CHSE Odisha Voc. Microbiology & Serology cheat sheet

Koch's postulates: the organism must be found in all diseased cases, be isolated in pure culture, reproduce disease when inoculated, and be re-isolated from the experimental host.

Biosafety levels: BSL-1 non-pathogenic teaching strains; BSL-2 moderate hazards (Salmonella, HBV) with biosafety cabinet for aerosols; BSL-3 airborne lethal agents (M. tuberculosis); BSL-4 no vaccine or therapy (Ebola).

Universal precautions treat all blood and body fluids as infectious; gloves, gown, and eye protection are required whenever splash or aerosol risk exists.

Hand hygiene is the single most effective break in the chain of infection; alcohol rub is ineffective against spores and non-enveloped viruses, so soap and water is used for C. difficile.

Sharps policy: never recap by hand, never bend or break needles; place in puncture-resistant yellow sharps container filled to no more than three-quarters capacity.

Spill of biological material: contain, cover with absorbent, apply 1% sodium hypochlorite for 30 minutes, remove with forceps and paper, then disinfect the area.

Autoclave and centrifuge are the main aerosol-generating hazards; wait 10 minutes after a centrifuge stops and open the lid slowly in a biosafety cabinet.

Immunisation, baseline serum storage, and post-exposure prophylaxis (HBV vaccine plus immunoglobulin) are standard laboratory worker protections.

Total magnification = objective magnification x eyepiece magnification; a 100x oil-immersion objective with 10x eyepiece gives 1000x.

Resolving power d = 0.61 lambda / (NA), so resolution improves with shorter wavelength and higher numerical aperture; oil immersion raises NA to about 1.25 by matching refractive index.

Numerical aperture of a dry objective is at most about 0.95; immersion oil (refractive index 1.515) prevents light loss by refraction at the slide-air interface.

Bright-field is the routine stain examination method; dark-field shows live spirochetes (Treponema pallidum) as bright spirals on a dark background.

Phase-contrast converts refractive index differences into brightness differences and is used for unstained living cells and motility checks.

Fluorescence microscopy uses fluorochrome-labelled antibodies (FITC, excitation 490 nm, emission 520 nm) for direct detection of pathogens in smears.

Electron microscopy resolution is about 0.1-0.2 nm; transmission EM shows internal ultrastructure, scanning EM shows surface topography.

Gram stain sequence: crystal violet 1 min, Gram's iodine 1 min, decolourise with acetone-alcohol 5-10 s, counterstain safranin 30 s; Gram-positive cells retain the violet-iodine complex.

Sterilisation kills or removes all microorganisms including spores; disinfection reduces pathogens on inanimate objects but does not necessarily kill spores.

Moist heat kills by protein denaturation; dry heat kills by oxidation. Autoclave standard cycle: 121 degrees C, 15 psi, 15-20 minutes for media; 134 degrees C for 3 minutes for discard loads.

Autoclave efficacy is monitored with Geobacillus stearothermophilus spore strips; dry-heat oven (160 degrees C, 2 hours) is checked with Bacillus atrophaeus spores.

Membrane filtration (0.22 micrometre pore) sterilises heat-labile liquids such as sera, vitamins, and antibiotics; 0.45 micrometre removes bacteria but not all spores.

Ethylene oxide is used for heat-sensitive plastics and endoscopes; it requires 4-12 hours aeration to remove toxic residues.

Culture media: nutrient agar is basal; MacConkey is selective for Gram-negatives (bile salts, crystal violet) and differential for lactose fermentation (pink colonies); blood agar is enriched and haemolytic.

Streak plate achieves single colonies; pour plate and spread plate allow colony counting. A viable count assumes one colony-forming unit arose from one cell.

Anaerobic culture uses anaerobic jar with gas pack, thioglycollate broth, or a glove box; obligate anaerobes die in ambient oxygen.

Bacterial shapes: cocci, bacilli, spirilla, vibrios, spirochetes; arrangements (chains, clusters, tetrads) reflect the plane of cell division.

Cell wall peptidoglycan is a glycan chain cross-linked by peptide bridges; Gram-positive walls are thick (20-80 nm) with teichoic acids, Gram-negative walls are thin with an outer membrane containing lipopolysaccharide.

Bacterial growth curve phases: lag, exponential (log), stationary, and death; generation time is the doubling time in log phase, typically 20 minutes for E. coli.

Growth rate constant k = (log Nt - log N0) x 3.32 / t; mean generation time g = 1/k.

Bacteria are classified by temperature: psychrophiles below 20 degrees C, mesophiles 20-45 degrees C, thermophiles 45-80 degrees C; most pathogens are mesophiles.

Obligate aerobes need oxygen; facultative anaerobes grow with or without it; microaerophiles need 5-10% oxygen; aerotolerant anaerobes ferment but tolerate oxygen; obligate anaerobes lack superoxide dismutase and catalase.

Spores (endospores) are dormant, heat-resistant structures formed by Bacillus and Clostridium via sporulation; they resist heat, radiation, and disinfectants and germinate when conditions improve.

Bacterial cell wall synthesis is inhibited by beta-lactams; protein synthesis at the 70S ribosome is blocked by aminoglycosides, tetracyclines, macrolides, and chloramphenicol.

Disinfectant efficacy depends on concentration, contact time, temperature, organic load, and pH; always clean organic matter before applying disinfectant.

High-level disinfectants (glutaraldehyde, ortho-phthalaldehyde, hydrogen peroxide) kill spores with sufficient contact time; low-level (quaternary ammonium, alcohol) do not.

Phenol coefficient compares a test disinfectant with phenol; a higher coefficient means greater activity. Alcohols are 60-90% effective and act by membrane damage and protein denaturation.

Antibiotics act on cell wall (penicillins, cephalosporins, vancomycin), ribosome (aminoglycosides, tetracyclines, macrolides), nucleic acid (rifampicin, fluoroquinolones), or folate synthesis (sulphonamides).

Beta-lactamase enzymes (including extended-spectrum beta-lactamases) hydrolyse penicillins and cephalosporins; clavulanate, tazobactam, and sulbactam are beta-lactamase inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) carries the mecA gene encoding PBP2a, which lowers affinity for beta-lactams; vancomycin is the standard alternative.

Antimicrobial resistance mechanisms: enzymatic inactivation, target alteration, efflux pumps, and reduced permeability; resistance genes spread by plasmids, transposons, and integrons.

Antibiotic stewardship: culture before starting therapy, use narrow-spectrum agents when possible, complete prescribed courses, and avoid antibiotics for viral infections.

Innate immunity is non-specific, present from birth, and has no memory; components include physical barriers, phagocytes, complement, natural killer cells, and inflammation.

Acquired immunity is specific and has memory; it is mediated by B lymphocytes (humoral, antibody) and T lymphocytes (cell-mediated).

Primary lymphoid organs: bone marrow and thymus, where lymphocytes mature. Secondary organs: spleen, lymph nodes, tonsils, and mucosa-associated lymphoid tissue, where responses are mounted.

Immunoglobulin classes: IgG is the most abundant and crosses the placenta; IgM is the first antibody in infection and the largest; IgA is secreted in mucosa; IgE mediates allergy; IgD is a B-cell receptor.

Antibody structure is a Y-shaped tetramer of two heavy and two light chains joined by disulphide bonds; the Fab region binds antigen, the Fc region mediates effector function.

Complement pathways: classical (IgG or IgM plus C1), alternative (microbial surfaces), and lectin (mannose-binding lectin); all converge on C3 convertase and the membrane attack complex C5b-9.

Cell-mediated immunity: helper T cells (CD4) recognise antigen on MHC class II and release cytokines; cytotoxic T cells (CD8) kill infected cells presenting antigen on MHC class I.

Hypersensitivity types: I immediate IgE (anaphylaxis), II antibody-mediated (transfusion reaction), III immune complex (serum sickness), IV delayed cell-mediated (tuberculin reaction).

An antigen is any substance that can be specifically bound by an antibody or T-cell receptor; an immunogen is an antigen that also provokes an immune response.

Immunogenicity is favoured by large molecular size, foreignness, chemical complexity, and a route of administration that reaches lymphoid tissue.

A hapten is a small molecule that is not immunogenic alone but becomes immunogenic when conjugated to a carrier protein.

Epitope (antigenic determinant) is the specific part of an antigen bound by an antibody; multivalent antigens cross-link antibodies and give visible reactions such as agglutination.

Antigen-antibody binding is non-covalent: hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals forces; it is reversible and follows the law of mass action.

Affinity is the strength of binding between one epitope and one Fab site; avidity is the overall strength of binding of a multivalent antibody to a multivalent antigen.

Cross-reactivity occurs when an antibody raised against one antigen binds a structurally similar antigen; it explains false-positive serology and heterophile antibody reactions.

Adjuvants (aluminium salts, Freund's adjuvant) enhance the immune response to an antigen; they are used in vaccine formulation and antibody production.

Agglutination detects antibodies against particulate antigens; latex agglutination and haemagglutination are common. Titre is the highest dilution showing a positive reaction.

Precipitation occurs with soluble antigen and antibody in optimal proportions; the prozone phenomenon (excess antibody) and postzone (excess antigen) cause false-negative results.

ELISA formats: direct (antigen detection), indirect (antibody detection), sandwich (antigen capture), and competitive. Enzyme conjugate converts substrate to a coloured product read at 450 nm.

Western blot separates proteins by SDS-PAGE, transfers to a membrane, and detects specific bands with labelled antibody; it is used as a confirmatory test for HIV.

Immunofluorescence: direct uses labelled antibody on a smear; indirect uses unlabelled patient antibody followed by labelled anti-human globulin.

Complement fixation test uses the ability of antigen-antibody complexes to consume complement; haemolysis of sensitised red cells indicates a negative result.

Rapid tests and immunochromatography use lateral flow of a colloidal-gold conjugate; results appear within 15 minutes but have lower sensitivity than ELISA.

Diagnostic test performance: sensitivity = true positives / (true positives + false negatives); specificity = true negatives / (true negatives + false positives).

Gram-positive cocci: Staphylococcus aureus (catalase positive, coagulase positive, clusters) versus Streptococcus pyogenes (catalase negative, bacitracin sensitive, beta-haemolytic, chains).

Gram-negative bacilli of enteric origin: E. coli (lactose fermenter, pink on MacConkey), Salmonella and Shigella (non-lactose fermenting, colourless), Vibrio cholerae (oxidase positive, alkaline peptone water enrichment).

Acid-fast bacilli: Mycobacterium tuberculosis resists decolourisation by acid-alcohol, stains red with Ziehl-Neelsen; slow growth (2-6 weeks) on Lowenstein-Jensen medium.

Viral diagnosis: PCR detects viral nucleic acid; serology detects IgM for recent infection and IgG for past infection or immunity; viral culture and immunofluorescence are also used.

Fungal pathogens: Candida albicans (germ tube positive), Cryptococcus neoformans (capsule, India ink), Aspergillus (septate hyphae, dichotomous branching), dermatophytes (keratinophilic moulds).

Parasites: Plasmodium species cause malaria (ring forms in thick film); Entamoeba histolytica causes amoebic dysentery; Giardia lamblia causes malabsorption; Ascaris, hookworm, and tapeworms are helminths.

Hepatitis B serology: HBsAg indicates infection, anti-HBs indicates immunity, HBeAg indicates high infectivity, anti-HBc IgM indicates recent infection.

HIV diagnosis: screening with ELISA for anti-HIV antibody, confirmation with Western blot or HIV-1/2 differentiation immunoassay; viral load and CD4 count guide treatment.

Collect specimens before starting antimicrobial therapy; use aseptic technique and sterile containers; label with patient identity, date, time, and site.

Blood culture: collect 10-20 mL per set in two bottles (aerobic and anaerobic), from two sites, at the time of fever spikes; do not delay for fever.

Sputum for tuberculosis: collect early-morning samples on three consecutive days in a sterile container; do not use saliva.

Urine culture: midstream clean-catch sample; a colony count of 100,000 CFU/mL of a single organism suggests infection, while lower counts may indicate contamination or infection in symptomatic patients.

Stool culture: use Cary-Blair transport medium for suspected Vibrio cholerae, Salmonella, or Shigella; examine fresh stool for parasites and ova.

CSF: collect in sterile tubes; process immediately for cell count, Gram stain, culture, and antigen or PCR testing; do not refrigerate CSF for culture.

Wound and pus: aspirate with a syringe or swab the deep portion of the wound; transport in Amies medium; note the site and appearance.

Reporting: state the organism identification, antimicrobial susceptibility pattern, and any relevant comment; critical results (e.g., positive blood culture) are phoned to the clinician and documented.

Water potability: coliform count is the indicator of faecal contamination; MPN method estimates coliforms per 100 mL; a standard of zero coliforms per 100 mL applies to drinking water.

Membrane filtration for water testing uses a 0.45 micrometre filter, incubated on selective medium at 37 degrees C or 44 degrees C; colonies are counted and confirmed.

Food spoilage is caused by bacteria, yeasts, and moulds; refrigeration slows growth; canning requires botulinum cook (121 degrees C, 15 psi) to destroy Clostridium botulinum spores.

Food-borne pathogens: Salmonella, Campylobacter, Listeria monocytogenes, E. coli O157:H7, and Staphylococcus aureus (enterotoxin).

Soil microbiology: nitrogen fixation by Rhizobium in root nodules and free-living Azotobacter; nitrification by Nitrosomonas and Nitrobacter; denitrification returns nitrogen to the atmosphere.

Industrial microbiology uses fermentation to produce antibiotics (Penicillium), alcohol (Saccharomyces), citric acid (Aspergillus niger), and enzymes; batch versus continuous fermentation are the main modes.

Sewage treatment stages: primary physical settling, secondary biological oxidation (activated sludge or trickling filter), and tertiary disinfection; BOD measures organic load.

Bioremediation uses microorganisms to degrade pollutants such as oil spills; composting relies on thermophilic decomposition of organic waste.