• Consulting a doctor for any medical concerns
• Using a health tracking app if you want to monitor vitals
• Sharing specific symptoms or questions here if you'd like general health information

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Microbiology & Immunology - Five Mark Questions

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Q1. Western Blotting Technique

Principle

Procedure

1. Sample Preparation: Protein sample is denatured with SDS (sodium dodecyl sulfate) and heated.
2. Gel Electrophoresis (SDS-PAGE): Proteins migrate through polyacrylamide gel based on molecular weight - smaller proteins move faster.
3. Transfer (Blotting): Proteins are electrophoretically transferred from gel onto nitrocellulose or PVDF membrane.
4. Blocking: Membrane is incubated with blocking solution (5% skimmed milk or BSA) to prevent non-specific antibody binding.
5. Primary Antibody Incubation: Membrane is incubated with specific primary antibody against the target protein.
6. Secondary Antibody: Enzyme-linked secondary antibody (anti-IgG linked to HRP or alkaline phosphatase) is added.
7. Detection: Substrate is added - enzyme reacts to produce a visible/chemiluminescent band at the protein's molecular weight.

Applications

• Diagnosis of HIV (confirmatory test - detects antibodies against gp41, gp120, p24)
• Diagnosis of Lyme disease (confirmatory)
• Detection of prion proteins (CJD diagnosis)
• Research: protein expression studies, post-translational modifications
• Confirmation of Hepatitis B/C infections

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Q2. Southern Blotting Technique

Principle

Procedure

1. DNA Extraction: DNA is extracted from cells/tissue.
2. Restriction Digestion: DNA is cut with restriction endonucleases into fragments of specific sizes.
3. Gel Electrophoresis: Fragments are separated by size on agarose gel.
4. Denaturation: Gel is treated with alkali (NaOH) to denature DNA into single strands.
5. Transfer: DNA fragments are transferred (by capillary action, vacuum, or electrophoresis) onto nylon membrane.
6. UV Crosslinking: DNA is fixed to membrane by UV or baking (80°C).
7. Hybridization: Radioactively (³²P) or chemiluminescently labeled probe (complementary DNA sequence) is added; it hybridizes to target DNA.
8. Washing: Excess probe is washed off.
9. Detection: Autoradiography or chemiluminescence reveals bands.

Applications

• Detection of gene mutations (point mutations, deletions, rearrangements)
• RFLP analysis (Restriction Fragment Length Polymorphism) - used in forensics and paternity testing
• Diagnosis of genetic diseases (sickle cell anemia, thalassemia)
• Detection of viral DNA (HPV typing, CMV)
• Gene mapping and cloning studies

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Q3. Schick's Test

Principle

Procedure

• Test dose: 1/50 MLD (Minimum Lethal Dose) of diphtheria toxin in 0.1 mL is injected intradermally into the left forearm.
• Control dose: Heat-inactivated toxin (same amount) is injected in the right forearm (to rule out hypersensitivity to non-toxic protein components).
• Reading: Observed at 24-48 hours and again at 5-7 days.

Interpretation

Applications

• Assess immunity status to diphtheria before vaccination
• Screen population for herd immunity levels
• Evaluate efficacy of diphtheria immunization programs
• Used in community surveys and epidemiological studies

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Q4. QBC (Quantitative Buffy Coat) Test

Principle

Procedure

1. A heparinized capillary tube pre-coated with acridine orange dye and a float is used.
2. Finger-prick blood (60 µL) is collected directly into the capillary tube.
3. The tube is centrifuged at 12,000 rpm for 5 minutes.
4. After centrifugation, the float compresses and expands the buffy coat layer for examination.
5. The tube is examined under a fluorescent microscope with UV light.
6. Infected RBCs fluoresce bright green/yellow with acridine orange.

Applications

• Rapid diagnosis of Plasmodium species (malaria) - all 4 species detected
• Diagnosis of filariasis (microfilariae detected in buffy coat)
• Diagnosis of trypanosomiasis (trypanosomes in buffy coat)
• Useful in field conditions and endemic areas - faster than peripheral smear
• Screening in blood banks

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Q5. Mantoux Test (Tuberculin Test)

Principle

Procedure

1. Tuberculin PPD (0.1 mL = 5 TU - Tuberculin Units) is injected strictly intradermally (not subcutaneous) on the volar surface of the left forearm using a 26-gauge needle (Mantoux method).
2. A small bleb (wheal) of 6-10 mm is raised.
3. The site is NOT rubbed after injection.
4. Reading is done at 48-72 hours (peak reaction).
5. Only induration (hard, palpable, raised area) is measured - NOT erythema. Measured transversely (perpendicular to long axis of forearm).

Interpretation

Applications

• Diagnosis of latent TB infection
• Epidemiological surveys (TB prevalence)
• Contact tracing of TB cases
• Pre-BCG screening (children <5 years)
• Occupational screening of healthcare workers
• Note: BCG vaccination can give false-positive results

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Q6. ELISA (Enzyme-Linked Immunosorbent Assay)

Principle

Types

• Direct ELISA: Antigen coated on plate, detected by enzyme-linked antibody
• Indirect ELISA: Detects antibody in patient serum (most common in diagnostics)
• Sandwich ELISA: Two antibodies capture and detect antigen (most sensitive)
• Competitive ELISA: Inhibition format, used for small antigens

Procedure (Indirect ELISA - for antibody detection)

1. Coating: Known antigen is coated on wells of a 96-well microtiter plate and incubated overnight.
2. Blocking: Wells are blocked with BSA or casein to prevent non-specific binding.
3. Patient Serum: Test serum (containing unknown antibodies) is added; if antibodies are present, they bind to antigen. Incubate 1-2 hours, wash.
4. Secondary Antibody: Enzyme-conjugated anti-human IgG antibody is added. Incubate, wash.
5. Substrate: Enzyme substrate (TMB for HRP, turns blue/yellow) is added.
6. Stop Solution: H₂SO₄ stops the reaction.
7. Reading: Absorbance measured at 450 nm using ELISA reader. Higher OD = more antibody present.

Applications

• Diagnosis of HIV (screening test)
• Hepatitis B/C detection (HBsAg, anti-HCV)
• Diagnosis of dengue (NS1 antigen, IgM/IgG)
• Detection of SARS-CoV-2 antibodies
• Diagnosis of typhoid, malaria, toxoplasmosis, filariasis
• Pregnancy tests (hCG detection)
• Food safety testing (allergens, contaminants)
• Drug monitoring (therapeutic drug levels)

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Q7. Widal Test

Principle

Procedure

1. Drop of patient serum placed on slide.
2. Known Salmonella antigen suspension (O and H) mixed with serum.
3. Rocked for 1 minute - observe for agglutination.

1. Patient serum is serially diluted (1:20 to 1:640) in saline in test tubes.
2. Equal volume of O and H antigen suspensions are added to separate sets of tubes.
3. Tubes are incubated at 37°C for 18-24 hours (H antigens) or 50°C for 4 hours in water bath (O antigens).
4. Read for agglutination. The highest dilution showing agglutination is the titer.

Interpretation

• O antigen titer ≥ 1:160 = significant (active/recent infection)
• H antigen titer ≥ 1:160 = significant (past infection or vaccination)
• Rising titers (4-fold rise) in paired sera (7-10 days apart) is most diagnostic
• AH (H agglutinin against paratyphi A) and BH (H agglutinin against paratyphi B) are also tested

Applications

• Diagnosis of typhoid fever (Salmonella typhi infection)
• Diagnosis of paratyphoid fever (S. paratyphi A, B, C)
• Epidemiological surveillance
• Limitations: False positives (other Salmonella infections, malaria, liver disease), false negatives in early disease or antibiotic use

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Q8. Antigen-Antibody Reactions and Diagnostic Applications

Types of Ag-Ab Reactions

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Q9. Immunity - Definition and Classification

Definition

Classification of Immunity

                    IMMUNITY
                       |
         ______________|______________
        |                             |
    INNATE                        ADAPTIVE
  (Non-specific)               (Specific/Acquired)
        |                             |
  - Skin/Mucosa               ________|________
  - Phagocytes               |                 |
  - NK cells             HUMORAL          CELL-MEDIATED
  - Complement           (B cells/         (T cells/
  - Lysozyme             Antibodies)       Cytokines)
  - Interferons               |
  - Fever                     |
                    __________|__________
                   |                     |
              NATURAL                ARTIFICIAL
                   |                     |
            _______|_______        _______|_______
           |               |      |               |
         ACTIVE          PASSIVE  ACTIVE        PASSIVE
      (Infection)     (Maternal  (Vaccination)  (Antiserum/
                      antibodies,             Immunoglobulin)
                      colostrum)

1. Innate (Non-specific) Immunity

• First line: Physical barriers - intact skin, mucous membranes, cilia, tears, saliva
• Second line: Internal defenses - fever, inflammation, phagocytes (neutrophils, macrophages), NK cells, complement system, interferons, lysozyme
• Responds immediately (0-4 hours), no memory, non-specific

2. Adaptive (Acquired/Specific) Immunity

• Develops after exposure to antigen; has specificity and memory
• Humoral immunity: Mediated by B lymphocytes → plasma cells → antibodies (IgG, IgM, IgA, IgE, IgD)
• Cell-mediated immunity (CMI): Mediated by T lymphocytes (CD4+ helper, CD8+ cytotoxic T cells, memory T cells)
• Response takes 5-14 days (primary), faster on re-exposure (secondary response due to memory cells)

Natural vs. Artificial

• Natural Active: After clinical/subclinical infection (e.g., immunity after measles)
• Natural Passive: Transfer of maternal IgG across placenta; IgA in breast milk
• Artificial Active: Vaccination (immunization)
• Artificial Passive: Administration of preformed antibodies (antitoxins, IVIG, hyperimmune sera)

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Q10. Structure of Antibodies (Immunoglobulins)

Basic Structure of an Antibody

• Two identical heavy (H) chains (molecular weight ~50,000 Da each)
• Two identical light (L) chains (molecular weight ~25,000 Da each) - either Kappa (κ) or Lambda (λ)
• Chains are held together by disulfide bonds and non-covalent interactions

Domains

• Variable (V) region: N-terminal; differs between antibodies; contains the antigen-binding site (paratope); has Complementarity Determining Regions (CDRs) - the actual contact residues
• Constant (C) region: C-terminal; same within an isotype; determines antibody class and effector functions

Regions Created by Papain Cleavage

• Fab (Fragment antigen-binding): Two arms of the Y; each contains VH + CH1 + VL + CL; binds antigen
• Fc (Fragment crystallizable): Stem of Y; contains CH2 + CH3; binds complement, Fc receptors on cells; determines isotype

Five Classes (Isotypes) of Antibodies

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Q11. Steps in Production of Antibodies

Steps

• Antigen enters the body and is captured by Antigen-Presenting Cells (APCs) - macrophages, dendritic cells, B cells
• APCs process the antigen and present antigen peptides on MHC Class II molecules

• CD4+ Helper T cells (Th cells) recognize the MHC II-peptide complex via their T cell receptor (TCR)
• Co-stimulatory signals (CD28-B7, CD40-CD40L) activate T cells
• Activated Th cells secrete cytokines (IL-4, IL-5, IL-6, IL-21)

• Specific B cells recognize the antigen via their B cell receptor (BCR = surface IgM/IgD)
• T cells provide help to B cells via direct contact (CD40-CD40L) and cytokines
• B cells are activated and enter the germinal center of lymph nodes

• Specific B cell clone that recognizes the antigen is selected
• This B cell clone undergoes rapid proliferation (clonal expansion)

• Somatic Hypermutation: Random mutations in V region genes improve antigen affinity (affinity maturation)
• Class Switch Recombination: B cells switch from making IgM to IgG, IgA, or IgE depending on cytokine environment

• B cells differentiate into:
  • Plasma cells: Short-lived (days-weeks) or long-lived (bone marrow); secrete large quantities of antibodies
  • Memory B cells: Long-lived; responsible for faster, stronger secondary immune response

• Primary response: After first antigen exposure; mainly IgM; low titer; slow onset (7-14 days)
• Secondary (anamnestic) response: After re-exposure; mainly high-titer IgG; faster (3-5 days); more affinity matured

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Q12. Vaccines - Definition and Classification

Definition

Classification

• Contain live but weakened (attenuated) microorganisms
• Produce strong, long-lasting immunity (humoral + cell-mediated)
• Examples: BCG (TB), OPV (oral polio), MMR (measles-mumps-rubella), yellow fever, varicella
• Disadvantage: May revert to virulence; contraindicated in immunocompromised

• Whole microorganism killed by heat, chemicals (formalin), or radiation
• Safer; need multiple doses (booster); mainly humoral immunity
• Examples: IPV (injectable polio), Hepatitis A, Rabies, whole-cell pertussis, influenza (flu shot)

• Chemically inactivated toxins (toxoids) - retain antigenicity, lose toxicity
• Examples: Diphtheria toxoid, Tetanus toxoid (DPT vaccine)

• Contain only specific antigens (purified proteins, polysaccharides)
• Examples: Hepatitis B (HBsAg), Influenza (split vaccine), Pertussis (acellular), HPV (L1 protein VLPs)

• Polysaccharide antigens conjugated to a carrier protein to enhance immunogenicity (especially in infants)
• Examples: Hib (Haemophilus influenzae b), Pneumococcal conjugate (PCV), Meningococcal conjugate

• Deliver mRNA encoding a pathogen protein; cells produce the protein and mount immune response
• Examples: Pfizer-BioNTech, Moderna COVID-19 vaccines

• Antigen gene inserted into expression vector (yeast, bacteria); antigen produced recombinantly
• Examples: Hepatitis B (recombinant), HPV vaccine (recombinant VLP)

• Plasmid DNA encoding antigen injected; antigen expressed in host cells
• Still largely experimental/veterinary use

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Q13. Toxins and Toxoids

Toxins

• Proteins secreted by living bacteria (mostly gram-positive)
• Heat-labile (destroyed at 60-80°C)
• Highly potent; antigenic; stimulate antitoxin production
• Can be converted to toxoids
• Examples:
  • Clostridium tetani → Tetanospasmin (tetanus toxin) - prevents inhibitory neurotransmitter release
  • Corynebacterium diphtheriae → Diphtheria toxin - inhibits protein synthesis (ADP-ribosylation of EF-2)
  • Vibrio cholerae → Cholera toxin - activates adenylyl cyclase → ↑cAMP → secretory diarrhea
  • Staphylococcus aureus → Exfoliative toxin, TSST-1, enterotoxins

• Lipopolysaccharide (LPS) of gram-negative bacteria cell wall
• Released on bacterial death/lysis
• Heat-stable; less potent than exotoxins; weakly antigenic
• Cannot be converted to toxoids
• Effects: fever (pyrogenic), hypotension, DIC, septic shock (via IL-1, TNF-α, IL-6 release from macrophages)

Toxoids

• Formalin (0.3-0.4% formaldehyde) at 37-40°C for 3-4 weeks (Ramon's method)
• Heat treatment

• Non-toxic but fully immunogenic
• Stimulate production of specific antitoxin antibodies
• Stable and safe to administer

• Diphtheria toxoid (component of DPT/DT vaccine)
• Tetanus toxoid (TT vaccine, DPT, Td)
• Cholera toxoid (experimental)

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Q14. Immunization Programme

Objectives

• Reduce morbidity and mortality from vaccine-preventable diseases
• Achieve eradication/elimination of certain diseases (polio, measles)

National Immunization Schedule (India - UIP)

Key Milestones

• Pulse Polio Programme: India certified polio-free in 2014
• Mission Indradhanush: Intensified immunization to reach unvaccinated children
• COVID-19 vaccination: India ran the world's largest vaccination drive (CoWIN platform)

Cold Chain

• Vaccines must be maintained at 2-8°C (refrigerator temperature)
• Some vaccines (OPV) need -20°C (freezer)
• Ice-lined refrigerators (ILR) and deep freezers used at health centers

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Q15. Role of Lymphocytes in Immunity

Types of Lymphocytes

• Mature in bone marrow
• Have surface BCR (B cell receptor = surface Ig) and co-receptors (CD19, CD21)
• Function: Humoral immunity - produce antibodies
• After antigen stimulation → differentiate into plasma cells (antibody factories) and memory B cells
• Memory B cells enable rapid secondary immune responses

• Mature in thymus (undergo positive and negative selection)
• Have TCR (T cell receptor) + CD3 complex on surface
• Subtypes:
  a) CD4+ Helper T cells (Th)
  • Recognize antigen on MHC Class II molecules (on APCs)
  • Secrete cytokines to help B cells and CD8+ T cells
  • Th1 cells: Secrete IFN-γ, IL-2 → activate macrophages → intracellular pathogens (TB, Leishmania)
  • Th2 cells: Secrete IL-4, IL-5, IL-13 → help B cells → allergies, parasites
  • Th17 cells: Secrete IL-17 → neutrophil recruitment → extracellular bacteria, fungi
  • T regulatory cells (Treg): Suppress excessive immune responses; prevent autoimmunity
  b) CD8+ Cytotoxic T cells (CTL)
  • Recognize antigen on MHC Class I molecules (on all nucleated cells)
  • Kill virus-infected cells, tumor cells by releasing perforin and granzymes (induce apoptosis), and Fas-FasL interaction
  • Key in antiviral immunity and tumor surveillance
  c) Memory T cells
  • Long-lived; respond faster on re-exposure to same antigen
  • CD4+ and CD8+ memory subsets

• Large granular lymphocytes
• Part of innate immunity but classified as lymphocytes
• Kill virus-infected cells and tumor cells WITHOUT requiring antigen presentation or prior sensitization
• Use perforin/granzyme mechanism; inhibited by normal MHC-I expression

• Innate-like lymphocytes with limited receptor diversity
• Rapid response; bridge innate and adaptive immunity

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Q16. Differences Between Endotoxin and Exotoxin

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Q17. Microbial Virulence Factors

Categories of Virulence Factors

• Allow bacteria to adhere to host tissues (first step in infection)
• Pili/Fimbriae: E. coli (Type 1 pili bind uroepithelium → UTI); N. gonorrhoeae
• Surface proteins: M protein (Streptococcus pyogenes), protein A (S. aureus)
• Biofilm formation: protects from antibiotics and immune cells

• Enable penetration into host cells and tissues
• Hyaluronidase ("spreading factor"): Breaks down hyaluronic acid in connective tissue; produced by Streptococci, Staphylococci, Clostridia
• Collagenase: Clostridium - destroys collagen in connective tissue
• Coagulase: S. aureus - clots plasma, forming fibrin barrier protecting bacteria from phagocytosis
• Type III secretion systems: Salmonella, Yersinia - inject effector proteins into host cells

• Exotoxins and endotoxins
• Specific examples:
  • Leukocidins: Kill WBCs (S. aureus Panton-Valentine leukocidin - PVL)
  • Hemolysins: Lyse red blood cells (alpha-toxin of S. aureus, streptolysin O/S)
  • Enterotoxins: Cause diarrhea (cholera toxin, ETEC toxin)

• Capsule: Most important - prevents phagocytosis; examples: Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella, Cryptococcus neoformans
• M protein (S. pyogenes): Resists phagocytosis
• Coagulase (S. aureus): Fibrin coat protects from phagocytes
• Leucocidins: Kill phagocytes

• Prevent phagosome-lysosome fusion: Mycobacterium tuberculosis, Legionella
• Escape from phagosome into cytoplasm: Listeria monocytogenes (using listeriolysin O)
• Survive within phagolysosomes: Coxiella burnetii

• Protein A (S. aureus): Binds Fc region of IgG → neutralizes opsonization
• IgA proteases: N. gonorrhoeae, H. influenzae, S. pneumoniae - cleave secretory IgA
• Antigenic variation: Neisseria (pili variation), Borrelia, Trypanosoma (VSG switching), Influenza (antigenic drift/shift)

• Capsule prevents complement deposition
• Factor H binding proteins: Capture host complement regulator
• Sialic acid on surface (Neisseria) mimics self

• Iron is essential for bacterial growth; host sequesters iron in transferrin/lactoferrin
• Bacteria produce siderophores (e.g., enterobactin, aerobactin) to scavenge iron
• Examples: E. coli, Pseudomonas aeruginosa (pyoverdin)

• Plasmids: Carry genes for toxin production, antibiotic resistance, adhesins
• Pathogenicity Islands (PAI): Large genomic regions acquired by horizontal gene transfer carrying clusters of virulence genes (e.g., Salmonella PAI-1 and PAI-2)

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