LW

Unit 1 Lecture 5

Heat fixation and slide preparation

  • Heat fixing is critically important to attach bacteria to the slide; a few seconds over a Bunsen burner fixes the cells so they don’t wash off during staining and rinsing.
  • If bacteria aren’t fixed, they will wash away during staining and washing steps, leaving nothing on the slide.
  • The process is quick and can be done in about 15–20 minutes per slide.

Gram staining and membrane structure

  • Bacteria have a double membrane in some cases, with an outer layer that contains lipids and lipoproteins, in addition to the inner membrane.
  • The outer membrane in Gram-negative bacteria contributes to differences in permeability and staining compared to Gram-positive bacteria.
  • Gram staining yields two main outcomes:
    • Gram-positive bacteria appear purple (stain retains the dye due to a thick, thick peptidoglycan layer).
    • Gram-negative bacteria appear red or pink (thin peptidoglycan layer with an outer membrane).
  • The outer layer lipids and lipoproteins contribute to the overall membrane properties and staining behavior.
  • Peptidoglycan thickness is a major distinguishing feature between Gram-positive and Gram-negative cells.
  • There are additional components in the cell envelope, such as lipoteichoic acids and lipids, that contribute to cell wall maintenance and function.

DNA organization and mobile genetic elements

  • The majority of bacterial DNA is organized in a nucleoid; there is a large loop of DNA that, if stretched out, would be much larger than the cell, so it is highly condensed and folded in an organized way (metaphor: like folding a rubber band into a knot, but more organized).
  • The nucleoid contains the essential genome required for life: reproduction, protein production, and general survival.
  • Plasmids are small, circular DNA molecules that exist in the cytoplasm and are not essential for life, but provide extra features that can help the bacterium.
  • Plasmids can carry genes that confer advantages (e.g., drug resistance, enhanced stress tolerance, or altered cell wall properties).
  • DNA transfer between bacteria can occur via several mechanisms:
    • Conjugation (cell-to-cell contact, often plasmid-mediated).
    • Transduction (bacteriophage-mediated DNA transfer).
    • Transformation (uptake of DNA from the environment) is implied by the discussion of DNA being taken up.
  • Bacteriophages are viruses that specifically infect bacteria; they can inject DNA into a bacterial cell, which may contribute to genetic exchange via plasmids or other DNA elements.
  • The presence of plasmids adds extra features that are not strictly necessary for life but can improve survival or virulence.
  • An analogy used: Peter Parker getting bitten by a spider, gaining extra powers; similarly, plasmids can grant bacteria extra capabilities that are not essential but beneficial.
  • This genetic flexibility can influence virulence and adaptation to changing environments or selective pressures (e.g., antibiotics).

Antibiotic resistance and virulence

  • Drug resistance can be carried on plasmids or other mobile genetic elements and can be transferred between bacteria, increasing resistance in populations.
  • If antibiotics are used inappropriately or for too short a time, resistant strains can be selected for, leading to difficult-to-treat infections (e.g., “superbugs”).
  • Historical note: antibiotics became widely used in the 1950s, with early overuse (e.g., in toothpaste, ice cream) contributing to the rise of resistant organisms. Modern infections may require longer or stronger courses to overcome resistant strains.
  • The emergence of highly drug-resistant pathogens is a critical public health concern.

Ribosomes and protein synthesis in prokaryotes vs eukaryotes

  • Bacteria (prokaryotes) have ribosomes composed of two subunits that come together in the cytoplasm to form the active ribosome for protein synthesis.
    • In eukaryotes, ribosome assembly begins in the nucleolus (a region within the nucleus) where ribosomal RNA (rRNA) and proteins are assembled into subunits; these components are then exported to the cytoplasm where the ribosome assembles into a functional unit.
  • In prokaryotes, ribosomes are synthesized in the cytoplasm; they are composed of a large subunit and a small subunit, which only come together when needed to translate mRNA into protein.
  • Prokaryotic ribosomes are typically referred to as 70S (composed of the 50S large subunit and the 30S small subunit), whereas eukaryotic ribosomes are 80S (60S large subunit and 40S small subunit).
  • Ribosomes contain ribosomal RNA (rRNA) and proteins; the specific rRNA components include small-subunit 16S rRNA (in bacteria) and large-subunit 23S rRNA and 5S rRNA (in bacteria).
  • Storage of macromolecules: bacteria may store glycogen, lipids, minerals, or water in specialized storage materials within the cytoplasm.

Bacterial size, shapes, and arrangements

  • Typical bacterial cell size is about 1 \, ext{µm} in length, though bacteria range from very small to relatively large (some approaching eukaryotic cell size in rare cases).
  • Some bacteria are large enough to be visible as comparatively big cells relative to viruses.
  • Common shapes:
    • Coccus (plural cocci): spherical
    • Bacillus (plural bacilli): rod-shaped
    • Coccobacillus: intermediate between cocci and bacilli
    • Spirillum/Spirilla: spiral-shaped
    • Spirochete: a special type with an axial filament inside the cell envelope implying internal flagella-like motility
  • Special arrangement patterns observed under microscopy:
    • Diplococci: pairs
    • Streptococci: chains
    • Staphylococci: grape-like clusters
    • Tetrads: groups of four
    • Palisades: arranged like a fence or rod-like clusters
  • Some species exhibit unique features (e.g., spiral bacteria with an axial filament inside the membrane).

Morphology and habitat interactions

  • Microbial habitats include bacteria, archaea, fungi, algae, and protozoa that often live in communities with abundant food, water, and appropriate environmental conditions.
  • Mutualism (a form of beneficial interaction): different species benefit from living close together (e.g., bacteria attaching to surfaces and forming communities that share resources).
  • Biofilms and dental plaque are classic examples of mutualistic communities that form when bacteria attach to surfaces (e.g., tooth enamel) and accumulate with other organisms and particulates to form a hard, protective layer.
  • Fimbriae (pili) can help bacteria attach to surfaces, enabling biofilm formation and community development.

Endospores, dormancy, and germination

  • Some prokaryotes can form endospores as a survival strategy in hostile conditions (low water, nutrient scarcity, high crowding, toxins).
  • Endospore formation entails shutting down metabolic activity and forming a protective coat around the nucleoid, resulting in a dormant, metabolically inactive cell.
  • When conditions improve, endospores germinate back into vegetative, actively growing bacteria. Water often triggers germination, leading to rehydration and metabolic reactivation.
  • Endospores are highly resistant to environmental stresses; they are difficult to kill with standard disinfection methods and require strong sterilization like autoclaving.
  • Autoclaving uses high pressure, high temperature, and moisture to kill vegetative cells and endospores; the process can eliminate spores given sufficient time and exposure.
  • Clinical implication: antibiotic treatment must be long and stringent enough to cover possible endospore-containing organisms to prevent relapse or persistent infection.

Practical and ethical implications discussed

  • The discussion highlights how improper antibiotic use can drive drug resistance and increase pathogenicity, leading to a public health challenge.
  • Understanding the differences in bacterial cell structure informs approaches to disinfection, sterilization, and infection control.
  • The conversation around plasmids and horizontal gene transfer emphasizes the importance of monitoring resistance genes and virulence factors in microbial populations.

Quick recap of key terms and concepts

  • Heat fixing: attaching bacteria to slides by brief heating to prevent wash-off during staining.
  • Gram-positive vs Gram-negative: differences in cell wall structure leading to different staining behaviors.
  • Outer membrane and lipoproteins: components that contribute to Gram-negative permeability and envelope characteristics.
  • Peptidoglycan: major component of bacterial cell walls, with thickness varying by Gram type.
  • Plasmids: extra-chromosomal DNA that can carry advantageous traits; transferable between cells.
  • Bacteriophages: viruses that infect bacteria and can mediate horizontal gene transfer.
  • Ribosomes: protein-synthesizing machinery; 70S in prokaryotes (50S + 30S), 80S in eukaryotes (60S + 40S).
  • rRNA components: 16S rRNA in the small subunit; 23S and 5S rRNA in the large subunit (bacteria).
  • Endospores: dormant, highly resistant cells formed under stress; germinate when conditions improve.
  • Biofilms/plaque: community-based growth on surfaces providing mutual benefits.
  • Spirochetes and axial filament: spiral bacteria with internal flagella-like structure.
  • Morphologies and arrangements: cocci, bacilli, coccobacilli, spirilla; diplococci, streptococci, staphylococci, tetrads, palisades.
  • Size reference: typical bacteria around 1 \, ext{µm}; some larger examples exist.
  • Practical sterilization: autoclaving combines heat, moisture, and pressure to kill vegetative cells and endospores.