Viruses & Bacteria
This is the only proper microbiology practical in the course, and half of it you can genuinely run at home. Growing bacteria needs agar, a jar and a warm cupboard. Staining them needs a laboratory, so that half arrives as described micrographs — and you classify every sample exactly as you would down a microscope.
Investigations column, block 44: "Show micrographs" and "Grow bacteria on agar plates and investigate under a strong microscope, using gram + and – stains". Resources: "Slides · Photographs · Diagrams · Charts · Video clips · Agar mix · Petri dishes · Microscope".
That is Investigations 1 and 2. Your booklet's core-concepts list then names three things you must be able to draw: the bacteriophage life cycle "by means of an annotated drawing", the microbial nutrient loop "in an annotated flow diagram", and the bacterium "using a labelled drawing". That is Investigation 3, and it is worth the most marks on the sheet. Investigation 4 is the booklet's own final question, built out with real South African context.
What you need
- 🟡 supermarket Agar-agar powder — sold with the baking or vegetarian products as a setting agent, about R60. Plain gelatine works as a fallback.
- 🟢 kitchen A stock cube or a teaspoon of sugar — the nutrient the bacteria will actually eat
- 🟢 kitchen Six or seven small jars with lids, or clear tubs with lids. They must seal.
- 🟢 kitchen Cotton buds, a kettle, and a warm spot (a cupboard near a geyser is ideal)
- 🟢 kitchen Sticky tape (to seal the lids down), a marker pen, and a magnifying glass if you have one
- 🌊 optional A small sample of rock-pool or harbour water from Algoa Bay
- 🔴 lab only Gram stains and a strong microscope — supplied as described micrographs, see Investigation 2
Seven described plates with colony counts and appearances are supplied, so every calculation, comparison and analysis question works from the sheet. Investigations 2, 3 and 4 need no apparatus at all — one is described micrographs, one is a pencil, and one is a data set.
But do try Investigation 1 if you can. Agar-agar is in most supermarkets, and there is a real difference between reading that your unwashed hand carries bacteria and looking at forty colonies that grew off your own thumb.
- 🔴 Once a plate is growing, seal it and never open it again. Tape the lid down. You do not know what you have cultured, and there is no way to find out at home. A sealed jar is safe; an open one is an unnecessary risk.
- 🔴 Dispose of them unopened. Into a plastic bag, tie it, into the bin. Do not tip them out, do not rinse them in the kitchen sink, do not compost them.
- Do not deliberately culture anything from a toilet, a wound, or an animal. Doorhandles, phones and hands are the point of the exercise and are quite interesting enough.
- Wash your hands after handling the sealed jars, and keep them away from food preparation surfaces while they incubate.
- The boiling water for the agar is the other genuine hazard — pour it, do not carry a full pot across a room.
Aim
To culture bacteria from everyday surfaces on a home-made nutrient agar, and to use the colonies to compare how much bacterial life different surfaces actually carry.
Method — making the plates
- Dissolve 1 stock cube (or 1 tsp sugar) and 2 tsp agar-agar powder in 500 mL of water.
- Boil it for two minutes. This does two jobs: it dissolves the agar, and it sterilises the mixture so that whatever grows later came from your swab and not from the jar.
- Pour about 1 cm depth into each clean jar. Put the lids on loosely while they cool — a sealed hot jar collects condensation and you will end up growing a swamp.
- Leave them to set and cool completely. They should be firm, like a very stiff jelly.
Method — the experiment
- Label the jars. Jar A is the control: do not swab it at all. Seal it immediately.
- For each of the others, wipe a cotton bud across the surface being tested, then wipe the bud gently in a zig-zag across the agar. Do not dig into it.
- Suggested sources: a doorhandle · a phone screen · the kitchen sink · your unwashed hand · the same hand after washing with soap for 20 seconds · (optional) 3 drops of rock-pool water.
- Seal every lid with tape. Write the date on each.
- Leave them somewhere warm for 4 days. Look at them daily through the glass and do not open them.
- On day 4, count the visible colonies in each jar and describe them: size, colour, shape, whether shiny or fuzzy.
Supplied results — seven plates, day 4
A measured run, incubated at about 28 °C. Use these if you did not culture your own. Note plates E and F carefully — they are the same hand twenty seconds apart.
| Plate | Source | Colonies | Appearance |
|---|---|---|---|
| A | Control — sealed, never swabbed | 0 | Clear. Agar unchanged. |
| B | Front doorhandle | 34 | Mostly small cream circles; 2 yellow |
| C | Phone screen | 51 | Many small cream; 1 large white fuzzy patch (a mould, not a bacterium) |
| D | Kitchen sink, near the drain | Too many to count | Growth has merged into a continuous cream-and-yellow film |
| E | Unwashed hand | 78 | Cream circular, several yellow |
| F | Same hand, after 20 s with soap | 9 | Cream circular, smaller |
| G | Algoa Bay rock-pool water, 3 drops | 26 | Small translucent; 3 orange; 1 spreading colony with a ragged edge |
Results — your own plates
| Jar | Source | Colonies counted | Description |
|---|---|---|---|
| A | Control (not swabbed) | ||
| B | |||
| C | |||
| D | |||
| E | |||
| F |
Analysis
- Complete Table 1.1, including the control. 6
- Explain why the agar mixture was boiled before pouring, and why plate A was left unswabbed. 4
- Plate A grew nothing. Explain what that single result tells you about every other plate in the experiment. 4
- Using plates E and F, calculate the percentage reduction in colonies achieved by twenty seconds of handwashing. Show your working. 4
- Plate F still grew 9 colonies. Explain why washing does not reach zero, and why that is not a failure of the method. 4
- Plate D is recorded as "too many to count" rather than an estimate. Explain why that is the honest result, and what a scientist would do differently to get a number. 4
- 🔴 One colony is not one bacterium. Explain what a single visible colony actually represents, given that a bacterium is 1–5 µm across and divides every 20–30 minutes. 5
- You can see colour, size and shape — but you cannot tell which species you have grown. Explain why not, and name the technique in Investigation 2 that gets you the first step further. 4
- Plate C grew one large fuzzy white patch, recorded as a mould rather than a bacterium. Using anything from the topic page, suggest two visible differences between a bacterial colony and a fungal one. 4
Gram staining needs crystal violet, iodine, an alcohol decolouriser, safranin, and a microscope that resolves down to about a micrometre. Those are school-laboratory items, and there is no honest kitchen version — a bacterium at 1–5 µm is at the very limit of a good light microscope and invisible without a stain.
So the results arrive as described micrographs, the same pattern Topic 14 uses for cell biology. You classify every sample and explain every result, which is the part CAPS actually assesses. No mark in this block is awarded for having personally held a slide.
Aim
To classify bacteria as Gram-positive or Gram-negative from their stained appearance, and to explain what that difference says about the cell wall.
Method (for reading and describing)
- A thin smear of the bacterial sample is dried onto a slide and fixed with gentle heat.
- Crystal violet is applied — the primary stain. At this point everything on the slide is purple.
- Iodine is added. It acts as a mordant, forming a large complex with the violet dye inside the cell so it cannot easily escape.
- 🔴 The critical step: a brief alcohol wash. This is what makes the test work, and over-washing ruins it.
- Safranin is applied last — a pink counterstain, so that any cell which lost the violet is still visible.
- The slide is examined at about 1000× magnification under oil.
Supplied — five described micrographs
Colour: deep purple. Shape: spheres, about 1 µm across. Arrangement: irregular clusters, like bunches of grapes.
Colour: pink. Shape: short straight rods, roughly 2 µm long. Arrangement: singly and in pairs.
Colour: pink. Shape: distinctly curved rods, comma-shaped, about 2–3 µm long. Arrangement: singly, many actively moving before fixing.
This is the shape of the genus Vibrio, which is common in warm coastal seawater and includes the species responsible for most shellfish-related food poisoning.
Colour: purple. Shape: long rods, 3–5 µm. Arrangement: end-to-end in chains.
Colour: purple and pink cells both clearly present. Shape: a mixture of spheres and rods, various sizes.
One more observation from the same run: a sixth slide was prepared from sample 1, but was left in the alcohol wash for a full minute instead of a few seconds. It finished pink.
Results
| Sample | Colour | Shape | Gram + or −? | Reason |
|---|---|---|---|---|
| 1 | ||||
| 2 | ||||
| 3 | ||||
| 4 | ||||
| 5 |
Analysis
- Complete Table 2.1 for all five samples. 10
- Explain the mechanism: why does a Gram-positive cell finish purple and a Gram-negative one pink? Refer to the cell wall. 6
- 🔴 The sixth slide was over-washed with alcohol and turned pink, even though it was the same sample as slide 1. Explain what went wrong, and what that tells you about the alcohol step. 5
- Explain what would happen if the technician skipped the safranin entirely. Would the test still work? Justify your answer. 4
- Sample 5 contains both purple and pink cells. Explain why that is a perfectly valid result rather than a mistake. 3
- Look at the bacterium diagram on the topic page. Its cell wall is labelled "rigid, plant-like". Explain how the Gram stain turns that label into a definitive feature rather than just another ring on a drawing. 4
- A doctor treating an infection asks the laboratory for a Gram result before prescribing. Explain why. 4
- Sample 3 came from seawater near a mussel farm. Using Investigation 4 and the topic page, explain why a mariculture operation would want to monitor for this genus. 4
Aim
To reproduce, from memory, the three diagrams your booklet's core-concepts list says you must be able to produce — and then to mark yourself against the criteria a paper would use.
Method
- Close the topic page. All three of these are memory tasks. Doing them with the answers open takes ten minutes and teaches nothing.
- Pencil, ruled label lines, no arrowheads on label lines (Topic 5 conventions). ⚠️ The flow diagram in part C is the exception — a flow diagram is made of directional arrows, so arrowheads there are correct and required.
- Do each one, then open the page and mark yourself in a different colour.
Part A — the bacteriophage, annotated with its life cycle
Draw the bacteriophage and label its parts. Then annotate the drawing with the six stages of the life cycle, in order, so the diagram explains the cycle rather than just showing the animal.
Part B — the bacterium, labelled
Draw a typical bacterium and label its structures. Add a note of its size range, and one line stating why it is prokaryotic.
Part C — the microbial nutrient loop, as an annotated flow diagram
CAPS says "explain and draw the microbial loop". Show the boxes, the arrows between them, and distinguish the normal food-chain flow from the viral kill-and-release path.
Analysis
- Mark Part A. Structure (6): protein capsule, nucleic acid, sheath, core, base, attachment fibres. Cycle (6): contact, attachment, injection, hijack, manufacture, release — in order. Convention (2): pencil, ruled label lines. 14
- Mark Part B. Structure (5): cell wall, plasma membrane, slime sheath, nucleoid, flagellae. Detail (3): size range, cytoplasm, and one prokaryotic justification. 8
- Mark Part C. Boxes (5) · arrows correct (4) · the two flows distinguished (2) · the loop actually closes (1). 12
- In Part A, what stays outside the bacterium during injection, and what goes in? 3
- In Part B, name the one structure that proves the cell is prokaryotic, and explain how. 3
- In Part C, explain why the loop is called a loop — what would happen to the diagram if the nutrient arrows were removed? 4
- Your booklet says the microbial loop passes no nutrients directly to the conventional food chain. Explain how it is nonetheless linked to it. 5
- Now cover all three again and redraw Part B only, against the clock, in under three minutes. Record your time, and list what you left out. 4
- The topic page's version of Part C uses two different arrow colours. Explain why a flow diagram with only one kind of arrow would be a worse answer. 3
Aim
To use stocking data from an abalone farm to work out why infections spread more easily in captivity than in the open sea — and to separate two variables that are easily confused.
Method
- Read the supplied trial data. Four tanks were stocked at different densities and run at two different water-exchange rates.
- Work out which tanks can be compared to isolate one variable at a time.
- Answer the questions. Note that one comparison in this data set is confounded — two things change at once — and finding it is worth marks.
Supplied — a six-week trial on a Western Cape abalone farm
Farmed Haliotis midae (perlemoen). All four tanks held the same age class at the same water temperature and were fed the same ration. A fungal and bacterial infection was present on the farm throughout the trial.
| Tank | Abalone per m² | Water exchange | Mortality |
|---|---|---|---|
| 1 | 60 | High | 3% |
| 2 | 120 | High | 7% |
| 3 | 120 | Low | 19% |
| 4 | 240 | Low | 41% |
⚠️ These figures are a teaching set. They are built to show a pattern that is real and well documented in aquaculture — mortality rises with crowding and falls with water exchange — but they are not measurements from a named farm and must never be quoted as such. Cite published DFFE or industry figures in any real report.
Analysis
- The booklet asks: why would infections and fungal diseases spread more easily in mariculture or aquarium facilities? Give at least two reasons. 4
- Which two tanks should be compared to isolate the effect of water exchange alone? State the pair and the difference in mortality. 4
- Which two tanks isolate the effect of stocking density alone? State the pair and the difference. 4
- 🔴 A farm manager compares tank 1 with tank 4 and concludes "crowding causes disease". Explain what is wrong with drawing that conclusion from that pair. 5
- Marine fungi are described as opportunists that target animals with weakened immune systems. Explain how crowding produces exactly the conditions they need. 5
- Explain why the same fungus can be harmless in the open sea and a serious problem in a tank. 4
- Using the data, recommend a stocking density and water-exchange rate to the farm, and state the trade-off your recommendation involves. 5
- CAPS names both the mariculture and aquarium industries. Explain why a public aquarium faces the same problem as an abalone farm, despite selling nothing. 4
Memo — worked answers and mark allocation (does not print)
Total: 170 marks — Investigation 1: 39 · Investigation 2: 40 · Investigation 3: 56 · Investigation 4: 35.
Investigation 1 — the agar plates
- Q1 (6): Marked on the learner's own plates; the control must be recorded even though it is empty.
- Q2 (4): Boiling sterilises the medium so nothing is already growing in it (2). The unswabbed plate is a control (1) against which the swabbed plates are compared (1).
- Q3 (4): A clear control proves the agar, jars and technique were not already contaminated (2). Therefore every colony on the other plates came from the swab, and the comparison between surfaces is valid (2). Had A grown anything, the whole experiment would be uninterpretable.
- Q4 (4): (78 − 9) ÷ 78 × 100 = 88.5% (3), working shown (1). Accept 88–89%.
- Q5 (4): Washing removes bacteria, it does not sterilise (2). Skin has its own permanent resident population living in creases and follicles that soap cannot reach, and the hand is recontaminated the moment it touches anything (2). A drop from 78 to 9 is a large real effect and the correct conclusion.
- Q6 (4): The colonies have merged, so individual ones can no longer be distinguished and any number would be invented (2). A scientist would dilute the sample by a known factor and plate that, then multiply back up (2).
- Q7 (5): A colony is a visible mound of millions of bacteria, all descended from a single original cell that landed at that point (3). A single bacterium at 1–5 µm is far too small to see; only after ~20 generations of doubling does the pile become visible (2). This is why microbiologists count "colony-forming units" rather than bacteria.
- Q8 (4): Colony colour and shape are not diagnostic — many unrelated species look identical on a plate (2). The next step is to look at the cells themselves under a microscope using a Gram stain, which sorts them by cell-wall type and shape (2).
- Q9 (4): Any two, 2 each: fungal colonies are typically fuzzy, fibrous or hairy rather than smooth and shiny; they are often much larger; they may spread irregularly rather than as neat circles; and they are frequently white, grey, green or black rather than cream or yellow.
Investigation 2 — Gram staining
- Q10 (10): 1 — purple spheres in clusters → Gram-positive cocci. 2 — pink short rods → Gram-negative. 3 — pink curved rods → Gram-negative (Vibrio shape). 4 — purple rods in chains → Gram-positive. 5 — both, a mixed community. (2 each: classification + a reason referring to the colour.)
- Q11 (6): Gram-positive cells have a thick peptidoglycan wall (2) which traps the crystal violet–iodine complex so the alcohol cannot wash it out — the cell stays purple (1). Gram-negative cells have only a thin peptidoglycan layer plus an outer membrane (2); the alcohol dissolves that outer membrane and the violet washes straight out, so the cell takes up the pink safranin instead (1).
- Q12 (5): Over-washing removed the violet from a Gram-positive cell, so it took the counterstain and read as a false negative (3). The alcohol step is the discriminating step and it is timed — too short and everything stays purple, too long and everything goes pink; the result is only meaningful if that step is controlled (2).
- Q13 (4): The test would still work for Gram-positives, which would be purple (1) — but Gram-negative cells would be colourless and effectively invisible (2), so you could not tell "Gram-negative" from "nothing on the slide" (1). The counterstain exists to make the negatives visible.
- Q14 (3): An environmental sample is a mixed natural community, not a pure culture (2). Rock-pool water contains many species at once, so both wall types are expected (1).
- Q15 (4): The label describes the wall's appearance; the Gram stain reveals its chemistry (2) — and since that chemistry sorts all bacteria into two great groups, the wall becomes a diagnostic, classifying feature rather than a structural detail (2).
- Q16 (4): The two groups have structurally different walls, and many antibiotics work by attacking wall construction (2). A drug effective against Gram-positives may do nothing to Gram-negatives, whose outer membrane blocks it — so the stain narrows the choice of drug before culture results arrive (2).
- Q17 (4): Vibrio species are common in warm coastal water and include the main causes of shellfish-related food poisoning (2). A mussel or abalone farm is both a food producer and a crowded, stressed population, so it must monitor both for consumer safety and for stock health (2).
Investigation 3 — the three drawings
- Q18 (14): As allocated. Mark the order of the six stages strictly — a drawing that shows all six out of sequence has not explained the cycle.
- Q19 (8): As allocated. Accept "cytoplasm" or "ribosomes" for the detail mark.
- Q20 (12): Boxes: phytoplankton, bacteria, zooplankton, viruses, nutrients (decomposition bacteria and the larger consumer chain also credit). The loop must close — nutrients must return to phytoplankton and bacteria, or it is a chain, not a loop.
- Q21 (3): In: the viral nucleic acids only (2). Out: the entire protein shell — capsule, sheath, base and fibres — remains attached to the outside of the cell wall (1).
- Q22 (3): The nucleoid (1) — a closed loop of DNA lying free in the cytoplasm rather than enclosed in a nuclear membrane (2). Accept "absence of a true nucleus" phrased either way.
- Q23 (4): Because the nutrients return to the start and are used again (2). Without those arrows it becomes a one-way chain ending at "nutrients", which would mean the surface ocean steadily loses its nutrients to deep water and eventually goes barren (2).
- Q24 (5): The loop passes nutrients only between microorganisms (1). It is linked because the same organisms sit in both (2): bacteria and phytoplankton are eaten by zooplankton and so feed the conventional chain, while also being lysed by viruses and so feeding the loop (2). The loop sustains the organisms the chain depends on.
- Q25 (4): One mark for a recorded time; three for an honest omissions list. There is no target time — the gap between attempt one and attempt two is the revision list.
- Q26 (3): Because two different processes are happening — ordinary feeding and viral lysis — and a single arrow style implies they are the same kind of transfer (2). Distinguishing them is what makes the diagram explain rather than merely connect (1).
Investigation 4 — disease in a tank
- Q27 (4): Two reasons, 2 each: animals are crowded together so a pathogen passes between hosts far more easily than in open water; and the stress of captivity weakens immune systems, which matters because parasitic marine fungi are opportunists targeting weakened hosts. Also credit: shared water recirculating a pathogen through the whole population, and reduced dilution compared with the open sea.
- Q28 (4): Tanks 2 and 3 (2) — both at 120 per m², differing only in water exchange. Mortality 7% vs 19%, so low exchange nearly tripled it (2).
- Q29 (4): Tanks 1 and 2 (2) — both high exchange, 60 vs 120 per m². Mortality 3% vs 7% (2).
- Q30 (5): Tanks 1 and 4 differ in two variables at once — density (60 → 240) and water exchange (high → low) (3). The comparison is confounded: the rise from 3% to 41% cannot be attributed to crowding alone, because the exchange rate also changed (2). The conclusion may well be partly right, but this pair of tanks is not evidence for it.
- Q31 (5): Crowding raises competition, physical contact and waste concentration, all of which are stressors (2). Sustained stress suppresses immune function (2), which is precisely the condition opportunistic fungi require — they cannot readily infect a healthy animal (1).
- Q32 (4): In the open sea the animals are dispersed, the water volume dilutes pathogens enormously, and the animals are not stressed (2). A tank removes all three protections at once, so the same organism meets crowded, stressed hosts in a small shared volume (2).
- Q33 (5): Any defensible recommendation supported by the data (3) — e.g. 120 per m² with high exchange, since it doubles stock over tank 1 for only 7% mortality. The trade-off must be named (2): higher density means more product but more disease risk, and high water exchange costs pumping energy and money.
- Q34 (4): A public aquarium has the same two conditions — captive animals held at densities far above natural, and the stress of captivity (2). It also has additional pressure: display animals are often long-lived, individually valuable and irreplaceable, and are frequently mixed from different source populations, which brings new pathogens into contact (2).
Marking note. Q3, Q7, Q12 and Q30 are the four that decide whether this sheet worked, and they test the same habit from four directions: knowing what your evidence does and does not establish. A clear control makes an experiment interpretable; a colony is not a bacterium; a mistimed wash produces a confident wrong answer; and a two-variable comparison proves nothing about either variable. A learner who has those four has learned more here than one who correctly labelled all three drawings.