Galdieria · Aim 1 · Heterotrophic screen
Bench lab book
Working notes for the heterotrophic screen: size each starved-pellet resuspension so every isolate inoculates at the same OD₆₁₀, keep the plate-reader layout to hand, and turn each Cytation5 export into ranked phycocyanin and growth curves. Live — type and it recalculates.
- STEP 01Phototrophic flaskMid-exp, OD₆₁₀ ≥ 0.8
- STEP 02→ 15–20 mL into FalconWash ×2–3, ~48 h dark starve, 45 °C
- STEP 03Measure OD₆₁₀Pull 1 mL for the colorimeter
- STEP 04Spin + resuspendPellet, remove sup, add V4 to calc'd volume
- STEP 05Split into flasksEqual volume each → all start OD₆₁₀ 0.10
Wash & starve
Before the screen, strip phototrophically grown cells of carried-over medium carbon (wash) and run their internal carbon and energy reserves down (starve — dark and carbon-free). This is what makes the no-carbon V4 control a true negative: any growth on sugar is then genuinely heterotrophic, not coasting on stored reserves.
All carbon-free 1× V4 — separate from the V4 that fills the flasks themselves (3 with carbon + 1 no-carbon control per isolate). Three washes is the safe default: residual phototrophic-medium carbon is exactly what would let the control grow and corrupt the heterotrophy call.
Step by step
- 1
Harvest. Spin late-exponential phototrophic culture (3,000–5,000 ×g, 5–10 min) and pour off the spent, carbon-containing supernatant.
- 2
Wash ×2–3. Resuspend fully in carbon-free V4 at culture pH (~2–4) and temperature, spin, discard — each cycle dilutes carried-over medium carbon ~20–40×. Never wash in water or neutral buffer; an acidophile will pH-shock.
- 3
Resuspend for the starve. Take the washed pellet up in carbon-free V4 at OD₇₅₀ ≈ 1–2, in a flask with generous headspace for aeration.
- 4
Starve. Hold dark and carbon-free at culture temperature (~45 °C), shaking 120–150 rpm — the screen's ~48 h, extended only if OD₇₅₀ is still climbing. Dark forces cells off photosynthesis onto stored carbon; reserve burn-down is aerobic, so keep it aerated.
- 5
Confirm it worked. Track OD₇₅₀ — a plateau (and a flat no-carbon control after inoculation) means reserves are spent. Under-starve → false heterotrophy positives; over-starve → lost viability and long lag.
- 6
Re-harvest. Spin again and discard the supernatant — this is the starved pellet the inoculation step below resuspends.
- 7
Resuspend & inoculate. Read OD, resuspend to the volume the inoculation calculator gives, and add the same small volume (≤ ~2–5% of the flask) to every flask so all isolates start at OD 0.10.
Methods blurb
Prior to the heterotrophic screen, phototrophically grown cultures were harvested in late-exponential phase by centrifugation (≈4,000 ×g, 10 min) and washed two-to-three times in carbon-free V4 medium (pH ~2–4) to remove residual organic carbon. Washed cells were resuspended in carbon-free V4 and starved in the dark at ~45 °C with shaking (≈120–150 rpm) for ~48 h (until OD₇₅₀ stabilised) to deplete intracellular carbon and energy reserves. Starved cells were re-pelleted, resuspended, and inoculated to a uniform starting OD₇₅₀ of 0.10 into 125 mL flasks containing 50 mL V4 with kiwifruit-derived sugar (10 g L⁻¹) or, for the no-carbon control, V4 without added carbon.
Starvation monitor
A separate upload for the daily 200 µL reads from the starving cultures (20 mL carbon-free V4, dark, 150 rpm, 45 °C). It tracks biomass and phycocyanin per isolate, flags whether each is still growing on reserves or has plateaued (ready to inoculate), and sizes the inoculation from the latest OD.
Reads from the starving cultures — 20 mL carbon-free V4, dark, 150 rpm, 45 °C, 200 µL sampled per day. Drop each day's Cytation5 export here (separate from the screen analysis below). It charts biomass and phycocyanin per isolate, flags whether each is still growing on its reserves, and once they've plateaued, sizes the inoculation.
Starved pellet → inoculation
After the 48 h starve, enter each isolate's measured OD₆₁₀. The calculator tells you what volume of sterile V4 to resuspend that pellet in, so that adding a fixed small volume to each flask lands every isolate at the same starting OD. It also checks you have enough biomass to fill all replicates.
| Isolate | Starved OD₆₁₀ | Resuspend pellet in | Available | Add / flask | Status | |
|---|---|---|---|---|---|---|
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough | |||
| 5.09 mL | 28.0 OD·mL | 1.0 mL ×3 | enough |
Resuspend each pellet in the listed volume of sterile carbon-free V4 (that puts it at OD 5.50), then pipette 1.0 mL into each flask — every isolate starts at OD 0.1. The leftover suspension is discarded. Confirm replicate OD CV < 10% before the run begins.
Plate layout — one isolate per row
7 isolates, one per row (A–G), three replicates each — every replicate read as a neat / 1:4 / 1:16 group across three wells. Row H holds three media blanks. Row labels follow the isolate names you set above. Scroll the plate sideways on a phone.
Plate layout with one isolate per row: 7 isolates in rows A to G, three media blanks in row H, each row holding three replicates laid left to right, every replicate a neat / 1:4 / 1:16 dilution group across three columns.
Read the plate — upload & analyse
Export each plate from the Cytation5 as .xlsx and drop it in. It reads the layout and the OD / chlorophyll / phycocyanin values, blank-corrects against the BLK wells, and back-calculates the neat / 1:4 / 1:16 dilution series. One file charts each isolate ranked by phycocyanin; several files (one per timepoint) plot growth curves over time. Everything runs in your browser; the files never leave your device.
Lab notes
The carbon-starve timecourse so far — figures plus dated bench notes (protocol decisions, what each run showed, what it means). Saved on-device and synced with the rest of the session; notes support light markdown.
| Isolate | Day 0 | Day 1 | Day 2 | Day 3 | Verdict |
|---|---|---|---|---|---|
| CM1.3Y | 8.5 | 7.3 | 8.8 | 7.3 | plateaued |
| CM1.1G | 9.0 | 8.1 | 9.3 | 7.7 | plateaued |
| CM1.2Y | 14.6 | 13.6 | 13.2 | 11.3 | declining |
| RK1.2 | 10.4 | 8.0 | 8.0 | 7.7 | plateaued |
| RK1.3 | 3.0 | 3.5 | 3.5 | 3.2 | plateaued |
| RTK37.1 | 6.0 | 4.6 | 4.8 | 4.8 | plateaued |
| KP1.2Y | 2.9 | 3.1 | 2.9 | 2.6 | declining |
OD₇₅₀ biomass — read protocol & corrections
Jun 11, 2026
Read protocol (every timepoint)
- 100 µl per well, Nunclon 96 flat bottom. Keep this fixed — day-to-day comparability depends on a constant path length.
- Pathlength correction OFF. The 977/900 method is corrupted by cell scattering in the near-IR and over-corrects turbid wells. Match volumes physically instead.
- Read raw OD₇₅₀, blank-subtract against carbon-free V4 at the same 100 µl.
Dilution & linearity (Cytation5, these strains)
- Multiple scattering makes dense neat wells under-read: ~22–33% at 150 µl, ~6–14% at 100 µl. A 1:9 back-calc (×9) is the cleanest true-density anchor; 1:3 (×3) agrees once it's pipetted right.
- Equal-volume discard scheme — pull the transfer volume off the final well too, so neat / 1:3 / 1:9 all sit at 100 µl. This fixed the 1:3: day-2 ratios came out ~3 (vs the under-diluted 1.1–2.4 when the middle well was left short).
- Pipetting the 1:3: fresh tip per transfer, wipe the tip after drawing culture, reverse-pipette, mix 4–5× in the destination, and make sure the full 100 µl V4 goes in.
Evaporation — the dominant artifact
- Flasks evaporate unevenly (2–16 mL/day off a 20 mL start; thermophiles in a warm incubator). Concentration inflates raw OD and fakes growth.
- Weigh every flask each timepoint and report total biomass = OD₇₅₀ × volume. Volume = (flask weight − ~75 g empty) ÷ 1.0 g/mL. Evaporation-proof.
- Best practice: top each flask back to a fixed volume with carbon-free V4 _before_ sampling. You then read at low concentration (suppression ~vanishes) and OD is directly comparable across days — day 2 done this way matched day 0 cleanly. Carbon-free holds osmolarity without re-feeding carbon.
Starvation monitor — readiness verdict (day 0–2)
Jun 12, 2026
Ran the day 0–2 timecourse through the starvation monitor's trend classifier (recent slope as %/day: >+5% growing, within ±5% plateaued, <−5% declining), on volume-corrected total biomass.
Verdict
strain rate status read CM1.1G +1.7%/d plateaued ready to inoculate CM1.3Y +1.9%/d plateaued ready KP1.2Y +0.8%/d plateaued ready * CM1.2Y -4.9%/d plateaued ready (edging down) RK1.3 +6.6%/d growing not done — keep starving RTK37.1 -11%/d declining over-starve risk RK1.2 -14%/d declining over-starve risk
Notes
- 4 at plateau (reserves spent → inoculate); RK1.3 still growing (under-starved — would give false heterotrophy positives); RK1.2 & RTK37.1 tipped into decline (viability / long-lag risk).
- ⚠️ On raw OD the monitor calls CM1.3Y *still growing* (+7.3%/d) — its day-2 flask had evaporated to 18 mL, inflating concentration. Volume-correction flips it to plateaued. Always normalise by weight (the monitor now has a toggle for this).
- * KP1.2Y has ~zero pigment, so its OD₇₅₀ isn't real biomass — verdict not meaningful.
- 3 timepoints only; the slopes will firm up from day 3.
Carbon-starve timecourse (day 0–2) — total biomass
Jun 12, 2026
Per day: 7 strains × neat / 1:3 / 1:9, all 100 µl, PLC off, blank-subtracted vs V4. Total biomass = OD₇₅₀ × flask volume at sampling (best-estimate OD = 1:9×9 for dense strains, neat for dilute). Started 20 mL/flask.
Total biomass — day 0 → 2
strain d0 d1 d2 d0→d2 CM1.3Y 8.46 7.35 8.77 +4% CM1.1G 9.00 8.05 9.29 +3% CM1.2Y 14.58 13.58 13.24 -9% RK1.2 10.44 8.04 8.01 -23% RK1.3 3.02 3.45 3.46 +15% RTK37.1 5.98 4.58 4.85 -19% KP1.2Y 2.86 3.14 2.91 +2%
Read
- Biomass is holding, not growing — textbook carbon starvation. Most strains flat (±4%) over 2 days.
- Day 0 ≈ Day 2 (both sampled at ~20 mL standard volume, low suppression) → standing biomass is stable. The day-1 dip is the evaporated/concentrated-state sampling artifact (CM1.1G at 6.3 mL), not biology.
- Decliners: RK1.2 (−23%), RTK37.1 (−19%), CM1.2Y (−9%) — losing standing biomass; watch whether they keep dropping.
- RK1.3 (+15%) is the only real-looking riser, but low-biomass and noisy.
Flags
- ⚠️ KP1.2Y flask evaporating catastrophically — down to 3.65 mL by day 2 (lost 16.4 of 20 mL), nearly dry, ~16 mL added back. Fix the lid/seal/position. RK1.2 & RK1.3 also lost ~10 mL.
- KP1.2Y OD₇₅₀ is not photosynthetic biomass — phycocyanin & chlorophyll at noise (~9 / 13 RFU vs hundreds–thousands elsewhere). Verify by cell count / scope before trusting the line.
- Day-0 numbers come from the method-matched re-read; treat day 2 (clean, topped-up sampling) as the reference protocol going forward.
carbon-free 1× V4 throughout · resuspend to the volume shown, then add the same volume to every flask · all manipulations in BSC · hamishlindsay.com