Microbiology and Molecular Biology (M20003) Lecture Notes

Microbiology and Molecular Biology (M20003) Lecture Notes

Part 1: Microbial Cell Morphology

Prokaryotic vs Eukaryotic Cells

  • Prokaryotic Cells:

    • No true nucleus (DNA is bound with proteins, forming an amorphous nucleoid).

    • Lack organelles.

    • Possess cytoplasmic membrane, cytoplasm, and ribosomes.

  • Eukaryotic Cells:

    • True nucleus containing DNA.

    • Contain membrane-enclosed organelles such as mitochondria, chloroplasts, plastids, and Golgi complex.

Electron Micrographs of Sectioned Cells

  • Heliobacterium modesticaldum: approximately $1 imes 3 ext{ μm}$

  • Methanopyrus kandleri: approximately $0.5 imes 4 ext{ μm}$

  • Saccharomyces cerevisiae (budding yeast): approximately $8 ext{ μm}$ in diameter

Bacterial Morphology

  • Shapes:

    • Bacillus (rod)

    • Cocci (spherical)

    • Spirillum (spiral)

    • Spirochaetes (corkscrew)

    • Vibrio (comma)

    • Filamentous (thread)

  • Arrangements:

    • Single cells (mono-)

    • Pairs (diplo-)

    • Chains (e.g., Streptococcus)

    • Clusters (e.g., Staphylococcus)

Flagella

  • Structure:

    • Helical, composed of protein flagellin.

    • Move by rotation (like a propeller).

  • Types and Movement:

    • Peritrichously Flagellated Organisms: Move in a straight line, slow and deliberate.

    • Polarly Flagellated Organisms: Move rapidly, spinning and dashing from place to place.

  • Motility Responses:

    • Chemotaxis (movement toward or away from chemicals)

    • Phototaxis (movement toward or away from light)

    • Magnetotaxis (movement in response to magnetic fields)

  • Speed:

    • Can propel cells through liquid at up to $60$ cell lengths/sec.

Fimbriae and Pili

  • Fimbriae:

    • Filamentous structures composed of protein, aiding in attachment to surfaces and biofilm formation.

    • Key in pathogens like Salmonella, Neisseria gonorrhoeae, and Bordetella pertussis for human tissue attachment.

  • Pili:

    • Longer than fimbriae, facilitating genetic exchange (conjugation) and adhesion to host tissues (e.g., by Neisseria species).

    • Type IV Pili: Assist in gliding motility along surfaces.

Other External Morphological Characteristics

  • Stalks, hyphae, and appendages.

  • Capsules: Protective layers aiding in survival and virulence.

Endospore Formation

  • Definition:

    • Certain bacterial species produce endospores as survival structures.

  • Distribution: Common in species found in soil (e.g., Bacillus species).

  • Function:

    • Enable survival during nutrient depletion, extreme temperatures, and drying.

    • Serve as the dormant stage in the bacterial life cycle.

    • Highly resistant to heat, desiccation, harsh chemicals, and radiation.

    • Easily dispersed by wind, water, or through animal guts.

  • Viability: Endospores can remain viable for millions of years.

Structure of Endospores

  • Core: Contains dehydrated cytoplasm, DNA, ribosomes, and enzymes.

  • Cortex: Protects the core and is mainly composed of peptidoglycan.

  • Spore Coat: Contains cross-linked proteins granting impermeability to most chemicals (e.g., acids and alcohols).

  • Activation: Requires only water to reactivate vegetative growth.

Bacillus anthracis

  • A Gram-positive, facultatively anaerobic saprophytic soil bacterium.

  • Known for causing anthrax and is part of Robert Koch's postulates.

  • Spore formation leads to infections primarily in animals and humans (cattle and farmers).

  • Forms of Infection:

    • Localized (skin), gastrointestinal, or pulmonary infections.

Anthrax as a Bioweapon

  • Causes severe systemic infection if acquired through mucous membranes of lungs.

  • Endospores can be disseminated in aerosols; resistant to heat and drying, making them highly lethal in biological warfare.

Bacillus cereus

  • Present in uncooked rice and may survive cooking.

  • Spores germinate at room temperature, producing enterotoxins causing food poisoning (nausea, vomiting, diarrhea).

  • Precaution: Cooked rice must be stored promptly to prevent germination.

Internal Cell Structure of Prokaryotes

Cell Components

  • Cytoskeleton: Composed of proteins that provide structure and aid in cell division.

  • Nucleoid: Area containing chromosomal DNA, not membrane-bound, taking up $¼ - ½$ of the cell volume.

  • Plasmids: Small, circular extrachromosomal DNA molecules often encoding non-essential but advantageous genes (like antibiotic resistance).

  • Ribosomes: Composed of $23S$, $16S$, and $5S$ rRNA, used in protein synthesis.

Plasmids

  • Usually circular and double-stranded DNA, sizes ranging from <1kb to several Mb.

  • Encode properties such as antibiotic resistance and can replicate independently; sometimes transferred between bacteria.

  • Application: Used in genetic engineering and biotechnology.

Cell Inclusions

  • Serve as energy reserves and structural building blocks (carbon polymers, phosphates, sulfur).

  • Magnetosomes: Allow orientation along Earth's magnetic field, helping with magnetotaxis.

Gas Vesicles

  • Hollow structures filled with gas, enabling buoyancy in water for cyanobacteria and plankton.

Cell Membrane and Cell Wall

Cytoplasmic Membrane

  • Phospholipid bilayer embedded with proteins.

  • Archaeal membranes may feature unique lipid structures allowing stability at high temperatures.

Membrane Composition

  • Bacteria:

    • Glycerol, ester linkages, fatty acids.

  • Archaea:

    • Glycerol/diglycerol (with and without phosphate), ether linkages, and distinctive lipids (e.g., phytanyl, biphytanyl).

Functions of Cytoplasmic Membrane

  • Separates cytoplasm from the environment, maintaining cell integrity.

Membrane Transport

  • Water and hydrophobic molecules pass freely; other molecules require transport proteins for movement (specific to molecules).

    • Types of transport proteins:

    • Symporter, antiporter, uniporter.

    • Translocases for larger molecules.

Cell Walls of Bacteria

  • Gram-positive: Thick peptidoglycan layer, staining purple during Gram staining.

  • Gram-negative: Two membranes separated by periplasmic space with a thin peptidoglycan layer, staining pink.

Gram Staining

  • Developed by Hans Christian Gram to differentiate bacteria into Gram-positive and Gram-negative.

  • Process:

    • Initially stained with crystal violet (purple).

    • Alcohol washing removes stain from Gram-negative bacteria.

    • Counterstain with safranin gives Gram-negative bacteria a pink color.

Characteristics of Bacterial Cell Walls

  • Gram-positive: presence of teichoic acids and wall-associated proteins.

  • Gram-negative: presence of lipopolysaccharides in the outer membrane.

  • Exceptions: Mycoplasmas lack cell walls, and Archaea lack peptidoglycan.

Cell Walls of Archaea

  • Composed of various polymers, including pseudomurein (similar to peptidoglycan), polysaccharides, proteins, and glycoproteins.

  • Most Common Structure: S-layer with interlocking glycoproteins.

Types of Microscopy

Light Microscopy

  • Resolution up to 1000x magnification, suitable for objects 0.2μm and larger.

  • Visualization of pigmented microorganisms for easier observation under bright-field microscopy.

Differential Staining Methods

  • Simple Stains: Basic dyes (crystal violet, methylene blue).

  • Gram Staining: Main differentiator for bacterial categories.

  • Specialized stains for endospores, capsules, and other structures.

Fluorescence Microscopy

  • Staining cells with fluorescent dyes (e.g., DAPI), requiring fluorescence microscopes.

  • Useful for total microbial counts due to the ability to visualize and distinguish cells.

Electron Microscopy

  • Scanning Electron Microscopy (SEM): Uses electron beams for surface imaging, producing 3D effects; capable of imaging down to $20 ext{ nm}$.

  • Transmission Electron Microscopy (TEM): High resolution down to $0.2 ext{ nm}$, ideal for observing internal structures, requires extensive specimen preparation.

Microbial Counts

Total Cell Count Using Microscopy

  • Counting tools (e.g., hemocytometers) allow volume calculations for cell densities expressed in cells/ml.

Viable Counts

  • Methods:

    • Spread-Plate Method: Preferred due to surface colony growth.

    • Serial dilutions in nutrient broth or saline solution necessary for accurate counting.

    • Viable counts assess cells capable of division and subsequent colony formation.

Limitations of Counting Methods

  • Microscopy: Dead cells cannot be distinguished, precision can vary, low-density samples may not represent actual numbers.

  • Viable Counts: Clumping can result in inaccurate readings; different microorganisms require specific conditions for growth, potentially underestimating total viable counts.

The Great Plate Count Anomaly

  • Observations indicate that direct microscopic counts can reveal many more organisms than plate counts due to viability and culturing issues.