Photosynthesis Experiment — Research Framing
Does the wavelength of light affect the rate of photosynthesis in Chlorella?
Independent Variable
Light wavelength (colour)
The colour of LED light each sample is incubated under. Six levels including a dark control.
Blue
Purple
Green
Red
Yellow
Dark
Dependent Variable
Change in buffer pH (ΔpH)
A proxy for net CO₂ consumption. Measured as absorbance at 562 nm and 595 nm using thymol blue + cresol red indicator, converted to pH via the calibration curve.
Control Condition
Dark (no light)
Provides a baseline for any metabolic activity (respiration) occurring independently of photosynthesis. Net photosynthesis = light ΔpH − dark ΔpH.
Controlled Variables
Incubation time (90 min)
Buffer composition
Buffer volume
Sphere number (10)
Sphere source/batch
Indicator volume (100 µL)
Indicator batch
Ambient temperature
Plate reader
Calibration file
Respiration Experiment — Research Framing
Does temperature affect the rate of respiration in Saccharomyces cerevisiae?
Independent Variable
Incubation temperature
Four temperatures spanning the expected enzyme activity range, including conditions above and below the yeast optimum.
0 °C
20 °C
40 °C
60 °C
Dependent Variable
Change in buffer pH (ΔpH)
A proxy for CO₂ production. Measured as absorbance at 450 nm and 630 nm using bromothymol blue + bromocresol purple indicator, converted to pH via the calibration curve.
Control Condition
20 °C (room temperature)
The reference condition. Provides a baseline rate against which the effect of warming or cooling is judged. The 0 °C condition also serves as a low-activity reference.
Controlled Variables
Incubation time
Buffer composition
Buffer volume (1000 µL)
Sphere number (8)
Sphere source/batch
Indicator volume (100 µL)
Indicator batch
Ambient room light
Plate reader
Calibration file
Biological Material
Buffer & Indicator
Sample Transfer
Incubation Conditions
Measurement & Analysis
DESIGN = built into protocol / equipment
PROCEDURE = depends on technique
1 · Biological Material
D01
Standardised sphere production
All alginate spheres are produced from the same well-mixed organism suspension using a consistent dropper protocol.
Standardisation means size and organism density variation between spheres should average out across the ten spheres per condition.
Addresses
U01
U03
D02
Fixed sphere number per condition
Each vial receives exactly the prescribed number of spheres: 10 Chlorella spheres for photosynthesis, 8 yeast spheres for respiration.
Standardises the total biological input across conditions. Averaging across multiple spheres reduces the influence of any single outlier.
Addresses
U01
U03
D03
Sphere observation at Step 2
Students visually inspect spheres for colour, integrity and uniformity before proceeding. Compromised spheres can be substituted.
Pale or fragmenting Chlorella spheres signal viability loss. This check filters out gross batch problems before time is invested.
Addresses
U02
D04
Phosphate-free buffer chemistry
The hydrogen carbonate buffer is prepared from plain sodium bicarbonate, deliberately excluding phosphate salts.
Phosphate chelates calcium and dissolves the alginate matrix. A phosphate-free buffer preserves sphere integrity across the incubation and between class sessions.
Addresses
U01
U02
D05
Between-class reconditioning
Between classes spheres are rinsed in distilled water, transferred to 1× culture medium and stored under moderate light at room temperature.
Maintains Chlorella viability across the 3–4 week program from a single sphere purchase, ensuring consistent biological material across classes.
Addresses
U02
2 · Buffer & Indicator
D06
Double rinse before T0
Spheres are rinsed twice with fresh buffer before the T0 measurement is taken.
Removes residual storage liquid that would otherwise alter the starting pH of every vial. The second rinse meaningfully reduces carry-over compared with a single rinse.
Addresses
U04
D07
Micropipette for buffer volume
The protocol specifies a micropipette (not a transfer pipette) to dispense exactly 1000 µL of fresh buffer into each vial.
Micropipette accuracy is ≈ ±1% versus ≈ ±5–10% for transfer pipettes. This brings buffer volume well below the dominant sources of uncertainty.
Addresses
U05
D08
Fresh tip per indicator addition
A clean micropipette tip is used for every 100 µL of indicator added to a well.
Prevents tip-side residue from carrying over between wells. Combined with the indicator being added after the sample, this minimises pre-equilibration with atmospheric CO₂.
Addresses
U06
D09
Calibration matches experiment
The calibration curve is generated using the same indicator batch, same plate reader and same wavelengths as the experiment itself.
If the calibration and experiment used different indicator preparations, every pH reading would carry a systematic offset. Matching them removes that offset.
Addresses
U07
U16
D10
Fresh buffer on the day
Buffer is prepared fresh for each class session, not stored from previous sessions.
Aged bicarbonate buffer drifts in pH as CO₂ exchanges with the atmosphere. Fresh preparation guarantees a known and consistent starting pH.
Addresses
U07
D11
Dual-indicator ratiometric system
Two pH indicators with overlapping ranges are combined: thymol blue + cresol red for photosynthesis, bromothymol blue + bromocresol purple for respiration.
A dual-indicator system measured at two wavelengths produces a more reliable absorbance-to-pH conversion across the working pH range than a single indicator.
Addresses
U07
U16
3 · Sample Transfer & Plate Loading
D12
Micropipette for sample transfer
Exactly 100 µL of incubated buffer is transferred from each tube to its assigned well using a P200 micropipette.
A P200 at 100 µL achieves ≈ ±1–3% transfer accuracy — small enough that pipetting noise sits well below the biological signal in a well-performing condition.
Addresses
U08
D13
Fresh tip per sample
A new pipette tip is used for every sample transfer rather than reusing the same tip across conditions.
Eliminates carry-over between conditions, which would otherwise blur the differences between, for example, a high-photosynthesis sample and the dark control.
Addresses
U09
D14
Indicator added after sample
The order is fixed: sample first, indicator added afterwards. The plate is then read promptly.
Minimises the window during which dissolved CO₂ can escape from solution into the atmosphere. Indicator-first loading would allow far more equilibration before reading.
Addresses
U10
D15
Lid replaced when possible
During respiration loading, the tube lid is replaced between samples whenever the sequence allows.
CO₂ escape is faster from warm (40°C, 60°C) samples. Keeping the lid on when possible reduces the systematic underestimate of respiration rate.
Addresses
U10
D16
Efficient loading sequence
Students load wells in a single uninterrupted sweep, then immediately load the plate into the reader.
Total time between opening the tubes and reading the plate is the dominant control on how much dissolved CO₂ is lost. A coordinated group can complete loading in under two minutes.
Addresses
U10
4 · Incubation Conditions
D17
Room-temperature incubation
All six photosynthesis conditions incubate in their light boxes at ambient room temperature, not in heated chambers.
Removes temperature as an intended variable. Differences between light conditions are interpreted as wavelength/intensity effects rather than thermal effects.
Addresses
U11
D18
Light intensity recorded & normalised
Peak irradiance (mW/m²) is measured for each LED with the UPRtek MK350N spectrometer and entered into the Analyser tool to normalise pH change per unit intensity.
LED brightness varies 5–6× between colours. Normalising by intensity helps separate the wavelength effect from the brightness effect, though this cannot fully resolve the confound.
Addresses
U12
D19
Wavelengths plotted against chlorophyll
LED peak wavelengths are overlaid on the chlorophyll a / b absorption spectrum in the companion visualisation tool.
Allows students to see directly which LEDs align with absorption peaks and which fall in absorption troughs — turning a numerical specification into a visual prediction.
Addresses
U13
D20
Pre-incubation bath check
Each water bath is measured with a thermometer before incubation begins. Hot or cold water can be added to correct any drift from setpoint.
Electronic bath controllers can drift over time. A pre-check shifts known offsets back toward setpoint before the biological clock starts running.
Addresses
U14
D21
Start & end temperature recorded
Bath temperatures are recorded at the start and the end of incubation, not just once.
Captures any drift across the incubation window. A bath that started at 38 °C and ended at 40 °C reveals more than a single reading would.
Addresses
U14
D22
Ice bath for 0 °C
The 0 °C condition uses an ice–water bath rather than an electronic refrigeration unit.
The ice–water phase equilibrium holds temperature at 0 °C by physical law, not electronic control. This makes 0 °C the most stable and reproducible of the four temperatures — and connects to the same equilibrium logic that stabilises the buffer's pH.
Addresses
U15
D23
Fixed incubation duration
All conditions in an experiment incubate for the same fixed time: typically 1.5–2 hours for photosynthesis and 45 minutes to 1 hour for respiration.
Holds time constant as a controlled variable. ΔpH then reflects rate × time with time held the same, so differences are interpretable as rate differences.
Addresses
U11
U14
D24
Light boxes partially enclosed
Each light condition is housed in a partially enclosed box that isolates its LED from neighbouring conditions and from ambient room light.
Ensures each sample receives the assigned wavelength rather than a mixture. The partial (not full) enclosure also allows passive ventilation to limit heat build-up.
Addresses
U11
U13
5 · Measurement & Analysis
D25
Same instrument and filters throughout
Calibration and experiment use the same Accuris MR9600 plate reader, the same filter set (562/595 nm for photosynthesis, 450/630 nm for respiration), and the same indicator batch.
A systematic offset in any of these would shift every pH reading by the same amount. Matching them removes the offset.
Addresses
U16
D26
Step 14 calibration check
Before running the Analyser, students verify that the loaded calibration file matches the experiment wavelengths (595 nm for photosynthesis, 450 nm for respiration).
Using a photosynthesis calibration on respiration data — or vice versa — produces wildly wrong pH values. The checklist catches this before time is wasted analysing bad data.
Addresses
U16
D27
T0 from a dedicated vial
The T0 (starting pH) is measured from a separate vial of freshly rinsed buffer that is not used for any of the experimental conditions.
Provides a clean baseline for every condition without sacrificing one of the four replicate vials. Each condition's ΔpH is computed relative to this common T0.
Addresses
U17
D28
Shared buffer stock across groups
All groups within a class draw their fresh buffer from the same prepared stock.
Ensures the buffer chemistry is identical across groups, so any between-group differences in T0 or ΔpH reflect technique or biology rather than buffer composition.
Addresses
U18
D29
Blank subtraction in Analyser
The Analyser tool subtracts a buffer-only blank reading from each sample well before converting absorbance to pH.
Removes the absorbance contribution of the indicator solution itself and corrects for small well-to-well differences in the plate, so the working absorbance reflects sample pH specifically.
Addresses
U06
U16
D30
Duplicate replicates per condition
Each condition occupies two adjacent rows in the 96-well plate. The Analyser averages the replicates and reports the replicate difference for each condition.
Averaging halves the random noise in the result. The replicate difference value is itself diagnostic — large differences point to transfer or pipetting issues for that condition.
Addresses
U06
U08
D31
Class-pool outlier flagging
When class data is pooled, the Analyser flags any individual group value that falls more than ±2 standard deviations from the class mean for that condition.
Surfaces likely execution errors — loading the wrong wells, missed T0, dropped tip — for class discussion, rather than letting them silently distort the group mean.
Addresses
U18
How this links to the Assumptions Catalogue
Each design choice above is an attempt to satisfy a particular assumption — a thing we need to be true for the experiment to work as intended. The corresponding U-numbers (U01 through U18) point you to the entry in the Experimental Assumptions Catalogue where you can examine how well each assumption holds in practice and what residual uncertainty remains.
The catalogue is the tool to use after you have analysed your results. By comparing the controls described here with the residual uncertainty described there, you can build a defensible argument about which differences in your data reflect biology and which reflect the limits of the experiment.