Unit 3: Cell Structure, function, microbial growth and nutrition - Subjective Questions

Prokaryotic cells lack a membrane-bound nucleus and organelles. Their structure includes:

Common Shapes:

• Cocci – spherical (e.g., Staphylococcus)
• Bacilli – rod-shaped (e.g., E. coli)
• Spirilla/Spirochetes – spiral-shaped
• Vibrio – comma-shaped

Structural Components:

• Cell envelope: consists of the cell wall, plasma membrane, and sometimes a capsule/glycocalyx.
• Cytoplasmic region: contains the nucleoid (DNA), ribosomes (70S), plasmids, and inclusion bodies.
• External structures: flagella (motility), pili/fimbriae (attachment/conjugation), and capsule (protection).

Key features:

• Size typically 0.5–5 µm.
• No true membrane-bound organelles.
• Genetic material is a single circular chromosome.
• Reproduce mainly by binary fission.

Prokaryotes are structurally simpler than eukaryotes but highly adapted for survival in diverse environments.

Several structures lie external to the bacterial cell wall, providing protection, attachment, and motility:

1. Capsule and Slime Layer (Glycocalyx):

• Capsule: well-organized layer firmly attached to the cell wall.
• Slime layer: loosely attached, diffuse layer.
• Functions: protection against phagocytosis, prevention of desiccation, adhesion to surfaces (biofilm formation), and virulence.

2. Fimbriae (Pili):

• Short, hair-like appendages made of pilin protein.
• Aid in attachment to host cells and surfaces.

3. Sex Pili (F-pili):

• Longer than fimbriae, involved in conjugation (transfer of genetic material).

4. Flagella:

• Long, whip-like appendages responsible for motility.
• Composed of the protein flagellin.
• Arrangements: monotrichous, lophotrichous, amphitrichous, peritrichous.

Significance: These structures help bacteria colonize, exchange genes, and move toward favorable environments (chemotaxis).

Bacterial (Plasma) Cell Membrane:

Structure:

• A phospholipid bilayer described by the fluid mosaic model.
• Contains proteins embedded within and on the surface.
• Bacterial membranes lack sterols but may contain hopanoids for rigidity.

Functions:

• Selective permeability barrier controlling nutrient entry and waste exit.
• Site of energy generation (electron transport chain, ATP synthesis).
• Contains enzymes for cell wall synthesis and lipid biosynthesis.
• Mesosomes (infoldings) may assist in secretion and DNA replication.

Cytoplasmic Matrix (Cytosol):

• A gel-like substance filling the cell.
• Composed mainly of water (~70%), proteins, nucleic acids, ions, and metabolites.

Contents include:

• Ribosomes (70S) for protein synthesis.
• Nucleoid carrying genetic material.
• Inclusion bodies (storage granules like glycogen, polyphosphate, sulphur).
• Plasmids and enzymes for metabolism.

Together they maintain cellular homeostasis and metabolic activity.

The bacterial cell wall is primarily made of peptidoglycan (murein), but its structure differs significantly:

Key points:

• Gram-positive walls have multiple peptidoglycan layers with teichoic and lipoteichoic acids.
• Gram-negative walls have an outer membrane containing LPS, which acts as an endotoxin.

These differences influence staining, antibiotic susceptibility, and pathogenicity.

Although both are prokaryotes, archaeal and bacterial cell walls differ chemically:

Bacterial Cell Wall:

• Composed of peptidoglycan (murein) containing N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG).
• Cross-linked by peptide bridges.

Archaeal Cell Wall:

• Lacks true peptidoglycan.
• Many archaea have pseudopeptidoglycan (pseudomurein) containing N-acetyltalosaminuronic acid instead of NAM.
• Some have walls made of polysaccharides, glycoproteins, or protein (S-layer).

Conclusion: Archaeal walls are chemically distinct, making them resistant to lysozyme and penicillin, and reflecting their evolutionary divergence from bacteria.

Nucleoid:

• An irregularly shaped region in the cytoplasm containing the bacterial chromosome.
• Consists of a single, circular, double-stranded DNA molecule.
• Not enclosed by a nuclear membrane.
• Associated with RNA and proteins (HU, H-NS) that help in DNA folding (supercoiling).

Functions of nucleoid:

• Stores the essential genetic information.
• Controls all cellular activities and replication.

Plasmids:

• Small, circular, extrachromosomal DNA molecules.
• Replicate independently of the chromosome.
• Carry non-essential but advantageous genes.

Types of plasmids:

• F (Fertility) plasmids – enable conjugation.
• R (Resistance) plasmids – confer antibiotic resistance.
• Col plasmids – produce bacteriocins.
• Virulence plasmids – encode toxins/pathogenicity factors.
• Degradative plasmids – enable breakdown of unusual substances.

Significance:

• Plasmids are key tools in genetic engineering and act as cloning vectors.
• They contribute to bacterial adaptation and evolution.

Microorganisms require various nutrients for growth, energy, and biosynthesis:

1. Macronutrients (required in large amounts):

• Carbon (C): backbone of all organic molecules.
• Hydrogen (H) & Oxygen (O): components of water and organic compounds.
• Nitrogen (N): for proteins and nucleic acids.
• Phosphorus (P): for nucleic acids, ATP, phospholipids.
• Sulphur (S): for amino acids (cysteine, methionine).
• Potassium, Magnesium, Calcium, Iron: for enzyme function and structural stability.

2. Micronutrients (trace elements):

• Zn, Mn, Cu, Co, Mo, Ni – act as enzyme cofactors.

3. Growth factors:

• Organic compounds that a microbe cannot synthesize itself, e.g., vitamins, amino acids, purines, and pyrimidines.

4. Water:

• Essential solvent for all metabolic reactions.

5. Energy source:

• Either light (phototrophs) or chemicals (chemotrophs).

These nutrients support metabolism, structural formation, and reproduction of microorganisms.

Microorganisms are classified nutritionally based on their carbon source, energy source, and electron source:

Based on Energy Source:

• Phototrophs: use light as energy source.
• Chemotrophs: use chemical compounds as energy source.

Based on Carbon Source:

• Autotrophs: use CO₂ as sole carbon source.
• Heterotrophs: use organic carbon compounds.

Based on Electron/Hydrogen Source:

• Lithotrophs: use inorganic electron donors.
• Organotrophs: use organic electron donors.

Four Major Combined Nutritional Types:

This classification helps understand the ecological roles and metabolic diversity of microbes.

Culture Media: A nutrient preparation used to grow, isolate, and maintain microorganisms in the laboratory.

Classification Based on Physical State:

• Liquid (Broth) media: no solidifying agent (e.g., nutrient broth). Used for growth studies and fermentation.
• Semi-solid media: contain low agar (0.5%); used for motility testing.
• Solid media: contain agar (1.5–2%); used for isolation and colony study (e.g., nutrient agar).

Classification Based on Chemical Composition:

• Defined (Synthetic) media: exact chemical composition is known (e.g., glucose-salts medium).
• Complex (Undefined) media: contain ingredients of unknown composition like peptone, yeast extract, beef extract.

Key Point: Agar is the ideal solidifying agent because it is not metabolized by most microbes, melts at ~100 °C, and solidifies at ~40 °C.

Culture media can be classified by function into the following types:

1. Basal (General purpose) media:

• Support growth of most microorganisms.
• Example: Nutrient agar, Nutrient broth.

2. Enriched media:

• Basal media supplemented with blood, serum, or egg to grow fastidious organisms.
• Example: Blood agar, Chocolate agar.

3. Selective media:

• Contain inhibitory substances that suppress unwanted microbes and favor specific ones.
• Example: MacConkey agar (bile salts inhibit Gram-positives).

4. Differential media:

• Distinguish organisms based on biochemical/metabolic differences (color change).
• Example: EMB agar, MacConkey agar (lactose fermentation).

5. Enrichment media:

• Liquid media that favor growth of a particular organism from a mixed population.
• Example: Selenite F broth for Salmonella.

6. Transport media:

• Preserve specimens during transport.
• Example: Stuart's medium, Cary-Blair medium.

7. Anaerobic media:

• Support growth of anaerobes by reducing oxygen.
• Example: Robertson's cooked meat medium.

Each type serves specific diagnostic and research purposes.

Isolation techniques are used to separate individual microbial species from mixed populations to obtain pure cultures:

1. Streak Plate Method:

• A loopful of sample is streaked over the agar surface in a pattern.
• Progressive dilution yields isolated colonies.
• Most commonly used technique.

2. Pour Plate Method:

• Diluted sample is mixed with molten agar and poured into a plate.
• Colonies develop on the surface and within the medium.
• Allows colony counting.

3. Spread Plate Method:

• A small volume of diluted sample is spread evenly on solidified agar using a sterile spreader.
• Colonies grow only on the surface.

4. Serial Dilution:

• Sample is diluted stepwise to reduce microbial density before plating.
• Used with pour/spread plate for enumeration.

5. Enrichment Culture:

• Selective conditions favor growth of the target organism.

Principle: All these methods aim to physically separate cells so each grows into a distinct colony arising from a single cell (a pure culture / CFU).

Streak Plate Technique: A method to obtain isolated pure colonies by mechanically diluting bacteria across an agar surface.

Principle:

• As the inoculating loop is streaked over the medium, the number of cells deposited progressively decreases.
• In the final streaks, individual cells are far enough apart to form discrete colonies, each derived from a single cell.

Procedure:

1. Sterilize the inoculating loop by flaming until red hot; allow it to cool.
2. Pick a loopful of the mixed culture aseptically.
3. Streak it across one section (Quadrant 1) of the agar plate.
4. Flame and cool the loop, then drag it from Quadrant 1 into Quadrant 2.
5. Repeat the process for Quadrants 3 and 4, reducing cell density each time.
6. Incubate the plate (usually at 37 °C for 24–48 hrs).

Result:

• Confluent growth in early quadrants and well-separated colonies in later quadrants.

Advantages:

• Simple, quick, and requires minimal equipment.
• Effective for obtaining pure cultures.

Preservation ensures microbial cultures remain viable, pure, and genetically stable over time. Methods are broadly categorized as:

A. Routine / Short-term Methods:

1. Periodic Subculturing (Serial Transfer):

• Cultures are transferred to fresh media at regular intervals.
• Simple but risks contamination and mutation.

2. Overlaying with Mineral Oil:

• Sterile mineral oil covers the agar slant to prevent dehydration and reduce metabolism.
• Stores cultures for months to a few years.

3. Refrigeration (4 °C):

• Slows metabolic activity for short-term storage.

B. Long-term Methods:

4. Freezing / Deep Freezing (−20 °C to −70 °C):

• Cultures stored with cryoprotectants like glycerol.

5. Liquid Nitrogen Preservation (−196 °C):

• Ultra-low temperature halts metabolism completely.

6. Lyophilization (Freeze-drying):

• Cells are frozen and water is removed by sublimation under vacuum.
• Best for very long-term storage.

Aim: Prevent genetic changes, contamination, and loss of viability during storage.

Liquid Nitrogen Preservation (Cryopreservation): A long-term method where microbial cultures are stored at ultra-low temperatures of −196 °C in liquid nitrogen.

Principle:

• At such low temperatures, all metabolic activity and enzymatic reactions cease, preventing growth, mutation, and death.

Procedure:

1. Cells are suspended in a medium containing a cryoprotective agent such as glycerol (10%) or DMSO to prevent ice crystal formation.
2. The suspension is sealed in ampoules or cryovials.
3. Cells are cooled slowly (about 1 °C/min) to prevent cell injury.
4. Ampoules are stored in liquid nitrogen tanks (liquid phase at −196 °C or vapor phase at ~ −150 °C).
5. To revive, cultures are thawed rapidly in a 37 °C water bath.

Advantages:

• Maintains viability for decades.
• Preserves genetic stability.
• Suitable for a wide range of organisms including those that cannot be lyophilized.

Limitations:

• Expensive equipment and continuous nitrogen supply required.
• Risk of injury from ice crystals if cooling is improper.
• Storage hazards (explosion, frostbite).

Application: Ideal for culture collections, research stocks, and industrial strains.

Lyophilization (Freeze-drying): A preservation technique in which microbial cultures are frozen and then dehydrated under vacuum by sublimation, converting ice directly into vapor.

Principle:

• Removal of water at low temperature halts all metabolic activity while preserving cell structure.

Procedure (Steps):

1. Preparation: A dense suspension of cells is prepared in a protective medium (e.g., skim milk, serum, or sucrose).
2. Freezing: The suspension is rapidly frozen at very low temperatures (−40 °C or below).
3. Primary drying (Sublimation): Under high vacuum, frozen water sublimes directly into vapor.
4. Secondary drying: Residual bound moisture is removed.
5. Sealing: Dried cultures in ampoules are sealed under vacuum and stored at low temperature.

Revival: Rehydration with sterile medium restores viability.

Advantages:

• Cultures remain viable for years to decades.
• Lightweight, easy to store and transport.
• Minimal risk of contamination.
• Maintains genetic stability.

Why it's the best method:

• It combines freezing and dehydration, giving maximum stability, long shelf life, and convenience — making it the preferred method for culture collections and commercial storage.

Both selective and differential media serve specialized purposes in microbiology but differ in function:

Note: Some media are both selective and differential, e.g., MacConkey agar selects Gram-negatives and differentiates lactose fermenters (pink) from non-fermenters (colorless).

Summary: Selective media screen out microbes; differential media identify and differentiate them.

Peptidoglycan (Murein): The rigid structural polymer of bacterial cell walls responsible for shape and strength.

Chemical Composition:

• Made of alternating sugar derivatives:
  • N-acetylglucosamine (NAG)
  • N-acetylmuramic acid (NAM)
• These form long glycan chains linked by β(1→4) glycosidic bonds.

Peptide Cross-links:

• Short chains of 4 amino acids (tetrapeptides) are attached to NAM.
• A typical tetrapeptide: L-alanine – D-glutamic acid – meso-diaminopimelic acid (or L-lysine) – D-alanine.
• Peptide bridges cross-link adjacent glycan chains.

Structure:

• Forms a mesh-like, multilayered network (sacculus) surrounding the cell.

Role in Cell Wall Integrity:

• Provides mechanical strength and resists osmotic (turgor) pressure, preventing cell lysis.
• Maintains the characteristic cell shape.

Clinical Relevance:

• Lysozyme cleaves the β(1→4) bond, destroying peptidoglycan.
• Penicillin inhibits cross-link formation (transpeptidation), weakening the wall.

Thus, peptidoglycan is essential for bacterial survival and a key antibiotic target.

Bacterial Flagella: Thread-like, helical appendages responsible for bacterial motility.

Structure (Three Main Parts):

1. Filament:

• Long, external helical structure made of the protein flagellin.
• Hollow core through which flagellin subunits are added at the tip.

2. Hook:

• A curved structure connecting the filament to the basal body.
• Acts as a universal joint.

3. Basal Body:

• Anchors the flagellum into the cell wall and membrane.
• In Gram-negative bacteria, has four rings (L, P, MS, C rings).
• In Gram-positive bacteria, only two rings (MS and C rings) are present.

Arrangements of Flagella:

• Monotrichous – single flagellum.
• Lophotrichous – tuft at one pole.
• Amphitrichous – flagella at both poles.
• Peritrichous – flagella all over the surface.

Mechanism of Motility:

• The flagellum rotates like a propeller, driven by the proton motive force (flow of H⁺ ions) across the membrane, not ATP directly.
• Counterclockwise rotation → smooth forward movement (run).
• Clockwise rotation → tumbling and reorientation (tumble).

Chemotaxis:

• Bacteria move toward attractants and away from repellents by alternating runs and tumbles.

Functions:

• Enable movement toward nutrients (positive chemotaxis).
• Help escape harmful substances.
• Contribute to colonization and virulence.

Thus, flagella are sophisticated molecular motors central to bacterial locomotion and survival.

Inclusion Bodies: Are storage granules or reserve deposits found in the bacterial cytoplasmic matrix, used to store nutrients and reduce osmotic stress.

Types of Inclusion Bodies:

1. Carbon/Energy Storage:

• Glycogen granules: store glucose polymers.
• Poly-β-hydroxybutyrate (PHB): lipid-like carbon reserve; also used in bioplastic production.

2. Phosphate Storage:

• Volutin granules (Metachromatic granules): store polyphosphate as an energy/phosphate reserve.
• Seen in Corynebacterium diphtheriae.

3. Sulphur Granules:

• Found in sulphur bacteria (e.g., Beggiatoa); store elemental sulphur as an energy source.

4. Gas Vacuoles:

• Provide buoyancy in aquatic photosynthetic bacteria (e.g., cyanobacteria).

5. Magnetosomes:

• Contain iron oxide (magnetite); allow bacteria to orient in magnetic fields (magnetotaxis).

6. Carboxysomes:

• Contain the enzyme RuBisCO for CO₂ fixation in autotrophs.

Functions:

• Storage of nutrients (C, P, S, N).
• Reduce osmotic pressure by keeping nutrients in insoluble form.
• Provide buoyancy, orientation, or metabolic support.

These inclusions reflect the metabolic versatility and adaptability of bacteria.

Pure Culture: A population of cells arising from a single parent cell, containing only one type of microorganism.

Importance of Maintaining Pure Cultures:

• Ensures accurate identification and characterization of organisms.
• Essential for reproducible research and experiments.
• Critical in industrial fermentation and antibiotic production.
• Needed for clinical diagnosis and vaccine development.
• Maintains strain integrity and genetic characteristics.

Challenges During Preservation:

• Contamination with foreign microbes.
• Genetic mutations during repeated subculturing.
• Loss of viability or desirable traits.

Methods to Minimize Contamination and Genetic Changes:

1. Aseptic Techniques:

• Use sterile media, equipment, and work under laminar flow.

2. Reducing Metabolic Activity:

• Refrigeration (4 °C) and mineral oil overlay slow growth and mutation.

3. Long-term Preservation Methods:

• Lyophilization (freeze-drying): removes water, halting metabolism; preserves genetic stability for decades.
• Liquid nitrogen storage (−196 °C): stops all metabolic and enzymatic reactions, preventing mutation.

4. Use of Cryoprotectants:

• Glycerol or DMSO prevent ice-crystal damage during freezing.

5. Minimizing Subculturing:

• Fewer transfers reduce the chance of mutations and selection of variants.

6. Regular Purity Checks:

• Periodic streaking and microscopy confirm culture purity.

Conclusion:

• By combining aseptic handling with low-temperature and dehydration-based methods, microbiologists preserve cultures in a viable, pure, and genetically stable state for research and industrial use.