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Microbiology Exam Questions - Comprehensive Answers

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1. MICROBIAL PATHOGENICITY AND DETERMINANTS OF VIRULENCE

Definitions

• Pathogenicity: The ability of a microorganism to cause disease in a susceptible host.
• Virulence: The degree or intensity of pathogenicity. It is a quantitative measure expressed as the LD50 (dose lethal to 50% of a test population).

Determinants of Virulence

A. Adherence Factors

• Pili/Fimbriae: Hair-like projections that allow attachment to host epithelial cells (e.g., N. gonorrhoeae pili, E. coli type 1 fimbriae).
• Adhesins: Surface proteins that bind specific host cell receptors.

B. Capsule (Antiphagocytic Factors)

• Polysaccharide capsule resists phagocytosis (e.g., Streptococcus pneumoniae, Klebsiella pneumoniae).
• M protein of Streptococcus pyogenes is another antiphagocytic factor.
• Protein A of S. aureus binds IgG Fc region, blocking opsonization.

C. Invasins / Invasion Factors

• Enzymes that facilitate spread through tissues:
  • Coagulase (S. aureus) - clots fibrin, forms protective coat
  • Hyaluronidase - breaks down connective tissue ("spreading factor")
  • Collagenase - digests collagen (Clostridium perfringens)
  • Streptokinase/Fibrinolysin - dissolves fibrin clots
  • IgA proteases - cleave secretory IgA (N. gonorrhoeae, H. influenzae)
  • Lecithinase - destroys cell membranes

D. Toxins

• Neurotoxins: Botulinum toxin (inhibits ACh release), tetanus toxin (inhibits inhibitory interneurons)
• Enterotoxins: Cholera toxin (stimulates adenyl cyclase), ETEC toxin
• Cytotoxins: Diphtheria toxin (inhibits EF-2, stops protein synthesis)
• Superantigens: S. aureus TSST-1, streptococcal pyrogenic exotoxins (activate T cells non-specifically)

E. Secretion Systems

• Type 3 SS ("needle and syringe"): Injects virulence proteins directly into host cell cytoplasm (e.g., Pseudomonas aeruginosa, Salmonella).
• Type 4 SS: Transports proteins or DNA (e.g., H. pylori CagA effector, Agrobacterium DNA transfer).
• Type 6 SS: Targets both eukaryotic cells and competing bacteria (V. cholerae, P. aeruginosa).
• Type 2 SS secretes AB toxins (cholera toxin).

F. Iron Acquisition (Siderophores)

• Iron is essential for bacterial growth. Most free iron is bound to transferrin/lactoferrin in the host.
• Siderophores (e.g., enterochelin, aerobactin) chelate iron from host proteins.

G. Biofilm Formation

• Bacteria encased in polysaccharide matrix on surfaces (e.g., P. aeruginosa in cystic fibrosis airways, biofilms on catheters and prostheses).
• Biofilm organisms are 100-1000x more resistant to antibiotics.

H. Antigenic Variation

• Switching surface antigens to evade host immune response (e.g., Borrelia recurrentis causes relapsing fever by switching outer surface proteins; N. gonorrhoeae switches pili/Opa/LOS antigens).

I. Pathogenicity Islands

• Clusters of virulence genes on bacterial chromosomes, acquired by horizontal gene transfer. Often flanked by insertion sequences.

Damage-Response Framework

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2. STERILIZATION AND DISINFECTION

Definitions

Classification of Sterilization Methods

A. Physical Methods

• Moist heat (steam) - see detailed section below
• Dry heat
• Pasteurization
• Boiling

• Ultraviolet (UV) light
• Ionizing radiation (gamma rays, X-rays)

• Membrane filters (0.22 μm pore size)
• Used for heat-labile solutions (sera, antibiotics, enzymes)

B. Chemical Methods

• Ethylene oxide gas
• Formaldehyde
• Glutaraldehyde
• Hydrogen peroxide
• Chlorine compounds
• Iodophors
• Phenolics
• Quaternary ammonium compounds
• Alcohols

METHODS OF STERILIZATION BY MOIST HEAT (Detailed)

Principle

Killing Kinetics

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1. Boiling (100°C)

• Kills most vegetative bacteria, fungi, and most viruses in 10-20 minutes.
• Does NOT kill bacterial spores or hepatitis B virus reliably.
• Activity level: High disinfection only.

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2. Pasteurization

• Kills: vegetative bacteria including Mycobacterium tuberculosis, Salmonella, Brucella, Listeria.
• Does NOT kill: spores, some thermophilic bacteria, hepatitis A virus.
• Uses: Milk, water, fruit juices, wine, beer, some plastic hospital equipment.

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3. Autoclave (Steam Under Pressure) - GOLD STANDARD

• Downward displacement (gravity displacement): Steam is lighter than air; air drains out through a valve at the bottom.
• Pre-vacuum autoclave: Chamber is evacuated before steam is introduced (better penetration).

• Cannot be used for heat-labile materials (plastics, lensed instruments, petroleum products immiscible with water).
• Oils and powders cannot be sterilized (steam cannot penetrate).

• Biological indicators: Spores of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) - killed at 121°C, 15 min.
• Chemical indicators: Bowie-Dick tape changes color.
• Temperature charts: Continuous recording.

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4. Tyndallization (Intermittent Sterilization / Fractional Sterilization)

• Heating at 80-100°C for 30 minutes on 3 consecutive days.
• First cycle kills vegetative organisms; spores survive but germinate.
• Second and third cycles kill germinated spores as new vegetative forms.
• Used for: heat-labile media that cannot tolerate autoclaving.

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Disinfectants - Classification and Mode of Action

• High-level: Kills all organisms except some spores (glutaraldehyde, H2O2, peracetic acid, moist heat). Used for semi-critical items (endoscopes).
• Intermediate-level: Kills most organisms including M. tuberculosis and fungi (alcohols, iodophors, phenolics).
• Low-level: Kills most bacteria/fungi and lipid viruses (quaternary ammonium compounds). Used for non-critical items (stethoscopes, blood pressure cuffs).

Tests for Efficiency of Disinfectants

1. Rideal-Walker (RW) Test (Phenol Coefficient Test):
   • Compares disinfectant effectiveness to phenol (standard).
   • Phenol Coefficient = MIC of phenol / MIC of disinfectant.
   • Limitation: Uses only one test organism (Salmonella typhi); not valid for non-phenolic compounds.
2. Chick-Martin Test: Modification of RW test; adds organic matter (feces) to more realistically simulate conditions.
3. Capacity Use-Dilution Test: Tests if the disinfectant remains effective after repeated use.
4. In-Use Test: Samples are taken from disinfectant solutions in actual use and cultured.
5. Kelsey-Sykes Test: Quantitative capacity test.

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3. POLYMERASE CHAIN REACTION (PCR) AND REAL-TIME PCR

Standard PCR

1. Denaturation (94-95°C): DNA double strand separates.
2. Annealing (50-65°C): Primers bind to complementary sequences flanking the target.
3. Extension/Elongation (72°C): Thermostable DNA polymerase (Taq polymerase) synthesizes new DNA strand.

• Template DNA
• Two specific oligonucleotide primers (forward and reverse)
• Thermostable Taq DNA polymerase (from Thermus aquaticus)
• dNTPs (deoxynucleotide triphosphates)
• Mg²+ (cofactor)
• Buffer

Real-Time PCR (Quantitative PCR / qPCR)

1. SYBR Green: Intercalating dye that fluoresces when bound to double-stranded DNA. Simple and inexpensive; works with any primer set. Limitation: binds to any dsDNA including primer dimers.
2. TaqMan Probes (Hydrolysis Probes):
   • A sequence-specific probe labeled with a fluorescent reporter (FAM) at the 5' end and a quencher (TAMRA) at the 3' end.
   • In intact probe, quencher suppresses fluorescence.
   • During extension, Taq polymerase's 5'-3' exonuclease activity cleaves the probe, separating reporter from quencher - fluorescence is released.
   • More specific than SYBR Green.

• Ct (Cycle Threshold): The cycle number at which fluorescence crosses a threshold above background.
• Low Ct = high initial amount of template (abundant target).
• High Ct = low initial amount (rare target).
• Using a standard curve of known concentrations, absolute or relative copy numbers can be determined.

• Quantitative (can determine viral load or bacterial copy numbers)
• Real-time monitoring
• Faster (no post-PCR gel electrophoresis needed)
• Lower risk of contamination (closed-tube system)
• Can detect small differences in template quantity

Applications of Real-Time PCR

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4. BACTERIAL GROWTH CURVE

The Four Phases

Log(N)
  |            ___________
  |           /           \
  |          /             \
  |         /               \___
  |________/
  |
  +--Lag--+--Log--+--Stationary--+--Decline--

• Log phase: Used for preparing cultures for biochemical/serological tests (predictable, uniform cell characteristics).
• Stationary phase: Maximum spore and toxin production; antibiotic production by Streptomyces.
• Guides fermentation industry (optimal harvest time).
• Determines appropriate sampling time in lab culture.

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5. ANAEROBIC CULTURE METHODS

Organisms Requiring Anaerobic Culture

• Clostridium (perfringens, tetani, botulinum, difficile)
• Bacteroides fragilis
• Fusobacterium, Prevotella, Porphyromonas
• Actinomyces, Peptostreptococcus

Methods

A. Mechanical Evacuation and Replacement

• Evacuation-replacement technique: Air is pumped out and replaced with O2-free gas mixture (N2 + H2 + CO2 - typically 80:10:10).

B. Chemical Oxygen Absorption - GasPak System

• McIntosh and Filde's anaerobic jar (GasPak):
  • Plates placed in airtight jar.
  • GasPak envelope generates H2 and CO2 when water is added.
  • H2 + O2 → H2O (catalyzed by palladium catalyst in lid).
  • Result: <1% O2, 5-10% CO2 in jar.
  • Indicator: Methylene blue (colorless = anaerobic conditions).

C. Thioglycollate Broth

• Contains sodium thioglycollate which absorbs oxygen.
• Resazurin indicator (pink = aerobic top layer; colorless = anaerobic bottom).
• Simple method for anaerobes; grows facultative, microaerophilic, and anaerobic organisms.

D. Anaerobic Glove Box / Workstation

• Complete anaerobic cabinet with airlock.
• All inoculation and examination done inside anaerobic atmosphere.
• Gold standard for strict anaerobes.

E. Candle Jar

• Creates ~5-10% CO2 (NOT anaerobic).
• Used for capnophilic organisms (N. gonorrhoeae, H. influenzae) - NOT true anaerobic technique.

F. Robertson's Cooked Meat (RCM) Medium

• Unsaturated fatty acids in meat particles absorb oxygen.
• Used for transport and culture of anaerobes, especially Clostridia.

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6. ANTIMICROBIAL SUSCEPTIBILITY TESTING

Purpose

Methods

A. Disc Diffusion (Kirby-Bauer Test)

• Organism inoculated onto Mueller-Hinton agar (standardized medium).
• Antibiotic-impregnated discs placed on surface.
• Antibiotic diffuses outward creating gradient.
• After 16-18 hr incubation, zone of inhibition measured.
• Zone size compared to CLSI/EUCAST breakpoints to determine S/I/R.

B. Broth Dilution

• Organism inoculated into tubes/wells with serial dilutions of antibiotic.
• MIC (Minimum Inhibitory Concentration): Lowest concentration that prevents visible growth.
• MBC (Minimum Bactericidal Concentration): Lowest concentration that kills 99.9% of organisms (subculture to antibiotic-free medium).
• Microdilution: Done in 96-well microtitre plates.

C. E-Test (Epsilometer Test)

• Plastic strip with continuous gradient of antibiotic (15 two-fold dilutions).
• Placed on inoculated agar surface.
• Elliptical inhibition zone forms; read MIC at point where zone intersects the strip.
• Combines accuracy of broth dilution with simplicity of disc diffusion.
• Gives precise MIC values.

D. Automated Systems

• VITEK, MicroScan, BD Phoenix: automated broth microdilution systems; provide results within 4-8 hours.

E. Molecular Methods

• PCR for resistance genes (mecA for MRSA, vanA for VRE, blaNDM for carbapenemase).
• MALDI-TOF MS: Used for identification; limited direct use for susceptibility (though some β-lactamase detection possible).

Breakpoints

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7. BACTERIAL TYPING METHODS

Phenotypic Methods

Genotypic Methods (Molecular Typing)

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8. PASTEURIZATION

• Named after Louis Pasteur.
• Purpose: Reduce pathogenic organisms without altering taste/nutritional quality.
• HTST (72°C for 15 seconds) is the most common commercial method.
• Organisms killed: M. tuberculosis (most heat-sensitive of relevant pathogens - the index organism for pasteurization), Brucella, Salmonella, Listeria, Staphylococcus, E. coli O157.
• Does NOT kill: Spores (Bacillus, Clostridium), Coxiella burnetii is relatively resistant (requires 72°C for 15 seconds minimally).

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9. PLASMIDS AND F PLASMID

Plasmid

• Extrachromosomal, circular, double-stranded DNA molecule.
• Self-replicating, independent of the bacterial chromosome.
• Non-essential for basic life but confer selective advantages.
• Carry genes for: antibiotic resistance (R plasmids), toxin production, virulence factors, fertility (F plasmid), colicins (Col plasmid), metabolism.

F (Fertility) Plasmid

• Also called the sex factor or F factor.
• ~94.5 kb in size.
• Carries genes for: synthesis of sex pili (F pili), conjugation machinery, transfer operon.
• Cells with F factor = F+ (donors) / Cells without = F- (recipients).

1. F+ × F-: F plasmid transfers to F- cell, converting it to F+. Chromosome rarely transfers.
2. Hfr (High Frequency Recombination): F plasmid integrates into bacterial chromosome. During conjugation, chromosomal DNA is transferred from origin of transfer (oriT) through the sex pilus. High frequency of chromosomal gene transfer; F plasmid rarely transferred completely.
3. F' (F prime): F plasmid excises imprecisely, carrying some bacterial chromosomal genes. Used in sexduction (F-duction) - transfers specific chromosomal genes to F- cells. Basis of complementation studies.

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10. METHODS OF GENETIC TRANSFER IN BACTERIA

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11. HOT AIR OVEN (Dry Heat Sterilization)

• Dry heat requires higher temperatures than moist heat because water is absent (no protein hydration).
• Mechanism: Oxidation of organic material; protein coagulation (less efficient than moist heat).

• Glassware (syringes, petri dishes, flasks)
• Metal instruments (scissors, forceps, scalpels)
• Non-aqueous liquids (oils, waxes, petroleum products - cannot be autoclaved as steam cannot penetrate)
• Powders (sulfonamides, starch, zinc oxide)

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12. TRANSPORT MEDIA

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13. BACTERIAL FLAGELLA AND DEMONSTRATION

Structure

• Thin, helical, proteinaceous appendages for motility.
• Composed of flagellin (protein) subunits assembled into a hollow cylinder.
• Structure: Filament → Hook → Basal body (anchored in cell membrane).
• Basal body has rings: L (outer membrane), P (peptidoglycan), S, M (cytoplasmic membrane) rings in Gram-negative; fewer rings in Gram-positive.

Types of Flagellation (arrangement)

Significance

• Motility
• Basis of H antigen (serotyping)
• Contributes to virulence (e.g., H. pylori's polar flagella help penetrate mucus)
• Strongly antigenic

Demonstration

1. Hanging drop preparation: Motility seen under darkfield/phase contrast microscopy. True motility (directional) vs. Brownian motion (random, non-directional).
2. Flagella staining: Flagella are too thin (~20 nm) to see directly. Special stains deposit a mordant (tannic acid, potassium alum) to increase thickness:
   • Leifson's flagella stain
   • Gray's flagella stain
   • Silver impregnation
3. Electron microscopy: Direct visualization.
4. Tube/plate motility test: Semisolid agar (0.2-0.5% agar); motile organisms spread from the line of inoculation.
5. Widal test: H antigens of Salmonella detected serologically.

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14. AUTOCLAVE

• Steam under pressure: 121°C, 15 psi, 15-20 minutes.
• Kills: ALL microorganisms including spores.
• Biological indicator: G. stearothermophilus spores.
• Control: Bowie-Dick test (for pre-vacuum autoclaves to test steam penetration).

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15. EXOTOXINS vs. ENDOTOXINS

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16. ROBERT KOCH'S CONTRIBUTIONS

1. Koch's Postulates (1876-1884): Criteria for proving causation of infectious disease: i. Organism found in all cases of disease. ii. Organism must be isolated from diseased host and grown in pure culture. iii. Pure culture must cause same disease when inoculated into healthy susceptible host. iv. Organism must be re-isolated from experimentally diseased host and shown to be identical to original.
2. Discovery of specific pathogens: M. tuberculosis (1882), V. cholerae (1883), Bacillus anthracis (1877).
3. Development of solid culture media: Introduced use of gelatin and then agar-agar (suggested by Frau Hesse) for solidifying culture media - allowed isolation of pure cultures.
4. Pure culture techniques: Developed methods (streak plate, pour plate) to isolate single colonies.
5. Staining techniques: Introduced bacterial staining with aniline dyes, enabling microscopic visualization.
6. Tuberculin: Developed purified protein derivative (PPD/tuberculin) for TB skin testing.
7. Koch's phenomenon: Described enhanced skin reaction on second exposure to TB antigens (basis of Mantoux test).

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