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MUHS Microbiology Paper 1 - Complete Topic Notes

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1. HIV (Human Immunodeficiency Virus)

• Envelope with glycoproteins gp120 (attachment) and gp41 (fusion)
• Core proteins: p24 (capsid - used in diagnosis), p17 (matrix)
• Enzymes: Reverse transcriptase, Integrase, Protease (all therapeutic targets)
• Two copies of single-stranded RNA

• Attaches to CD4 receptor + CCR5 or CXCR4 co-receptors on T helper cells, macrophages, dendritic cells
• Reverse transcriptase converts RNA to DNA -> integrates into host genome as provirus
• Progressive CD4+ T cell depletion -> immunodeficiency
• AIDS defined as CD4 count <200 cells/µL or AIDS-defining illness

• Acute retroviral syndrome: fever, rash, malaise, lymphadenopathy, pharyngitis, weight loss, diarrhea (2-4 weeks post exposure)
• Latent phase: asymptomatic, years
• AIDS: opportunistic infections (PCP, CMV, Toxoplasma, MAC, Cryptococcus), malignancies (Kaposi's sarcoma, NHL)

1. ELISA (4th generation: detects p24 Ag + anti-HIV Ab) - Screening
2. Western Blot - Confirmatory
3. CD4 count - Immunological staging
4. Viral load (HIV RNA by RT-PCR) - Monitoring treatment

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2. Hepatitis B

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3. Biomedical Waste Management

• Incineration: Yellow category (anatomical, cytotoxic, chemical waste). Destroys all including spores.
• Autoclaving: Red category (contaminated recyclable waste)
• Shredding + Autoclaving: Sharp waste (white category), then sent to recyclers
• Microwaving: Alternative to autoclaving
• Chemical disinfection: Liquid waste

• Segregation at source is the most important step
• Do NOT recap needles (causes needle-stick injury)
• Sharps containers must be filled only to 3/4 capacity
• Never mix sharp waste with other categories
• Sharp injuries: Wash with soap & water, report, post-exposure prophylaxis for HIV/HBV

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4. Widal Test

• O antigen (somatic, heat stable) - S. typhi O, S. paratyphi A, B
• H antigen (flagellar, heat labile) - S. typhi H, S. paratyphi A H, B H

• Serial dilutions of patient's serum (1:20 to 1:640 or beyond)
• Added to known antigens
• Incubated at 37°C overnight
• Agglutination observed

• O agglutinin: ≥1:80 (significant), ≥1:160 (highly significant)
• H agglutinin: ≥1:160 (significant)
• Fourfold rise in paired sera (10-14 days apart) is most diagnostic

• O agglutinins appear earlier (1st week) and indicate active/recent infection
• H agglutinins appear later and persist longer (may indicate past infection or vaccination)
• Should be done in 2nd week of illness for best sensitivity
• Negative in 1st week, unreliable in treated patients

• False positives: Other Salmonella species, liver diseases, malaria, connective tissue diseases, prior vaccination
• False negatives: Early disease, antibiotic treatment, immunocompromised patients
• Not useful alone for diagnosis - must correlate with clinical picture

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5. Complement System

• C3 convertase cleaves C3 into C3a and C3b
• C3b binds to microbial surface -> C5 convertase formation
• C5 cleaved to C5a + C5b
• C5b + C6 + C7 + C8 + C9 -> Membrane Attack Complex (MAC)

1. Opsonization: C3b coats microbes -> phagocytosis by neutrophils and macrophages (via CR1 receptors)
2. Inflammation (Anaphylatoxins): C3a, C4a, C5a -> mast cell degranulation, histamine release, vascular permeability, neutrophil chemotaxis
3. Cell Lysis: MAC creates pores in cell membrane -> osmotic lysis (especially effective against Neisseria)

• C1q, C4, C2 deficiency -> SLE-like syndrome
• C3 deficiency -> recurrent pyogenic infections (most severe)
• C5-C9 (MAC) deficiency -> recurrent Neisseria infections (meningococcal, gonococcal)
• Properdin deficiency -> Meningococcal infections

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6. Active and Passive Immunity

Active Immunity

• Produced by the host's own immune system in response to antigen
• Develops after natural infection or vaccination
• Slow onset (days to weeks)
• Long lasting (years to lifelong) due to memory B and T cells
• No immediate protection

Passive Immunity

• Ready-made antibodies transferred from another source
• No immune memory formed
• Rapid onset (immediate protection)
• Short duration (weeks to months) as antibodies are catabolized

Comparison Table

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7. ELISA (Enzyme-Linked Immunosorbent Assay)

1. Direct ELISA

• Antigen coated on plate
• Enzyme-linked primary antibody added
• Detects antigen directly
• Simple but less sensitive

2. Indirect ELISA (most common for serology)

• Antigen coated on plate
• Patient's serum (primary antibody) added
• Enzyme-linked secondary antibody added
• Detects antibodies in patient serum (e.g., HIV antibody testing, Weil Felix)

3. Sandwich ELISA (most sensitive)

• Capture antibody coated on plate
• Antigen (from sample) added -> binds to capture antibody
• Enzyme-linked detection antibody added
• Detects antigen
• Used to detect: HBsAg, HIV p24 antigen

4. Competitive ELISA

• Known antigen competes with test sample antigen for antibody
• Reduced color = more antigen in sample (inverse relationship)

• HIV screening (4th gen: detects both p24 Ag + anti-HIV Ab)
• HBsAg detection
• Anti-HBs, Anti-HCV
• Dengue NS1 antigen, dengue IgM/IgG
• TORCH infections
• Typhoid (Typhidot)

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8. Hepatitis A

• Mostly subclinical/mild, especially in children
• Prodromal phase: anorexia, nausea, fatigue, fever, RUQ pain
• Icteric phase: jaundice, dark urine, pale stools
• Self-limiting - does NOT cause chronic hepatitis or cirrhosis
• Fulminant hepatic failure rare (<0.1%) but can occur in older patients

• Improved sanitation and hygiene
• Safe water supply
• HAV vaccine: Inactivated vaccine; 2 doses; provides long-lasting protection
• Post-exposure prophylaxis: Normal human immunoglobulin (within 2 weeks)

• HAV: fecal-oral, no chronic state, no carrier state, no HCC
• HBV: parenteral/sexual, chronic state possible, carrier state, HCC risk

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9. Blood Cultures

• Brain Heart Infusion (BHI) broth - most common
• Nutrient broth
• Biphasic media (Castaneda bottle): liquid + solid phase in same bottle; subculture without opening
• Automated systems: BACTEC, BacT/ALERT (detect CO2 production by growing bacteria)

1. Collect before antibiotic therapy if possible
2. Take 2-3 sets from different sites (increases sensitivity)
3. Strict aseptic technique (skin prep with alcohol + iodine)
4. Volume: 10-20 mL in adults (3-5 mL in children)
5. Blood:broth ratio = 1:10 (dilutes antibodies and antibiotics)
6. Inoculate aerobic + anaerobic bottles
7. Incubate at 37°C and observe for turbidity, gas, hemolysis
8. Subculture to solid media; identify by colony morphology, biochemical tests, sensitivity

• For typhoid: best in 1st week (positive ~80-90%)
• For endocarditis: multiple sets over 24 hours
• Before antibiotic therapy whenever possible

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10. Sterilization

Methods of Sterilization

A. Physical Methods

• Most widely used, reliable, cheap, non-toxic
• Steam under pressure: 121°C for 15 minutes (standard cycle)
• Kills by denaturation of proteins and disruption of cell membranes
• Drop of 1.7°C increases required exposure time by 48%

• Hot air oven: 160°C for 1 hour OR 170°C for 30 min
• Used for glassware, oils, powders (items damaged by moisture)
• Less efficient than moist heat; requires longer exposure

• Used in laboratory for wire loops; also for biomedical waste (anatomical waste)
• Burns material completely

• UV radiation: Poor penetration, used for surface sterilization (OTs, BSCs)
• Ionizing radiation (gamma rays): Used for industrial sterilization of disposable items (syringes, sutures)

• Membrane filters (0.22 µm pore size) for heat-sensitive liquids (serum, antibiotics)
• Does NOT remove viruses

B. Chemical Methods

• For temperature/pressure-sensitive items (electronics, plastics, catheters)
• Exposure 4 hours + aeration 12 hours to remove toxic residue
• Highly efficient but flammable, explosive, carcinogenic

• Oxidizing agent; replaced many ethylene oxide applications
• No toxic by-products

Sterilization vs Disinfection

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11. Immunoglobulins (Antibodies)

• 4 polypeptide chains: 2 heavy (H) chains + 2 light (L) chains, joined by disulfide bonds
• Y-shaped molecule
• Variable region (Fab): Antigen-binding site (unique for each antibody)
• Constant region (Fc): Effector functions (complement activation, opsonization, FcR binding)
• Hinge region: Flexibility

• IgG: Only Ig that crosses the placenta (neonatal protection)
• IgM: First Ig in primary immune response; largest; most efficient complement activator (classical pathway)
• IgA: Most abundant Ig produced in total (mainly in secretions); protects mucosal surfaces
• IgE: Type 1 hypersensitivity; elevated in parasitic infections and atopy

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12. Bacterial Growth Curve

Phase 1: Lag Phase

• No increase in cell number
• Bacteria adapting to new environment
• Active synthesis of enzymes, RNA, proteins
• Duration depends on: inoculum size, medium composition, previous growth conditions
• Metabolically active but not dividing

Phase 2: Log (Exponential) Phase

• Rapid doubling at constant rate (generation time)
• Cell number increases geometrically (2^n)
• Most metabolically active; uniform cell population
• Generation time: varies (E. coli ~20 min, M. tuberculosis ~18-24 hours)
• Most sensitive to antibiotics (antibiotics like penicillin act on dividing cells)
• Used for research (cells most representative of species characteristics)

Phase 3: Stationary Phase

• Growth rate = Death rate (no net increase)
• Nutrients depleted, toxic metabolites accumulate
• Some bacteria form spores (Bacillus, Clostridium) at this stage
• Some bacteria produce exotoxins at this stage

Phase 4: Decline (Death) Phase

• Death rate exceeds growth rate
• Bacteria die due to nutrient depletion, toxic waste accumulation
• Some bacteria survive (spore-formers, acid-fast bacilli)

Generation Time

• Time for one doubling: E. coli = 20 min; Staph aureus = 30 min; M. tuberculosis = 18-24 hrs
• Formula: g = t / n (g = generation time, t = time elapsed, n = number of generations)

Continuous Culture (Chemostat)

• Artificial maintenance of log phase by continuously adding fresh media and removing old
• Used in industrial fermentation

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13. Helicobacter pylori (H. pylori)

• Most common chronic bacterial infection worldwide
• Transmission: fecal-oral, oral-oral, contaminated water
• Higher prevalence in developing countries (India: ~60-80%)

• Colonizes gastric antrum and body
• Urease -> ammonia -> damages gastric epithelium
• CagA (cytotoxin-associated gene A) - virulence factor; associated with ulcers and gastric cancer
• VacA (vacuolating cytotoxin) - induces vacuolation and cell death
• Induces chronic inflammation -> gastritis -> peptic ulcer -> gastric cancer

1. Chronic gastritis (most common)
2. Peptic ulcer disease (duodenal ulcer 95%, gastric ulcer 70%)
3. Gastric adenocarcinoma (WHO Class I carcinogen)
4. MALToma (mucosa-associated lymphoid tissue lymphoma)
5. Non-ulcer (functional) dyspepsia

• Proton Pump Inhibitor (omeprazole/pantoprazole) + Amoxicillin + Clarithromycin for 14 days
• Quadruple therapy (PPI + Bismuth + Metronidazole + Tetracycline) if clarithromycin resistance suspected
• Test of cure: Urease breath test or stool antigen test (4 weeks after completing treatment)

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Sources: Jawetz, Melnick & Adelberg's Medical Microbiology 28th Ed; Medical Microbiology 9e (Murray); Sherris & Ryan's Medical Microbiology 8th Ed; Robbins & Kumar Basic Pathology; Henry's Clinical Diagnosis by Laboratory Methods

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MUHS Microbiology Paper 1 - Topics 4 to 13 (Complete Answers)

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4. Widal Test

Definition

Note: The CDC does not recommend Widal test alone for typhoid diagnosis as it is difficult to interpret in endemic populations and false positives are common with previous Salmonella infection or vaccination. (Red Book 2021)

Principle

Antigens Used

Procedure - Tube Agglutination (Standard/Quantitative)

1. Prepare serial doubling dilutions of patient's serum: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
2. Add equal volumes of each antigen to respective tubes
3. Incubate at 37°C for 18-24 hours
4. Read for agglutination

Procedure - Slide Agglutination (Rapid/Qualitative)

• One drop of undiluted serum + one drop of antigen on a slide
• Mix and observe for agglutination within 1-2 minutes
• Only screening; must be confirmed by tube method

Interpretation (Significant Titers)

• Most diagnostic: Fourfold or greater rise in titer in paired sera (2nd sample taken 10-14 days after 1st)
• Best time to perform: 2nd week of illness (antibodies begin rising in 1st week, peak in 2nd-3rd week)

O vs H Agglutinins - Key Differences

Limitations

• Other Salmonella species (cross-reactions due to shared O antigens)
• Liver diseases (cirrhosis, hepatitis)
• Malaria, dengue
• Connective tissue diseases (SLE, RA)
• Previous vaccination or past typhoid infection (raises H titers)

• Early disease (1st week)
• Early antibiotic treatment
• Immunocompromised patients
• Partially treated cases

Gold Standard for Typhoid

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5. Complement System

Definition

Three Activation Pathways

A. Classical Pathway

• Trigger: Antigen-antibody complex (IgM or IgG bound to antigen)
• Initiating component: C1q binds to Fc region of IgM/IgG
• Sequence: C1 (C1q-C1r-C1s) -> cleaves C4 and C2 -> C4b2a = Classical C3 convertase
• Requires calcium ions (Ca²+) for C1 complex stability
• Also activated by CRP (C-reactive protein) binding microbial antigens

B. Alternative Pathway

• Trigger: Microbial surfaces directly (LPS/endotoxin, complex polysaccharides, fungal cell walls)
• No antibody required - part of innate immunity
• Components: Factor B, Factor D, Properdin (Factor P stabilizes convertase)
• Sequence: C3 undergoes spontaneous low-level hydrolysis -> C3b + Bb = C3bBb = Alternative C3 convertase
• Properdin stabilizes C3bBb on microbial surfaces

C. Lectin Pathway (MBL Pathway)

• Trigger: Mannose-binding lectin (MBL) or ficolins recognize carbohydrate patterns (mannose, fucose) on microbial surfaces
• No antibody required
• Components: MBL + MASP-1 + MASP-2 (MBL-associated serine proteases)
• MASP-2 cleaves C4 and C2 -> same C4b2a C3 convertase as classical pathway

Central Step: C3 Cleavage

C3 --[C3 convertase]--> C3a + C3b
C3b + C3 convertase --> C5 convertase
C5 --> C5a + C5b
C5b + C6 + C7 + C8 + poly-C9 --> MEMBRANE ATTACK COMPLEX (MAC)

Three Major Functions

• C3b coats microbial surfaces (opsonization)
• Phagocytes (neutrophils, macrophages) have CR1 receptors for C3b
• Enhances phagocytosis dramatically

• C3a, C4a, C5a are anaphylatoxins
• Cause mast cell degranulation -> histamine release
• Increase vascular permeability, vasodilation
• C5a is the most potent chemotactic factor for neutrophils

• C5b-C6-C7-C8 + multiple C9 molecules form a transmembrane pore
• Osmotic lysis of cells, especially Gram-negative bacteria like Neisseria

Complement Regulation

• C1-inhibitor (C1-INH): Inhibits C1r, C1s (deficiency = Hereditary Angioedema)
• Factor I + Factor H: Cleave C3b to inactive iC3b (alternative pathway regulation)
• CD59 (Protectin) + CD55 (DAF): Protect host cells from MAC (deficiency = PNH)

Complement Deficiency Diseases

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6. Active and Passive Immunity

Types of Immunity

ACTIVE IMMUNITY

• Develops following natural infection with pathogen
• Examples: Immunity after measles, chickenpox, mumps, typhoid
• Strong, long-lasting

• Deliberate introduction of antigen (vaccine) to stimulate immune response without causing disease
• Types of vaccines:
  • Live attenuated: BCG (TB), OPV (Salk - oral polio), MMR, Varicella, Rotavirus, Yellow fever
  • Killed/Inactivated: IPV (Salk - injectable polio), Pertussis, Rabies (Semple), Influenza, Typhoid Vi (parenteral)
  • Toxoids: Tetanus toxoid (TT), Diphtheria toxoid (DT)
  • Subunit/Recombinant: Hepatitis B vaccine (recombinant HBsAg), HPV vaccine, Pertussis (acellular)
  • Polysaccharide: Pneumococcal (PPSV23), Meningococcal, Typhoid Vi

• Onset: Slow (days to weeks)
• Duration: Long (years to lifelong)
• Memory: Yes (immunological memory)
• Use: Prevention

PASSIVE IMMUNITY

• IgG: Transferred across placenta via FcRn receptors (from 12 weeks, maximum in 3rd trimester). Protects neonate for 3-6 months
• Secretory IgA: Provided in breast milk (colostrum especially rich) - mucosal protection

• Administration of ready-made antibodies (immunoglobulins/antisera)

• Onset: Immediate
• Duration: Short (2-4 weeks to 3-6 months, depending on half-life of IgG)
• Memory: None
• Use: Treatment of established disease / emergency prophylaxis

Comparative Table

Herd Immunity

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7. ELISA (Enzyme-Linked Immunosorbent Assay)

Definition

Principle

1. One component (antigen or antibody) is bound to a solid phase (plastic microwell plate)
2. The complementary component from the test sample binds to it
3. An enzyme-linked antibody is added to detect bound complex
4. Enzyme substrate added -> color change proportional to amount of antigen/antibody

Commonly Used Enzymes

• Horseradish peroxidase (HRP) - substrate: TMB (tetramethylbenzidine) -> blue color
• Alkaline phosphatase - substrate: pNPP -> yellow color

Types of ELISA

1. Direct ELISA

• Antigen coated on plate
• Enzyme-labeled primary antibody added directly
• Simple, fast, but less sensitive
• No signal amplification

2. Indirect ELISA (Most common for antibody detection)

• Antigen coated on plate
• Patient serum (primary antibody) added -> binds antigen
• Enzyme-labeled secondary antibody (anti-human IgG) added -> binds primary antibody
• Substrate added -> color reaction
• Uses: HIV antibody screening, HBsAb, HCV Ab, TORCH serology
• Advantage: Signal amplification; one secondary antibody works for many assays

3. Sandwich ELISA (Most sensitive; for antigen detection)

• Capture antibody coated on plate
• Test sample (antigen) added -> captured by first antibody
• Enzyme-labeled detection antibody added (recognizes different epitope on same antigen)
• Substrate added -> color
• Uses: HBsAg, HIV p24 antigen, Dengue NS1 antigen, cytokine quantification
• Very high sensitivity and specificity

4. Competitive ELISA

• Known enzyme-labeled antigen competes with unlabeled test antigen for limited antibody
• Inverse relationship: More test antigen = less color
• Used for haptens and small molecules

Sandwich ELISA - Step by Step Diagram

[Plate] -> Coat with capture antibody
        -> Add patient sample (antigen)
        -> Add enzyme-labeled detection antibody
        -> Add substrate -> COLOR (OD reading)

4th Generation HIV ELISA (Combo Assay)

• Detects both p24 antigen AND anti-HIV antibody simultaneously
• Reduces window period to ~2 weeks
• Recommended for HIV screening

Advantages of ELISA

• High sensitivity and specificity
• Can be fully automated (ELISA reader)
• Can test hundreds of samples simultaneously
• Quantitative results possible
• No radioactivity (safer than RIA)

Applications in Microbiology

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8. Hepatitis A

Virus Properties

• Family: Picornaviridae, Genus: Hepatovirus
• Size: 27 nm
• Structure: Non-enveloped, icosahedral capsid, single-stranded positive-sense RNA virus
• Single serotype worldwide
• Stable in environment (acid-stable, resists temperatures up to 60°C for 1 hour; inactivated by boiling 100°C for 5 min, autoclaving, formalin, UV)

Epidemiology & Transmission

• Route: Fecal-oral (most important)
  • Contaminated water and food
  • Raw/partially cooked shellfish (filter feeders concentrate virus from contaminated seawater)
  • Food handlers who are infected
  • Close personal contact; institutional settings (schools, nurseries, daycare)
• No parenteral/sexual transmission (viremia is transient)
• HAV accounts for ~25% of acute hepatitis worldwide
• Higher prevalence in developing countries with poor sanitation

Incubation Period

• 15-50 days (mean 28 days) (Medical Microbiology 9e)

Pathogenesis

• HAV itself is NOT directly cytopathic
• Liver injury is immune-mediated - cytotoxic CD8+ T cells are the key mediators of hepatocellular damage
• Virus shed in feces for 2-3 weeks before and 1 week after onset of jaundice (most infectious in pre-icteric phase)

Clinical Features

• Fever, malaise, anorexia, nausea, vomiting, right upper quadrant pain
• Dark urine (bilirubinuria) appears at end of this phase

• Jaundice (scleral icterus first, then skin)
• Hepatomegaly, liver tenderness
• Pale stools

• Symptoms resolve, jaundice clears over 2-6 weeks

• Children: often asymptomatic or subclinical
• Adults: more symptomatic
• Does NOT cause chronic hepatitis - always self-limiting
• No carrier state
• Fulminant hepatic failure in ~0.1% (rare; more common in elderly and those with pre-existing liver disease)
• No associated HCC or cirrhosis

Laboratory Diagnosis

Treatment

• Supportive only - rest, adequate hydration, nutrition
• Avoid hepatotoxic drugs (alcohol, paracetamol overdose)
• No specific antiviral therapy
• Most recover completely within 3-6 months

Prevention

• 2-dose schedule (0 and 6-12 months)
• Available since 1995; reduced HAV rates by >95% in vaccinated populations
• Recommended for travelers to endemic areas, food handlers, MSM, chronic liver disease

• Effective if given within 2 weeks of exposure
• Short-term protection (3-6 months)

Comparison: Hepatitis A vs Hepatitis E (Both Fecal-Oral)

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9. Blood Cultures

Definition & Purpose

Indications

• Suspected bacteremia/septicemia
• Enteric fever (typhoid, paratyphoid)
• Infective endocarditis
• Fever of unknown origin (FUO)
• Immunocompromised patients with fever
• Meningitis, osteomyelitis, septic arthritis
• Neonatal sepsis

Media Used

Collection Procedure

1. Timing: Ideally before antibiotic therapy; collect at peak of fever (temperature rise) when bacteremia is highest
2. Number of sets: Minimum 2-3 sets from different venepuncture sites (increases sensitivity; helps distinguish true bacteremia from contamination)
3. Skin preparation: Clean with 70% alcohol, then 2% tincture iodine or chlorhexidine; allow to dry
4. Volume: Adults: 10-20 mL per set; Children: 3-5 mL; Neonates: 1-2 mL
5. Inoculation: One aerobic bottle + one anaerobic bottle per set
6. Blood:broth ratio = 1:10 (dilutes bactericidal antibodies, antibiotics, and complement in blood)
7. Incubate at 37°C

Incubation and Subculture

• Manual bottles: Observe daily for turbidity, gas production, hemolysis, clot formation
• Subculture to solid media (Blood agar, MacConkey agar) at 24 hours, 48 hours, Day 5, Day 7 (for Brucella - up to 6 weeks)
• Automated systems flag positive bottles automatically
• Routine incubation: 5-7 days (except Brucella/Fungi: 4-6 weeks)

Identification After Growth

• Gram stain of positive broth
• Subculture on appropriate solid media
• Colony morphology, biochemical tests (API strip, VITEK)
• Antimicrobial susceptibility testing (AST/ABST)

Timing for Specific Infections

Common Blood Culture Isolates

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10. Sterilization

Definition

Difference from Disinfection

Methods of Sterilization

A. PHYSICAL METHODS

• Most widely used, reliable, cheap, non-toxic, and efficient method
• Kills by denaturation and coagulation of proteins
• Standard cycle: 121°C for 15 minutes at 15 lbs/inch² (103 kPa) pressure
• Flash sterilization: 134°C for 3 minutes
• Caution: A drop of 1.7°C increases required exposure time by 48%
• Uses: Surgical instruments, dressings, culture media, rubber goods, glassware
• Cannot use for: Heat-sensitive items (plastics, rubber that melts, electronics)

• Temperature: 160°C for 1 hour OR 170°C for 30 minutes OR 180°C for 20 minutes
• Kills by oxidation of cellular components
• Requires longer exposure and higher temperature than moist heat (dry heat less efficient)
• Uses: Glassware, metal instruments, oils, powders, waxes - items damaged by moisture
• Cannot be used for: Rubber, fabrics, dressings

• Most effective method - complete destruction by burning
• Wire loops in microbiology lab
• Biomedical waste (anatomical waste, infectious waste)
• Not used for instruments (destroys them)

• UV radiation (253.7 nm wavelength):
  • Kills by causing thymine dimer formation in DNA
  • Poor penetration (only surfaces and air)
  • Used for: OT air sterilization, biosafety cabinets (BSC), surface decontamination
  • Not for instruments or liquids
• Ionizing radiation (Gamma rays from Cobalt-60):
  • Kills by causing strand breaks in DNA and free radical formation
  • Excellent penetration through materials
  • Used for: Industrial sterilization of disposable items - syringes, catheters, sutures, gloves, prosthetics
  • Food sterilization

• Membrane filters: 0.22 µm (removes bacteria and fungi)
• Used for heat-sensitive liquids: serum, antibiotics, toxins, vaccines, culture media supplements
• Limitation: Does NOT remove viruses or mycoplasma (0.1 µm filters needed for viruses)
• Types: Millipore filter (cellulose acetate), Seitz filter (asbestos), Candle filter (ceramic - Chamberland, Berkfeld)

B. GASEOUS/CHEMICAL METHODS

• Used for: Temperature/pressure-sensitive items (plastics, rubber, electronics, catheters, endoscopes, cardiac catheters, disposable items)
• Exposure: 4 hours + aeration 12 hours (to remove toxic residue before use)
• Kills by alkylating DNA (highly effective against all microbes including spores)
• Limitations: Flammable, explosive, carcinogenic to laboratory animals; strictly regulated
• Replaced by plasma sterilization where possible

• Plasma gas sterilization: H₂O₂ vaporized, then activated by microwave/radio-frequency energy to form reactive free radicals
• No toxic by-products (H₂O + O₂)
• Has replaced many EO applications
• Cannot be used with materials that absorb H₂O₂

• Excellent antimicrobial activity (oxidizing agent)
• End products: acetic acid + oxygen (non-toxic)
• Used for sterilization of instruments

• High-level disinfectant/sterilant when used for prolonged contact (10 hours = sterilization; 20-30 min = high-level disinfection)
• Used for: Endoscopes, instruments that cannot be autoclaved
• Toxic - handle with care, use in ventilated area

Summary Table

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11. Immunoglobulins (Antibodies)

Definition

Basic Structure

• 4 polypeptide chains: 2 identical Heavy (H) chains + 2 identical Light (L) chains
• Chains linked by disulfide bonds (inter and intra-chain)
• Y-shaped molecule

Regions of Immunoglobulin

Five Immunoglobulin Classes

IgG

• Most abundant in serum (75-80% of total serum Ig)
• 4 subclasses: IgG1 (most common), IgG2, IgG3, IgG4
• Monomer
• Only Ig that crosses the placenta (via FcRn receptors) - provides neonatal immunity for first 3-6 months
• Half-life: ~23 days (longest)
• Functions: Secondary immune response, opsonization, complement activation (classical), ADCC (antibody-dependent cellular cytotoxicity), neutralization of toxins and viruses

IgM

• First Ig to appear in primary immune response
• Largest immunoglobulin (molecular weight ~900,000 Da)
• Pentamer (5 monomers joined by J chain) - 10 antigen-binding sites (but usually ~5 functional due to steric hindrance)
• Present mainly in intravascular compartment
• Most efficient activator of classical complement pathway
• Functions: Primary immune response, ABO blood group agglutination, rheumatoid factor (IgM anti-IgG), cold agglutinins
• Half-life: ~5 days

IgA

• Most abundant Ig produced in total by the body (mainly secreted at mucosal surfaces)
• Serum IgA: Monomer; 10-15% of serum Ig
• Secretory IgA (SIgA): Dimer joined by J chain + Secretory Component (SC)
  • SC protects SIgA from proteolytic digestion in GI tract
  • Found in: saliva, tears, nasal secretions, breast milk (especially colostrum), bronchial secretions, intestinal secretions
• 2 subclasses: IgA1 (serum) and IgA2 (secretions - shorter hinge, more resistant to bacterial proteases)
• Function: Mucosal immunity - prevents adherence of pathogens to mucosal epithelium (immune exclusion); protects newborn gut (via breast milk)
• Half-life: ~6 days

IgE

• Lowest concentration in serum (normal <0.0003 mg/mL)
• Monomer (like IgG) but with extra CH domain instead of hinge
• Binds with very high affinity to FcεRI receptors on mast cells and basophils
• When antigen (allergen) cross-links IgE on mast cells -> mast cell degranulation -> histamine, leukotrienes, prostaglandins
• Functions:
  • Type I Hypersensitivity (allergy, anaphylaxis)
  • Defense against helminths (parasitic worms) - elevated in parasitic infections
• Half-life: ~2 days (in serum); much longer when bound to mast cells

IgD

• Trace amounts in serum
• Present on surface of naive B lymphocytes (as B cell receptor - BCR - along with IgM)
• Function: B cell activation/maturation - serves as antigen receptor on B cells; signals for B cell activation when antigen binds
• Half-life: ~3 days

Summary Table of Immunoglobulin Classes

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12. Bacterial Growth Curve

Definition

The Four Phases

Phase 1: LAG PHASE

• No increase in cell numbers (population remains stationary)
• Bacteria are metabolically active but NOT dividing
• Cells adapting to new environment:
  • Synthesizing new enzymes and RNA
  • Repairing any damage
  • Accumulating sufficient intermediary metabolites and co-enzymes
• DNA replication begins, but no cell division yet
• Duration depends on:
  • Age of inoculum (older culture = longer lag)
  • Size of inoculum
  • Previous growth conditions vs new medium composition
  • Temperature change

Phase 2: LOG (EXPONENTIAL) PHASE

• Cells dividing at maximum constant rate by binary fission
• Cell number doubles at regular intervals (generation time/doubling time)
• Population grows geometrically: N = N₀ × 2ⁿ (where n = number of generations)
• Cells are: uniform in size, physiology, and biochemistry; most representative of species
• Most metabolically active phase
• Most sensitive to antibiotics (especially cell wall synthesis inhibitors like penicillin/beta-lactams that act on dividing cells)
• Used for:
  • Preparing cultures for experiments
  • Determining generation time
  • Industrial fermentation

g = t / n; where n = (log N - log N₀) / log 2 = 3.32 × log(N/N₀)

Phase 3: STATIONARY PHASE

• Growth rate = Death rate (no net increase in population)
• Reasons:
  • Nutrients becoming depleted (especially carbon, nitrogen source)
  • Toxic metabolic products accumulating (acids, alcohol, H₂O₂)
  • Oxygen depletion (for aerobes)
  • pH changes
• Biologically important events during stationary phase:
  • Spore formation (e.g., Bacillus, Clostridium) as survival mechanism
  • Production of many exotoxins (e.g., C. perfringens alpha toxin, S. aureus TSST-1)
  • Production of secondary metabolites (antibiotics in industrial fermentation)
  • Some regulatory changes in gene expression

Phase 4: DECLINE (DEATH) PHASE

• Death rate exceeds growth rate - population falls
• Nutrients completely exhausted, toxins at lethal levels
• Irreversible damage to cellular components
• Survival of resistant forms: spores (Bacillus, Clostridium), acid-fast bacilli, cysts
• Rate of death varies: some bacteria show logarithmic death (parallel to log growth)

Graphic Representation

Log viable count
    |      ___________
    |     /           \
    |    /             \
    |   /               \
    |__/                 \___
    Lag  Log  Stationary  Decline
    |----|----|-----------|------> Time

Continuous Culture (Chemostat)

• Method to maintain bacteria in perpetual log phase
• Fresh medium continuously added + old medium + bacteria continuously removed
• Growth rate controlled by nutrient concentration (growth-limiting nutrient)
• Used in: Industrial fermentation (production of antibiotics, vaccines, enzymes), research

Measurement of Bacterial Growth

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13. Helicobacter pylori

Classification & Morphology

• Family: Helicobacteraceae
• Gram-negative, curved/spiral-shaped (helical) rod ("S-shaped" or "seagull wing" morphology)
• Microaerophilic (requires 5-10% O₂, 5-10% CO₂)
• Motile: 2-6 sheathed polar flagella (lophotrichous)
• Urease-positive at very high levels - characteristic feature

Key Virulence Factors

Epidemiology

• Most common chronic bacterial infection worldwide: infects ~50% of global population
• India: prevalence ~60-80% (developing countries > developed)
• Transmission: Fecal-oral (contaminated water, food) and oral-oral routes
• Humans are the only significant reservoir
• Usually acquired in childhood; persists lifelong if untreated
• No seasonal pattern

Pathogenesis

1. H. pylori colonizes gastric antrum (and body in heavy infection)
2. Urease -> ammonia -> damages protective mucus layer and epithelium
3. CagA + VacA -> epithelial cell damage, pro-inflammatory cytokines (IL-8), neutrophil infiltration
4. Chronic active gastritis -> loss of gastric glands
5. Progression: Gastritis -> Gastric atrophy -> Intestinal metaplasia -> Dysplasia -> Gastric adenocarcinoma
6. OR: Antral gastritis -> increased gastrin -> increased acid -> Duodenal ulcer
7. Chronic antigen stimulation of mucosa-associated lymphoid tissue -> MALToma

Diseases Caused

1. Chronic gastritis (most common - asymptomatic usually)
2. Peptic ulcer disease:
   • Duodenal ulcer: ~95% associated with H. pylori
   • Gastric ulcer: ~70% associated with H. pylori
3. Gastric adenocarcinoma - WHO Class I (definite) carcinogen
4. MALToma (Mucosa-Associated Lymphoid Tissue lymphoma) - eradication of H. pylori alone can treat early-stage MALToma
5. Non-ulcer (functional) dyspepsia

Diagnosis

Invasive Tests (require endoscopic biopsy)

Non-Invasive Tests

Treatment

First-Line: Standard Triple Therapy (14 days)

• PPI (omeprazole 20 mg BD or pantoprazole 40 mg BD)
• Amoxicillin 1 g BD
• Clarithromycin 500 mg BD

Second-Line: Bismuth Quadruple Therapy (10-14 days)

• PPI + Bismuth subcitrate + Metronidazole + Tetracycline

Concomitant Therapy (all four without bismuth):

• PPI + Amoxicillin + Clarithromycin + Metronidazole

Test of Cure

• Perform 4 weeks after completing treatment
• Best tests: UBT or Stool Antigen Test
• Serology NOT used (antibody titers decline slowly over months)

Important Points for MUHS Exam

• H. pylori is a WHO Class I carcinogen for gastric cancer
• Eradication of H. pylori heals peptic ulcers and prevents recurrence (recurrence rate without eradication: 60-80%/year; with eradication: <5%)
• Early-stage MALToma may completely regress with H. pylori eradication alone
• Clarithromycin resistance is increasing worldwide - major treatment challenge
• Stains for histology: Giemsa (routine), Warthin-Starry silver (best specificity), Genta stain

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Sources: Medical Microbiology 9e (Murray et al.); Sherris & Ryan's Medical Microbiology 8th Ed; Jawetz, Melnick & Adelberg's Medical Microbiology 28th Ed; Robbins & Kumar Basic Pathology; Roitt's Essential Immunology; Janeway's Immunobiology 10e; Textbook of Family Medicine 9e; Red Book 2021 (AAP)