen, but the co- e.g., corneal and lens proteins, testicular stimulatory signal is blocked. antigens and antigens from brain. B7 molecules on APC bind to CTLA-4 molecules on T cells instead of CD28 molecules. Mechanisms of Autoimmunity Autoimmunity results due to breakdown of one or more of the mechanisms of immunological tolerance. 1. Breakdown of T Cell Anergy Q In the presence of tissue necrosis and local inflammation express co-stimulatory molecules (B7) –– Multiple Sclerosis, Rheumatoid Arthritis Phenotypic Skewing: and Psoriasis Self-reactive T cells interacting with APCs 2. Failure of AICD presented with self-antigens, undergo full activation. Failure of the auto reactive activated T cells to undergo activation induced cell death (AICD) Secrete non-pathogenic cytokines and chemokine –– SLE (Systemic Lupus Erythematosus) 3. Loss of Treg Cells Autoimmunity can result following the loss of regulatory T cell-mediated suppression of self- reactive lymphocytes. 4. Providing T Cell Help to Stimulate Self-Reacting B Cells Antibody response to self-antigens occurs only Apoptosis By AICD: when potentially self-reactive B cells receive Q help from T cells. Activation-induced cell death Activation of T cells induces upregulation of Fas ligand which subsequently interacts with the death receptor Fas leading to apoptosis. 5. Release of Sequestered Antigens Sequestered antigens -never been exposed to the tolerance mechanisms during development Regulatory T Cells (Treg Cells): of immune system. Treg cells can down regulate the self-reactive Injury to the organs leads to release of such T cells through secreting certain cytokines sequestered antigens which are very well (e.g., IL-10 and transforming growth factor β capable of mounting an immune response. [TGF-β]) or killing by direct cell to cell contact. 226 Cerebellum Quick Revision Notes Eg.  Spermatozoa and ocular antigens release Chronic mucocutaneous candidiasis can cause post vasectomy orchitis and post- Purine nucleoside phosphorylase (PNP) deficiency traumatic uveitis. Disorders of complement 6. Molecular Mimicry Q Complement component deficiencies Complement regulatory protein deficiencies Combined immunodeficiencies (B and T cell defects) Severe combined immunodeficiencies Cytokine receptor mutation Some microorganisms share antigenic determinants (epitopes) with self-antigens. Adenosine deaminase (ADA) deficiency Immune response against such microbes would –– Wiskott–Aldrich syndrome produce antibodies that can cross-react with –– Ataxia telangiectasia self-antigen. –– Nezelof syndrome Common Autoimmune Diseases Disorders of phagocytosis 1. Graves’ disease Chronic granulomatous disease 2. Hashimoto’s disease 3. Diabetes mellitus type 1 Myeloperoxidase deficiency 4. Goodpasture’s syndrome Chediak–Higashi syndrome 5. Rheumatoid fever Leukocyte adhesion deficiency 6. Addison’s disease Ankylosing spondylitis Lazy leukocyte syndrome 7. Antiphospholipid antibody syndrome (APS) Job’s syndrome or Hyper-IgE syndrome 8. Aplastic anemia 9. Guillain-Barré syndrome (GBS) Humoral Immunodeficiency (B Cell 10. Idiopathic thrombocytopenic purpura Defects) 11. Multiple sclerosis Bruton Disease (X-linked Agammaglobulinemia) Q 12. Sjögren’s syndrome X-linked  primarily in males IMMUNODEFICIENCY DISORDERS  Immunodeficiency is a state where defence Due to absence of an enzyme Bruton’s tyrosine mechanisms of body are impaired  enhanced kinase susceptibility to microbial infections and certain  forms of cancer. Failure of pre-B cells to differentiate into immature B cells in bone marrow Classification of primary immunodeficiency  diseases Total absence of B cells and plasma cells  Humoral immunodeficiency (B cell defects) Depressed serum levels of all classes of Bruton disease (X-linked agammaglobulinemia) immunoglobulins. Common variable immunodeficiency  Isolated IgA deficiency Pre-B cells are found in normal numbers in bone marrow and the T cell-mediated Hyper-IgM syndrome responses are also normal Cellular immunodeficiencies (T cell defects) DiGeorge syndrome (thymic hypoplasia)  227 Microbiology ADA is an enzyme required for purine degradation  ADA deficiency  Accumulation of deoxyadenosine  Toxic to rapidly dividing immature T cells > B cell Cellular Immunodeficiency (T Cell  Defects) SCID DiGeorge Syndrome (Thymic Aplasia) Q Deletion chromosome 22q11  Developmental malformation affecting the third and fourth pharyngeal pouches in embryonic life  thymus, parathyroid glands, and portions of the face and aortic arch become defective  congenital defect in thymic development  Defect in T cell maturation Disorders of phagocytosis Clinical features  Chronic Granulomatous Disease (CGD) Q Thymic defects  Infants are extremely vulnerable to viral, fungal, intracellular bacterial 2 Forms  and protozoan infections In X-linked form (70%)  membrane component Parathyroid gland hypoplasia  neonatal tetany of phagocyte oxidase is defective and hypocalcemia In autosomal recessive form (30%)  Anomalies of heart and great vessels  Fallot’s cytoplasmic component of phagocyte oxidase is tetralogy defective. Characteristic facial appearance. Combined Immunodeficiencies (B And T Cells Defects Severe Combined Immunodeficiencies (SCID) Q Defects in both humoral and cell-mediated immune responses. Types  X-linked (60%)  seen in males Autosomal recessive (40%) Autosomal recessive SCID  Adenosine deaminase (ADA) deficiency: Most common type of autosomal recessive SCID. 228 Cerebellum Quick Revision Notes TRANSPLANT IMMUNOLOGY Characterized by thrombosis of graft vessels and ischemic necrosis of the graft. Transplantation  transfer of a graft or transplant (cells, tissues, or organs) from one site to another. Mediated by circulating preformed antibodies (present before transplantation) that are Individual from whom the transplant is taken specific for antigens on the graft endothelial Donor cells Individual to whom it is transplanted  Exposure to foreign HLA antigens can occur as Recipient a consequence of previous blood transfusions, pregnancy, or organ transplantation Autograft (Autogenic Graft): Graft from self Eg. include transferring healthy skin to a burned area in burn patients and use of healthy blood vessels of the same person to replace blocked coronary arteries Isograft (syngraft): Graft from genetically identical person Eg. monozygotic twins Allograft (homograft or allogenic graft): Graft from genetically unrelated member of same species Acute Graft Rejection Q Eg. kidney or heart transplant Occurs within days or weeks after Xenograft (heterograft): Graft from different transplantation. species Due to an active immune response of the host stimulated by alloantigens in the graft. Mediated by  –– T cells (Cytotoxic T cells or helper T cells) –– Antibodies specific for alloantigens in the graft Types of Graft rejection Graft Time taken for Immune mechanisms rejection rejection involved Hyperacute Minutes to Preformed antibodies hours (Anti ABO and/or anti- HLA) Chronic Graft Rejection Acute Weeks to Cytotoxic T cell Occurs over months or years months mediated Antibody mediated T cells that react against graft alloantigens secrete cytokines  stimulate proliferation Chronic Months to years Chronic DTH mediated of fibroblasts and vascular smooth muscle cells Antibody mediated in the graft Delayed type hypersensitivity (DTH) reaction Hyperacute Rejection Manifested as fibrosis and by gradual narrowing Occurs within minutes to hours of transplantation of graft blood vessels (graft arteriosclerosis)  229 Microbiology Q Capsulated Bacteria PAKIYB. M.C.V Pneumococcus Bacillus anthrax Kleibsella H. influenza Yersinia Bordetella N. meningococci Mechanism of Graft rejection CI. perfringes and butyricum V. parahemolyticus Spore Producing Bacteria BSC Chemistry B. anthrax and subtilis Sporosarcina Clostridia Coxiella Bumetti Motile with polar flagella: Very Protective Solution HCL Vibrio Pseudomonas Spirochetes Graft versus host (GVH) reaction H.pylori Graft mounts an immune response against the host (i.e., recipient) and rejects the host Campylobacter (In contrary to the usual situation of graft Legionella rejections, in which the recipient mounts an immune response against the graft antigens) Motile with peritrichous flagella: Cute Baby SLEP Runt disease in animals Clostridia all except cl.perfringens & cl.tetany Conditions for GVHD Bacillus except b.antrax 1. Graft must contain immunocompetent T cells Salmonella except salmonella gallenarum – pullorum  e.g., stem cells or bone marrow or thymus Listeria monocytogen transplants E.Coli 2. Recipient is immunologically suppressed  cannot Proteus mount immune response against the graft. Gram Positive Cocci BACTERIOLOGY Staphylococcus Streptococcus 230 Cerebellum Quick Revision Notes Coagulase +ve Coagulase -ve Antigenic Structure More virulent Less Virulent Form golden yellow colonies on Form white colonies solid media Usually pathogenic Usually not pathogenic S. AUREUS Culture media Q Protein A binds IgG molecules, non-specifically, Virulence factors through Fc region leaving specific Fab sites free to Q combine with specific antigen  When suspension of such sensitized cells is treated with homologous (test) antigen  Q Antigen combines with free Fab sites of IgG attached to staphylococcal cells  This is known as coagglutination Extracellular Enzymes Coagulase test is done by two methods – 1. Slide coagulase test  detects bound coagulase (Clumping factor) 2. Tube coagulase test  detects free coagulase  231 Microbiology CONS Staphylococcus epidermidis  i. Food poisoning Q Q Novobiocin sensitive 2-6 hours Most common cause of prosthetic valve meat and fish, or milk and milk products endocarditis (early onset, upto 12 months). Site of action: The toxin stimulates the vagus Staphylococcus Saprophyticus  nerve and vomiting center of the brain. Novobiocin resistant. It also stimulates the intestinal peristaltic activity Common cause of UTI in sexually active young Q women. Resistance in S. aureus to β lactam antibiotics ii. Toxic shock syndrome (TSS) Q Multisystem disease Young women Most cases occur in menstruating women who use tampons 1. Production of β lactamase enzyme: Characterised by high fever, hypotension, β lactamase or penicillinse enzymes cleave the vomiting, diarrhoea and scarlatiniform rash β lactam ring Plasmid coded iii. Staphylococcal scalded skin syndrome (SSSS) Produced by > 90% of strains of S.aureus. Exfoliative skin diseases. Can be overcome by addition of β lactamase inhibitors such as clavulanic acid or sulbactam. 0uter layer of epidermis gets separated from the underlying tissues. 2. By alterations of PBP: Q Shown by MRSA strains Q Mediated by mecA gene Chromosomally coded 232 Cerebellum Quick Revision Notes STREPTOCOCCUS Virulence factors Q Group B Streptococci: Streptococcus pyogenes Culture media Selective Media a. Crystal violet blood agar b. PNF medium (polymyxin-B, neomycin and fusidic acid) Q Transport medium  Pike’s medium Biochemical Rx i. “Strep throat” – Pharyngitis with streptococcus pyogenes  233 Microbiology CAMP positive -> Area of increased hemolysis (arrow head) around staphylococcus streaks (by streptococcus agalactiae) Characters S. pyogenes S. agalactiae Q Lancefield Group A Group B grouping Bacitracin Sensitive Resistant sensitivity test CAMP test Negative Positive β-hemolytic 0.5–1 mm, pin Mucoid, slightly colonies point larger (2 mm) Streptococcus Pneumoniae (Pneumococcus) Diplococcus pneumoniae  flame-shaped or lanceolate appearance Streptococcus pneumonia Capsuled  –– capsule encloses each pair. –– Type specific (95 capsular serotypes) Group B Streptococci: Streptococcus agalactiae Single most common cause of neonatal sepsis and meningitis Q 1. Hippurate hydrolysis test positive Q 2. CAMP positive 3. Bacitracin resistant Q 4. PYR test is negative CAMP reaction CAMP (Christie, Atkins and Munch-PeVerson) In the center of blood agar plate single Straight-line streak of staphylococcus aureus is made. Perpendicular to staphylococcus Streak, streptococcal streaks are inculcated. 234 Cerebellum Quick Revision Notes Treatment Penicillin G  DOC Culture media If resistant to penicillin, then alternative options are: Blood Agar Cephalosporins (e.g., ceftriaxone) or vancomycin After incubation for 18 hours  Alpha hemolysis for meningitis isolates On further incubation  Draughtsman or Quinolones such as IV levofloxacin for non- carrom coin appearance / central umbonation meningeal infections Prophylaxis  Pnemoccocal vaccine There are two types of pneumococcal vaccines available: 1. 23-valent pneumococcal polysaccharide vaccine (PPSV23) 2. Pneumococcal conjugate vaccine (PCV13) Biochemical Reaction Streptococci Viridans The viridans streptococci are opportunistic pathogens but can, on occasion, cause disease. Two clinically important phenomena are associated with viridans streptococci  1. Dental caries 2. Subacute endocarditis Viridans Features S. pneumoniae streptococci Gram-positive Gram-positive cocci Arrangement cocci in long in pairs chains Morphology Lanceolate shaped Round/oval Capsule Present Absent  235 Microbiology Draughtsman or N. meningitidis N. gonorrhoeae On blood agar Q Minute colony Q carrom coin colony Capsulated Non capsulated Bile solubility Soluble in bile Insoluble in bile Lens-shaped/half-moon- Kidney-shaped (diplococci Optochin Sensitive Resistant shaped (diplococci with with adjacent sides Inulin Fermented Not fermented adjacent sides flattened) concave) Ferments glucose and Ferments only glucose Enterococci maltose Virulance factor - Capsule Virulance factor - Pilli Habitat - genital tract Habitat - nasopharynx (urethra, cervix), rarely pharynx Culture media Approach to GPC Biochemical Reaction GRAM NEGETIVE COCCI NEISSERIA Pathogenesis of N. Meningitidis Pyogenic meningitis: Young children (3–5 years of age). Rashes: A non-blanching rash (petechial or purpuric) Septicemia Q Waterhouse-Friderichsen syndrome : severe form of fulminant meningococcemia, characterized 236 Cerebellum Quick Revision Notes by large purpuric rashes (purpura fulminans), Capsulated (polyglutamic) shock, DIC, bilateral adrenal hemorrhage and Spore forming bacillus (Oval, Central, non- multi-organ failure. bulging) Pathogenesis of N. Gonorrhoeae Bamboo stick appearance Q Gonorrhoea In men: acute urethritis, prostate, seminal vesicles and epididymis, abscesses and multiple discharging sinuses (‘watercan perineum’) In women: Cervicitis, Acute salpingitis, PID, Peritoneal spread may produce a perihepatic Q