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Introduction to General Microbiology - MBBS Level

Comprehensive Lecture Notes

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1. Introduction to General Microbiology

What is Microbiology?

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Classification of Microorganisms

Prokaryotes (Bacteria)

• Primary distinguishing feature: absence of a nuclear membrane
• DNA is a single circular chromosome (~1 mm if stretched linearly), organized into a nucleoid
• Haploid - only one chromosome
• Size: typically 0.5-5 μm in diameter
• Gene count varies: 468 genes (Mycoplasma genitalium) to 7,825 (Streptomyces coelicolor)

Eukaryotes

Non-cellular Infectious Agents

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2. Microbiological Laboratories and Their Equipment

Types of Microbiology Laboratories

Essential Laboratory Equipment

Microscopes (covered in detail in Section 4)

• Light (brightfield) microscope
• Darkfield microscope
• Phase-contrast microscope
• Fluorescence microscope
• Electron microscope (TEM and SEM)

Sterilization Equipment

• Autoclave (steam sterilizer): Uses saturated steam at 121°C for 15-20 minutes at 15 psi. Kills all microorganisms including spores. Gold standard for moist heat sterilization.
• Hot air oven: Dry heat at 160-180°C for 1-2 hours. For glassware, oils, powders.
• UV lamps: Surface decontamination of laminar flow hoods.
• Bunsen burner: Flame sterilization of inoculating loops.

Culture Equipment

• Incubators: Maintain temperature (37°C for most pathogens)
• CO₂ incubators: For fastidious organisms requiring 5-10% CO₂
• Anaerobic jars / chambers: For anaerobic culture
• Water bath: Precise temperature for enzyme reactions
• Refrigerators/freezers: Storage of specimens and media at 4°C, -20°C, -80°C

Other Equipment

• Laminar flow hood (BSC - Biological Safety Cabinet): Protects both specimen and worker
• Centrifuge: Sedimentation of cells, concentration of specimens
• pH meter / spectrophotometer
• Vortex mixer
• Inoculating loops and needles
• Petri dishes, tubes, flasks

Culture Media

• Nutrient broth/agar: General purpose (non-selective)
• Selective media: Inhibit unwanted organisms (e.g., MacConkey - inhibits Gram-positives)
• Differential media: Distinguish between organisms by color change (e.g., Blood agar - hemolysis patterns)
• Enrichment media: Enhance growth of target organism (e.g., Selenite F broth for Salmonella)

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3. Safety Rules for Working in a Microbiological Laboratory

General Safety Principles

Personal Protective Equipment (PPE)

1. Lab coat - Always worn; removed before leaving lab
2. Gloves - Nitrile or latex; changed between specimens
3. Eye protection - Goggles or safety glasses when splash risk
4. Face mask/respirator - For BSL-3 organisms (e.g., TB)
5. Closed-toe shoes - Mandatory in all labs

Key Safety Rules

Before Work

• Tie back hair; remove jewelry
• Check equipment for damage before use
• Know location of fire extinguisher, eyewash station, emergency exits
• Never work alone when handling BSL-3 or higher pathogens

During Work

• No eating, drinking, or mouth pipetting - ever
• Work with cultures inside biosafety cabinet when open vessels are involved
• All work surfaces must be kept clean; decontaminate spills immediately with 70% alcohol or 10% bleach
• Use the closed-cap technique when transporting specimens
• Label all specimens and cultures clearly
• Handle sharps (needles, broken glass) with forceps, never fingers; dispose in sharps containers
• Minimize aerosol generation (centrifuge with sealed rotors)

After Work

• Discard all biological waste in biohazard bags or autoclave waste bins
• Decontaminate work surface with appropriate disinfectant
• Remove gloves using the "glove-in-glove" technique
• Wash hands thoroughly before leaving (even if gloves were worn)
• Sterilize reusable equipment before cleaning

Accidents and Spills

• Skin/mucous membrane exposure: Wash with soap and water ≥15 minutes; report to supervisor
• Eye splash: Flush with eyewash for ≥15 minutes
• Spill of infectious material: Cover with disinfectant-soaked cloth for 15-30 min; then clean up wearing PPE
• Needle stick: Wash, report, follow post-exposure prophylaxis protocol

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4. Bacterial Morphology

Size of Bacteria

Basic Shapes (Morphotypes)

1. Cocci (spherical)

2. Bacilli (rod-shaped)

3. Spiral Forms

Bacterial Cell Structure

Surface Structures

• Monotrichous - single polar flagellum (Vibrio cholerae)
• Lophotrichous - tuft at one pole
• Amphitrichous - flagella at both poles
• Peritrichous - flagella all around (Salmonella, E. coli)

Cell Envelope

• Thick peptidoglycan layer (20-80 nm) - multilayered
• Teichoic acids (major antigen)
• No outer membrane
• Retains crystal violet - appears purple on Gram stain

• Thin peptidoglycan layer (2-7 nm)
• Outer membrane containing lipopolysaccharide (LPS/endotoxin)
• LPS components: Lipid A (toxic), Core polysaccharide, O-antigen (somatic antigen)
• Periplasmic space between inner and outer membranes
• Decolorized by alcohol - appears pink/red after safranin counterstain

• Phospholipid bilayer with embedded proteins
• Site of electron transport chain in bacteria (unlike eukaryotes where this occurs in mitochondria)
• Lacks sterols (except Mycoplasma which incorporates cholesterol from the host)

Internal Structures

Endospores

• Form when nutrients are depleted
• Extremely resistant to heat, desiccation, UV, disinfectants
• Contain calcium dipicolinate (5-15% dry weight) - contributes to heat resistance
• Structure layers (outside to inside): Exosporium → Spore coat → Cortex → Spore wall → Core

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5. Microscopic Research Methods

Types of Microscopes

1. Brightfield (Light) Microscopy

• Oil immersion (100×) × Ocular (10×) = 1000× total - necessary to see bacteria

• Best resolution: ~0.2 μm
• Oil between lens and slide reduces light dispersion, improves resolution
• Limitation: refractive indices of organisms and background are similar; staining required

2. Darkfield Microscopy

• Special condenser prevents direct light reaching specimen; only oblique scattered light enters the lens
• Organisms appear bright against black background
• Resolving power: 0.02 μm (10× better than brightfield)
• Best for thin spirochetes: Treponema pallidum (syphilis), Leptospira
• Cannot study internal structure (light passes around, not through)

3. Phase-Contrast Microscopy

• Different densities within the cell cause light beams to travel at different speeds, shifting their phase
• Annular rings in condenser and objective lens amplify phase differences
• Creates 3-dimensional image of internal structures
• No staining needed
• Useful for examining live, unstained organisms

4. Fluorescence Microscopy

• Fluorochromes absorb UV/blue light and emit longer-wavelength visible light
• Light source: high-pressure mercury, halogen, or xenon vapor lamp
• Examples of fluorochromes:
  • Auramine-rhodamine - stains mycobacteria (TB screening)
  • Acridine orange - stains nucleic acids
  • FITC (fluorescein isothiocyanate) - conjugated to antibodies
• Organisms appear brightly illuminated against dark background
• Can screen rapidly at low magnification
• Direct immunofluorescence (DIF): Fluorochrome-labeled antibody applied directly to specimen
• Indirect immunofluorescence (IIF): Two-step; unlabeled primary antibody + fluorochrome-labeled secondary antibody

5. Electron Microscopy

• Magnetic coils direct a beam of electrons (not light); much shorter wavelength
• Can visualize individual viral particles (not just inclusion bodies)
• Specimens coated with metal ions for contrast

• Today used primarily as a research tool, not routine diagnostics
• Nucleic acid amplification tests (NAATs) have replaced EM in clinical diagnosis

Specimen Preparation Methods

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6. Simple Staining Methods

Principle of Staining

• Basic stains: Colored cation + colorless anion (e.g., methylene blue⁺ chloride⁻)
  • Bacteria are rich in nucleic acids with negatively charged phosphate groups
  • These attract and bind positively charged basic dyes
  • Most commonly used for routine staining
• Acidic stains: Colorless cation + colored anion (e.g., sodium⁺ eosinate⁻)
  • Repelled by the negatively charged bacterial surface
  • Used to stain the background (negative staining)

A. Simple (Direct) Staining

1. Prepare smear on clean glass slide
2. Air dry, then heat-fix (pass through flame 2-3×) or methanol-fix
3. Flood slide with stain for 1-3 minutes
4. Rinse gently with water
5. Blot dry (do not rub)
6. Examine under oil immersion

B. Gram Stain (Differential Stain)

Procedure (4 steps):

Interpretation:

Mechanism of Gram reaction:

• In Gram-positive bacteria: alcohol dehydrates the thick peptidoglycan, closing the pores and trapping the crystal violet-iodine complex → remains purple
• In Gram-negative bacteria: alcohol dissolves the lipid outer membrane, creating pores in the thin peptidoglycan → CV-I complex washed out → takes up pink safranin counterstain

Organisms that do not Gram-stain reliably:

• Mycobacteria - high lipid content (waxy mycolic acid) in wall; use acid-fast stain
• Mycoplasma - no cell wall
• Spirochetes - too thin (use darkfield)
• Rickettsia, Chlamydia - intracellular, special stains

C. Acid-Fast Stain (Ziehl-Neelsen Stain)

Procedure:

Interpretation:

D. Negative Staining

• Background is stained with an acidic dye (nigrosin or India ink); bacteria remain unstained (colorless)
• Because acidic dye is repelled by negatively charged bacterial surface
• Not a heat-fixed smear; cells remain in natural state (no shrinkage artifacts)
• Uses:
  • Demonstrate capsules (unstained halo around organism)
  • Identify delicate organisms easily distorted by heat fixing
  • Cryptococcus neoformans in CSF (India ink - capsule = clear halo)

Summary Table - Staining Methods

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Key Points for MBBS Exams

1. Gram stain - most important routine stain; decolorization step is the critical differential step
2. Resolving power of light microscope = 0.2 μm; only bacteria and larger can be seen (not viruses)
3. Oil immersion (100× objective) needed to see bacteria; total magnification = 1000×
4. Darkfield - for spirochetes (Treponema pallidum); 10× better resolution (0.02 μm)
5. Acid-fast - for mycobacteria; heat drives carbolfuchsin into waxy wall; acid-alcohol cannot remove it
6. Endospores are the most resistant biological entities; require autoclave (121°C, 15 min) to destroy
7. Prokaryote = no nuclear membrane; 70S ribosomes; single circular chromosome
8. LPS (endotoxin) is a component of the Gram-negative outer membrane; Lipid A is the toxic moiety
9. Fluorescence microscopy - auramine-rhodamine for TB screening (faster than ZN)
10. All biohazardous waste must be autoclaved before disposal; sharps in sharps containers

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