Bubble Counting on a Hydrilla Sprig
In a practical examination a candidate is given a fresh sprig of Hydrilla, a 250 mL beaker, 1% sodium bicarbonate solution, a 60 W lamp on a movable stand, a thermometer and a stopwatch. The lamp is placed 20 cm from the beaker and bubbles from the cut end are counted for one minute; the count is repeated after moving the lamp to 10 cm, 5 cm, 2 cm and 1 cm. The water is held at 30 °C throughout. Counts per minute: 12, 22, 38, 40 and 41. The candidate then repeats the 2 cm reading using 0.1% bicarbonate (19 bubbles) and 5% bicarbonate (24 bubbles). Finally she repeats the 20 cm reading with water that has been boiled and cooled, and gets 3 bubbles in the first minute falling to 0 in the fourth minute. The examiner asks her to identify, for each step, which factor is limiting the rate, and to say what the bubbles actually prove.
1. Identify the limiting factor in each part of the experiment and justify each answer from the data rather than from theory alone.
Read the worked answer
Read the data as a response curve with a plateau.
20 cm → 10 cm (12 → 22). Halving the distance raises light intensity about fourfold and the rate nearly doubles: light is the limiting factor here. The rate falls short of a fourfold rise because at 20 cm light is very low and other factors already exert a partial drag.
10 cm → 5 cm (22 → 38). Still rising steeply, so light is still limiting, but the response is flattening.
5 cm → 2 cm → 1 cm (38 → 40 → 41). Light saturation. Adding more light changes nothing, so light is no longer limiting; the plateau is set by the dark reaction's supply of CO2 (and by the enzyme capacity, with temperature fixed at 30 °C).
2 cm with 0.1% bicarbonate (19). Same saturating light, tenfold less CO2, and the rate drops - this proves that at the plateau CO2 concentration is the limiting factor.
2 cm with 5% bicarbonate (24). The rate falls again, but this is not limitation, it is inhibition. A tenfold rise above 1% does not increase the rate; it depresses it, so the cause is an adverse effect - a shift in pH of the medium, osmotic stress on the tissue, or CO2 toxicity - not a shortage of substrate. The distinction between limitation and inhibition is the crux of the question: more of a limiting factor increases rate up to saturation, more of an inhibitor decreases it.
2. Explain the boiled-and-cooled water result fully. Why does the rate start at 3 and reach 0, and why is this control essential to the interpretation?
Read the worked answer
Boiling drives off dissolved gases, and on cooling the water re-equilibrates only with the atmosphere, so it contains almost no dissolved CO2 and little dissolved O2. The sprig therefore has no carbon source for the Calvin cycle, and photosynthesis cannot run even though light and temperature are adequate.
The 3 bubbles in the first minute are not photosynthetic oxygen: they are the residual dissolved gases in the tissue and the water coming out of solution as the sprig is handled, plus a small amount from the last stored CO2 in the tissue. That finite pool is used up, so by the fourth minute the count is 0. This is exactly the shape expected when the CO2 supply is removed.
The control is essential for two reasons. First, it shows that the bubbles in the bicarbonate series are generated by the plant and are CO2-dependent - that is, they are photosynthetic O2, not air simply degassing from the solution. Second, it sets the baseline: any bubble count in a treatment must be read against this near-zero control. Without it, the candidate cannot claim that the lamp and the bicarbonate are driving photosynthesis at all; she can only claim that bubbles appeared.
3. The examiner remarks that bubble counting is a crude measure of the rate of photosynthesis. Explain why, and propose two improvements that would make the result defensible.
Read the worked answer
Bubble counting measures gas volume, not oxygen, and it measures net exchange, so several errors accumulate:
- bubble size varies with the cut surface, the rate of release and the surface tension, so counting bubbles is not counting equal volumes of gas;
- oxygen is partly retained in solution and in the intercellular spaces, so the visible bubbles under-record the gas evolved;
- the sprig also respires, and its respiration is not measured, so the count is net photosynthesis;
- any dissolved air coming out of solution adds a non-photosynthetic component;
- the cut end is a wound, and its condition changes during the experiment.
The 3-bubble boiled-water value is precisely the size of this artefact.
Improvements:
- Collect the gas over 10 minutes in an inverted graduated tube or a syringe and record volume per unit time, then test the gas with a glowing splint - volume measurement eliminates the bubble-size error and identifies the gas.
- Measure dissolved oxygen directly with a Winkler titration or an oxygen electrode, which detects the O2 still in solution and allows a dark-respiration blank to be subtracted.
Alternative supporting measures are dry-mass increase of the sprig or ¹⁴CO2 fixation, both of which integrate over the whole treatment rather than at one cut end.