Unit 3: Cell Structure, function, microbial growth and nutrition

Prokaryotes (bacteria and archaea) are cells lacking a membrane-bound nucleus, first classified as a distinct group after electron microscopy revealed their organisation in the mid-20th century. Their small size (typically 0.5–5 µm) gives a high surface-area-to-volume ratio that drives rapid nutrient exchange and fast growth. Every later section of this unit refers back to the following defining features.

I. Overview of Prokaryotic Cell Structure

The prokaryotic cell is a compartmentless unit whose functions are carried out at the membrane and in a crowded cytoplasm rather than in organelles.

• No membrane-bound nucleus: genetic material lies free in the cytoplasm as a nucleoid, not enclosed by a nuclear envelope.
• No membrane organelles: no mitochondria, ER or Golgi; respiration and photosynthesis occur on the plasma membrane or its infoldings.
• 70S ribosomes: smaller than the 80S eukaryotic type; composed of 30S and 50S subunits — the target of aminoglycoside and macrolide antibiotics.
• Cell wall present in most: provides shape and osmotic protection; chemistry differs between bacteria and archaea.
• Common shapes: coccus (sphere), bacillus (rod), spirillum/spirochaete (helical), vibrio (comma).
• Haploid genome: usually a single circular chromosome; reproduction by binary fission.

II. The Cell Envelope: Wall, Membrane and Matrix

The envelope encloses and organises the cell, from the rigid outer wall to the fluid cytoplasm within.

A. Archaeal and bacterial cell wall

The wall is the rigid layer that resists internal turgor pressure; its polymer chemistry separates the domains.

1. Bacterial wall — peptidoglycan (murein): a mesh of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) chains cross-linked by peptide bridges.
   • Gram-positive: thick peptidoglycan (20–80 nm) with teichoic acids; retains crystal violet, stains purple.
   • Gram-negative: thin peptidoglycan plus an outer membrane of lipopolysaccharide (LPS), whose lipid A is endotoxin; stains pink with safranin counterstain.
   • Anchor: penicillin blocks transpeptidase cross-linking, so growing Gram-positive cells lyse.
2. Archaeal wall — no peptidoglycan: built from pseudomurein (with N-acetyltalosaminuronic acid replacing NAM), polysaccharide, or a protein/glycoprotein S-layer.
   • Consequence: archaea are insensitive to penicillin and to lysozyme, which cleaves the NAG–NAM bond.

B. Cell membranes and cytoplasmic matrix

The membrane is the selectively permeable boundary; the matrix is the reaction medium it encloses.

• Membrane structure: phospholipid bilayer described by the fluid-mosaic model, with embedded transport and electron-transport proteins.
• Lipid linkage: bacteria use ester-linked fatty acids; archaea use ether-linked isoprenoid chains, sometimes forming a rigid monolayer that survives extreme heat and acid.
• Functions: site of ATP synthesis, nutrient transport, and, via infoldings (mesosomes), septum formation.
• Cytoplasmic matrix: a colloidal, protein-dense fluid holding ribosomes, the nucleoid, enzymes and reserves.
• Inclusion bodies: storage granules such as poly-β-hydroxybutyrate (carbon), polyphosphate (volutin), and sulfur globules; gas vacuoles aid buoyancy.

C. Components external to the cell wall

These surface structures mediate attachment, movement and protection beyond the wall.

• Glycocalyx: polysaccharide coat; a firm capsule (e.g. Streptococcus pneumoniae) resists phagocytosis, while a diffuse slime layer aids biofilm adhesion.
• Flagella: rotating helical filaments of flagellin driven by proton-motive force; arrangements include monotrichous, lophotrichous, amphitrichous and peritrichous.
• Fimbriae: short numerous hairs for adhesion to surfaces and host tissue.
• Pili (sex pili): longer than fimbriae; the F-pilus joins cells for conjugation and plasmid transfer.

III. The Prokaryotic Genetic Material

Prokaryotic DNA occupies two forms: an essential chromosome and optional accessory elements.

A. Plasmids and nucleoid

The nucleoid carries the core genome; plasmids carry dispensable but often advantageous genes.

• Nucleoid: an irregular region of supercoiled, circular double-stranded DNA (~1 mm long in E. coli, compacted ~1000-fold) with no bounding membrane; associated with HU/nucleoid-associated proteins.
• Plasmids: small, self-replicating circular DNA molecules independent of the chromosome.
  • Replication: possess their own origin (ori); copy number ranges from one to hundreds.
  • Types: F (fertility) plasmids enable conjugation; R (resistance) plasmids carry antibiotic-resistance genes; Col plasmids encode bacteriocins.
  • Significance: transfer of R plasmids spreads multidrug resistance and makes plasmids key vectors in recombinant DNA work.

IV. Microbial Nutrition

Growth requires raw materials and an energy source; microbes are classified by how they obtain each.

A. Common nutritional requirements and nutritional types of microorganisms

All cells need carbon, energy and electrons plus mineral nutrients; the source of each defines the nutritional type.

• Macronutrients: C, O, H, N, S, P for building macromolecules; K, Ca, Mg, Fe as cofactors and structural ions.
• Micronutrients (trace): Mn, Zn, Co, Cu, Mo — enzyme cofactors needed in tiny amounts.
• Growth factors: organic compounds a cell cannot synthesise — amino acids, purines/pyrimidines, vitamins.
• Classification by energy source: phototrophs (light) versus chemotrophs (chemical bonds).
• Classification by electron source: lithotrophs (inorganic donors) versus organotrophs (organic donors).
• Classification by carbon source: autotrophs (CO₂) versus heterotrophs (organic carbon).
• Combined types:
  • Photoautotroph: cyanobacteria — light energy, CO₂.
  • Chemoheterotroph: most bacteria and fungi — chemical energy, organic carbon.
  • Chemolithoautotroph: Nitrosomonas — oxidises NH₃, fixes CO₂.

V. Culture Media and Isolation

Growing microbes in the laboratory needs a nutrient medium and a technique to separate one cell type from a mixture.

A. Culture media

A culture medium is a prepared nutrient mixture supporting microbial growth in vitro, classified by chemical definition, physical state and purpose.

• By chemical composition:
  • Defined (synthetic): every component and concentration known — e.g. glucose–salts medium for autotrophs.
  • Complex: contains digests of unknown exact composition — peptone, beef extract, yeast extract.
• By physical state: liquid broths; solid media set with 1.5–2% agar (a seaweed polysaccharide that melts at ~95 °C, gels at ~40 °C, and is not degraded by most microbes); semi-solid with ~0.5% agar for motility.
• By function:
  • Selective: favours target organisms and suppresses others — MacConkey agar (bile salts inhibit Gram-positives).
  • Differential: distinguishes colonies by appearance — blood agar showing haemolysis; EMB giving a green sheen for E. coli.
  • Enrichment: liquid medium increasing the proportion of a desired organism before isolation.

B. Culture media and isolation techniques

Isolation converts a mixed population into a pure culture — a clone descended from a single cell.

• Streak plate: a loop is dragged across agar in successive sectors, diluting cells until single colonies form; the standard routine method.
• Pour plate: a diluted sample is mixed with molten agar and set, yielding surface and embedded colonies; allows counting.
• Spread plate: a small volume is spread over set agar with a sterile glass spreader, giving surface colonies for counting.
• Principle of purity: each isolated colony (CFU) is assumed to arise from one cell, so subculturing it gives a genetically uniform strain.

VI. Preservation and Maintenance of Microbial Cultures

Stock cultures must stay viable, pure and genetically stable; methods range from short-term transfers to long-term dormancy.

A. Preservation and Maintenance of microbial cultures: routine methods and liquid nitrogen preservation

Routine methods keep cultures alive short-term; liquid nitrogen halts metabolism for years.

1. Routine (short-to-medium term):
   • Periodic subculture: transfer to fresh medium at set intervals; simple but risks contamination and mutation.
   • Refrigeration (4 °C): slows metabolism on agar slants; weeks to months.
   • Overlaying with mineral oil: limits oxygen and drying on slants, extending life to years.
2. Liquid nitrogen preservation (long term):
   • Principle: storage at −196 °C (vapour phase −150 °C) stops all metabolic and enzymatic activity.
   • Procedure: suspend cells with a cryoprotectant — 10% glycerol or DMSO — to prevent intracellular ice crystals; cool slowly, then plunge into liquid nitrogen.
   • Merit: preserves fastidious and viability-sensitive organisms for decades with high survival.

B. Freeze-drying (lyophilization)

Lyophilization removes water by sublimation from the frozen state, leaving a stable dry powder for long-term storage.

• Principle: water passes directly from ice to vapour under vacuum, avoiding the liquid phase and heat damage.
• Steps:
  • Pre-freezing: the cell suspension (often with a protective additive such as skimmed milk or sucrose) is frozen.
  • Primary drying: sublimation of free ice under high vacuum.
  • Secondary drying: removal of bound water; the ampoule is then sealed.
• Advantages: ampoules store for years at room temperature, are light and easy to distribute, and best suit culture-collection banking.
• Limitation: unsuitable for some sensitive cells; a fraction of the population dies during freezing and desiccation.