Question 7 of 8: Pure Culture Technique, Fermentation vs. Respiration, and Media Diversity

Question 7: Pure Culture Technique, Fermentation vs. Respiration, and Media Diversity (20 marks)

Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.

(a) Pure cultures and isolation methods. A pure culture is a population descended from a single cell, containing only one species (strain) of microorganism, free of any contaminating organisms. Pure cultures matter because virtually all rigorous microbiological work — identifying an organism, characterizing its physiology, testing its response to a treatment, producing a defined fermentation product — requires knowing that every observed effect is attributable to a single, known organism; a mixed culture confounds results (which organism did what?) and can allow an undesirable contaminant to outcompete or contaminate an intended production strain. Three standard isolation methods: streak-plate method — a loopful of mixed culture is spread across an agar surface in a series of dilution streaks, progressively diluting cells until, in the final streaks, individual cells are physically separated far enough apart that each grows into a visually distinct, isolated colony that can be picked and subcultured. Spread-plate (or pour-plate) method — a diluted sample is spread evenly across (or mixed into molten agar poured onto) a plate, so that after solidification and incubation, well-separated colonies again arise from single cells, at a density controlled by the dilution factor. Serial dilution (often paired with either plating method, or used alone with liquid media in a most-probable-number/single-cell-per-tube approach) — a sample is diluted through a series of tubes until, statistically, only a single cell (or none) is present per aliquot, so that growth in a given tube can be attributed to a single founder cell.

(b) Fermentation vs. respiration; aerobic vs. anaerobic respiration; media diversity. Respiration and fermentation are both ATP-generating catabolic strategies, but they differ in electron-acceptor chemistry: respiration passes electrons from the substrate through a membrane-bound electron transport chain to a terminal electron acceptor external to the substrate itself, generating a proton-motive force that drives ATP synthesis via ATP synthase (oxidative phosphorylation) — this yields substantially more ATP per mole of substrate. Fermentation, by contrast, uses no electron transport chain and no external terminal acceptor at all; ATP is generated solely by substrate-level phosphorylation, and the electrons removed from the substrate during catabolism are disposed of by reducing an internally generated organic product (e.g. pyruvate to ethanol/lactate) purely to regenerate the oxidized coenzyme (NAD⁺) needed to keep glycolysis running — which is exactly why fermentations like the ethanol/glucose reactions elsewhere on this paper always co-produce a reduced organic byproduct. Aerobic respiration uses O₂ as the terminal electron acceptor (yielding the largest possible ATP yield per substrate, since O₂ has the largest electrochemical pull on the electron transport chain); anaerobic respiration substitutes a different, less electronegative terminal acceptor (nitrate, sulfate, CO₂, or others, depending on the organism) when O₂ is unavailable, still using an electron transport chain and still generating a proton-motive force, but with a smaller net free-energy yield and hence less ATP than the aerobic case. Media diversity exists because no single formulation can simultaneously support the enormous range of microbial nutritional requirements (autotroph vs. fastidious heterotroph), physical tolerances (aerobic/anaerobic, pH, temperature, osmotic strength), and diagnostic/selective goals a laboratory needs to address; general-purpose, enrichment, selective, differential, and chemically-defined media each solve a different piece of that problem, so a working lab necessarily stocks many types rather than one universal medium.