Microbiology

Question 1 [12]

(a) Each division doubles the population. After $n$ generations $N = N_0 \cdot 2^{n}$. Number of generations in time $t$ is $n = t/g$, so $N(t) = N_0 \cdot 2^{t/g}.$

• $N_0$ = initial cell number; $t$ = elapsed time; $g$ = generation (doubling) time. (3: relation 1, substitution 1, symbols 1)

(b) $t = 3\text{ h} = 180$ min, $g = 20$ min $\Rightarrow n = 180/20 = 9$. $N = 5\times10^3 \cdot 2^{9} = 5\times10^3 \cdot 512 = 2.56\times10^6 \text{ cells.}$ (3: n=9 →1, 2^9=512 →1, answer →1)

(c) From $N = N_0 e^{\mu t}$ and $N = N_0 2^{t/g}$: $e^{\mu t} = 2^{t/g} \Rightarrow \mu t = \frac{t}{g}\ln 2 \Rightarrow \mu = \frac{\ln 2}{g}.$ With $g = 20\text{ min} = 1/3\text{ h}$: $\mu = \dfrac{0.693}{1/3} = 2.08\ \text{h}^{-1}$. (3: equate 1, derive 1, numeric μ 1)

(d) Curve: lag (flat), log (steep straight line on log axis), stationary (plateau), death (decline). (2 for correct labelled phases) Assumption that breaks: the model assumes unlimited resources / constant g. In stationary phase nutrients deplete and toxic wastes accumulate, so division rate ≈ death rate; $g$ is no longer constant. (1)

Question 2 [10]

(a) 1. Crystal violet (primary stain); 2. Gram's iodine (mordant, fixes dye as CV–I complex); 3. Alcohol/acetone (decolouriser); 4. Safranin (counterstain). (4, 1 each)

(b) Gram-positive cells have a thick peptidoglycan layer that dehydrates and traps the large CV–I complex; Gram-negative cells have thin peptidoglycan plus an outer lipid membrane that alcohol dissolves, releasing the complex, so they lose the purple and take up safranin (pink). (4: thick PG retains 2, thin PG + outer membrane loses 2)

(c) Without decolourisation, all cells keep the crystal violet, so the Gram-negative organism would appear purple (a false Gram-positive result). (2)

Question 3 [10]

(a)

• Conjugation: direct cell-to-cell transfer via a pilus/conjugation bridge; DNA usually plasmid. (2)
• Transformation: uptake of free (naked) DNA fragments from the environment. (2)
• Transduction: transfer of bacterial DNA by a bacteriophage (virus) vector. (2)

(b) Conjugation via a plasmid is most likely: resistance genes are frequently carried on conjugative plasmids (R-plasmids) that transfer readily between different species by pilus contact, giving rapid horizontal spread. (4: identify conjugation/plasmid 2, cross-species justification 2)

Question 4 [10]

(a) RNA $\xrightarrow{\text{reverse transcriptase}}$ DNA $\to$ mRNA $\to$ protein (i.e. RNA→DNA→RNA→protein). Unique enzyme: reverse transcriptase (also integrase for insertion). (3: flow 2, enzyme 1)

(b) (1) Virus enters host; reverse transcriptase copies the ssRNA into complementary DNA, then a double-stranded DNA copy (provirus). (2) Integrase inserts this DNA into the host chromosome. (3) The provirus is replicated with host DNA and inherited by daughter cells. (4, ~1.3 each; award full for correct RT→dsDNA→integration→inheritance chain)

(c) Retroviruses require reverse transcriptase to convert their RNA genome into DNA; blocking it stops proviral DNA formation and the whole cycle. An ordinary DNA virus already has a DNA genome and uses the host DNA/RNA polymerases, so it never needs reverse transcriptase — the drug has no target. (3)

Question 5 [10]

(a) Any 3 contrasts (2 marks each):

(6)

(b) Prions are misfolded proteins (PrPˢᶜ) that catalyse the misfolding of normal host protein, propagating without any nucleic acid — hence "infectious protein." DNase destroys DNA only, so it cannot degrade a protein; and prions are extraordinarily heat-stable, resisting boiling that denatures ordinary proteins/inactivates nucleic-acid pathogens. (4: no nucleic acid/self-templating 2, DNase & heat resistance 2)

Question 6 [8]

(a) Random mutation (or acquired gene) gives a few cells resistance. When antibiotic is applied, susceptible cells die while resistant cells survive and reproduce (natural selection), so the resistant proportion rises. Resistance genes then spread horizontally, e.g. by conjugation of R-plasmids, to other cells/species. (5: variation 1, selection 2, vertical/horizontal spread 2)

(b) Any two (1.5 each): flaming the loop/bottle neck — kills contaminating microbes on equipment; working near a Bunsen flame / laminar flow — creates convection updraught / sterile air excluding airborne contaminants; sterilising media by autoclaving — removes pre-existing organisms. (3)

[
  {"claim":"Q1b: N after 3h = 2.56e6","code":"N0=5e3; g=20; t=180; n=t/g; N=N0*2**n; result = (N == 2560000)"},
  {"claim":"Q1b: 9 generations in 180 min","code":"result = (180/20 == 9)"},
  {"claim":"Q1c: mu = ln2/g with g=1/3 h gives about 2.08 per hour","code":"mu=log(2)/(Rational(1,3)); result = (abs(float(mu)-2.0794)<0.01)"},
  {"claim":"2^9 = 512","code":"result = (2**9 == 512)"}
]