Microscopy — magnification and resolution

Magnification is how much larger the image is than the object. Resolution is the smallest distance between two points that can still be distinguished — and it is limited by the wavelength of the radiation used. Magnifying beyond the resolution limit simply gives a larger blur.

• Light microscope: resolution ~200 nm (limited by the wavelength of visible light); magnification up to ~×1500. Living material can be examined; stains such as methylene blue and iodine add contrast. Laser scanning confocal microscopes give sharp optical sections and fluorescence.
• TEM: electrons pass through the specimen; resolution ~0.1 nm; reveals internal ultrastructure. SEM: electrons are reflected from the surface, giving a 3-D image. Both require dead, dehydrated specimens in a vacuum — so artefacts are always a risk.
• Eyepiece graticule and stage micrometer: the graticule must be calibrated at each objective magnification against the micrometer, because the true value of one graticule division changes as you change objective.

Your turn — magnification

Ultrastructure and the prokaryotic contrast

Each organelle earns its keep, and the exam wants the link between structure and function.

• Nucleus — nuclear envelope with pores (mRNA is far too large to cross the membrane); nucleolus makes rRNA and assembles ribosomes.
• Rough ER — ribosome-studded; synthesises and transports proteins. Smooth ER — synthesises lipids and steroids.
• Golgi apparatus — modifies (e.g. glycosylation), sorts and packages; produces lysosomes, which contain hydrolytic enzymes.
• Mitochondrion — cristae give a large surface area for the electron transport chain; the matrix contains Krebs enzymes, circular DNA and 70S ribosomes.
• Cytoskeleton — microfilaments (actin: cell movement and cytokinesis), microtubules (tracks for organelle transport; form the spindle) and intermediate filaments. Cilia and flagella have a 9+2 microtubule arrangement; centrioles are 9 triplets.

Prokaryotic cells: no nucleus and no membrane-bound organelles; circular DNA free in the cytoplasm and not bound to histones; a peptidoglycan (murein) cell wall; 70S ribosomes rather than 80S; plus plasmids, a capsule and flagella. The 70S/80S difference is precisely why some antibiotics inhibit bacterial protein synthesis without harming us.

Carbohydrates, lipids and proteins

Condensation joins monomers and releases water; hydrolysis uses water to break the bond. Learn which bond goes with which molecule: glycosidic (sugars), peptide (proteins), ester (lipids), phosphodiester (nucleic acids).

• Starch and glycogen — polymers of α-glucose. Amylose is a 1,4-linked helix; amylopectin and glycogen add 1,6 branches, giving many free ends for rapid hydrolysis. Compact and insoluble, so they have no osmotic effect.
• Cellulose — β-glucose, with alternate molecules inverted 180°. Straight chains hydrogen-bond into microfibrils of enormous tensile strength.
• Triglyceride — glycerol + 3 fatty acids, 3 ester bonds, 3 water molecules released. Unsaturated fatty acids have C=C double bonds that kink the tail and lower the melting point. Phospholipids are amphipathic → the bilayer.
• Proteins — primary (sequence, peptide bonds) → secondary (hydrogen bonds → α-helix or β-pleated sheet) → tertiary (ionic, disulfide, hydrogen bonds and hydrophobic interactions) → quaternary (several polypeptides; haemoglobin has 4, each with an iron-containing haem prosthetic group).

Tests: Benedict’s → brick-red for reducing sugars (boil with acid first for non-reducing); iodine → blue-black for starch; biuret → purple for peptide bonds; emulsion test → white emulsion for lipids. Use a colorimeter to make Benedict’s quantitative.

DNA, replication and ATP

A nucleotide is a pentose sugar + phosphate + base. Nucleotides are joined by phosphodiester bonds into a sugar-phosphate backbone. The two DNA strands are antiparallel and held by hydrogen bonds: A–T (2) and C–G (3).

Semi-conservative replication: DNA helicase breaks the hydrogen bonds and unwinds the double helix. Each strand is a template. Free nucleotides pair with the exposed bases, and DNA polymerase forms the phosphodiester bonds. Because DNA polymerase works only in one direction along the template, one new strand is made continuously (leading) and the other in fragments (lagging), which are joined by DNA ligase. Each daughter molecule keeps one original strand.

Meselson and Stahl confirmed this: after one generation in ¹⁴N, all the DNA was of intermediate density (ruling out conservative replication); after two, half was intermediate and half light (ruling out dispersive).

ATP is a phosphorylated nucleotide. Hydrolysis of the terminal phosphate by ATP hydrolase releases about 30.5 kJ mol⁻¹ — a small, immediately usable packet. It is an energy currency, not an energy store: it is constantly recycled from ADP + Pi by ATP synthase.

Induced fit, inhibition and cofactors

Enzymes lower the activation energy of a reaction by forming an enzyme-substrate complex. Under the induced-fit model, the active site is not initially complementary: substrate binding moulds the active site around it, and this distortion strains the bonds in the substrate, which is how the activation energy is reduced.

• Temperature — rate rises with kinetic energy (Q₁₀ ≈ 2), then falls sharply as hydrogen and ionic bonds break and the enzyme denatures. pH — extremes alter the charges on R groups, breaking ionic and hydrogen bonds and changing the active site shape.
• Competitive inhibitor — similar shape to the substrate, binds the active site. Its effect is reduced by increasing substrate concentration, and V_max is still reached.
• Non-competitive inhibitor — binds an allosteric site and changes the shape of the active site. Increasing substrate does not reverse it, and V_max is permanently lowered.
• End-product inhibition — the product of a pathway acts as a non-competitive inhibitor of an earlier enzyme: elegant negative feedback (e.g. ATP inhibiting phosphofructokinase in glycolysis).

Know the three helpers: a cofactor is a non-protein component needed for activity (e.g. Cl⁻ for amylase); a coenzyme is an organic cofactor that moves between enzymes, carrying chemical groups (NAD, coenzyme A); a prosthetic group is permanently bound to the enzyme (the Zn²⁺ of carbonic anhydrase).

Distinguishing the inhibitors

The fluid mosaic model and water potential

The membrane is a phospholipid bilayer studded with proteins, glycoproteins and glycolipids (cell recognition), with cholesterol between the tails to regulate fluidity and reduce permeability to ions.

• Simple diffusion — small, non-polar molecules pass straight through, passively, down the gradient.
• Facilitated diffusion — charged or large particles use channel or carrier proteins. Passive, but the rate plateaus once all the proteins are occupied.
• Osmosis — water moves from a higher (less negative) to a lower (more negative) water potential, through the bilayer and through aquaporins.
• Active transport — a carrier protein is phosphorylated by ATP, changes shape, and moves the solute against its gradient. Bulk transport (endocytosis and exocytosis) also requires ATP.

Investigating permeability (the beetroot practical): raising the temperature increases the kinetic energy of the phospholipids, so the membrane becomes more fluid, and above ~40 °C the membrane proteins begin to denature. Both effects increase permeability, so more red betalain pigment leaks out and the absorbance measured by the colorimeter rises. Organic solvents such as ethanol dissolve the lipids and do the same.

Your turn — water potential

The cell cycle, mitosis, meiosis and stem cells

Interphase (G1 → S → G2) occupies most of the cycle: the cell grows, replicates organelles, and copies its DNA in S phase. Checkpoints (G1/S, G2/M and the spindle assembly checkpoint) verify that DNA is undamaged and correctly replicated before the cell is allowed to proceed. Failure of these checks is central to cancer.

• Prophase — chromosomes condense (each with two sister chromatids); the nuclear envelope breaks down; centrioles migrate and the spindle forms.
• Metaphase — chromosomes align on the equator, attached by centromeres.
• Anaphase — centromeres divide; spindle fibres shorten and pull the chromatids to opposite poles (ATP-dependent).
• Telophase — chromatids decondense; nuclear envelopes re-form. Then cytokinesis: in animal cells the membrane pinches in; in plant cells a cell plate forms.

Meiosis gives four genetically different haploid cells. Variation arises from crossing over at chiasmata in prophase I and the independent assortment of homologous pairs in metaphase I (2ⁿ combinations), and then from random fertilisation.

Stem cells: totipotent (any cell type, plus placenta) → pluripotent (any body cell) → multipotent (a limited range, e.g. bone marrow) → unipotent. Erythrocytes and neutrophils arise from multipotent haematopoietic stem cells. Uses include bone-marrow transplants and, in future, repair of damaged tissue.

Your turn — mitotic index

Interpreting a mitotic index

Beetroot and temperature

Which class of molecule?

Tap a card, then tap the class it belongs to.

Enzyme helpers and inhibitors

Tap an item on the left, then its partner on the right.

The big ideas to take away

That is the whole of OCR Module 2 — Foundations in biology. Press Finish to see your score.