Contributors: Tank, Chester
Protocol
Overnight LB culture (from 10 Aug) was washed twice into M9 salts (no carbon source, no supplements) before use — no vortexing, gentle inversion only, to avoid stressing the cells. Washed-suspension OD600 (against M9 blank) = 2.5; inoculum volume calculated as V = 1000 × target-OD ÷ OD600 per 20 mL culture at a target starting OD of 0.05 (≈ 400 µl).
FeSO₄·7H₂O stock was prepared fresh on the day of use — Fe²⁺ oxidises to Fe³⁺ within hours in neutral aqueous solution, forming a brown precipitate, so it can’t be stocked long-term.
7 parallel 20 mL cultures were set up to isolate which M9 supplements are actually required:
| # | Condition | Purpose |
|---|---|---|
| 1 | LB | Positive control — strain viability |
| 2 | M9 + glucose, no supplement | Negative control — baseline |
| 3 | M9 + glucose, full supplement | Main experiment (MgSO₄, CaCl₂, thiamine, casamino acids, FeSO₄) |
| 4 | M9 + full supplement, no carbon source | Negative control — supplements alone shouldn’t support growth |
| 5 | M9 + glucose, no thiamine | Is thiamine essential? |
| 6 | M9 + glucose, no FeSO₄ | Is iron limiting? |
| 7 | M9 + glucose, no casamino acids | Are amino acids essential? (lost — see below) |
Cultures grown at 37 °C, 200 rpm; OD600 sampled at T = 0, 2, 4, 6, 7, 8 h against matched uninoculated blanks (to control for FeSO₄’s own colour change over time).
Results
| Hour | 1 — LB | 2 — no supplement | 3 — full supplement | 4 — no carbon |
|---|---|---|---|---|
| 0 | 0.050 | 0.103 | 0.053 | 0.063 |
| 2 | 0.284 | 0.126 | 0.177 | 0.081 |
| 4 | 1.061 | 0.138 | 0.700 | 0.146 |
| 5 | 1.120 | 0.130 | 0.993 | 0.182 |
| 6 | 2.040 | 0.120 | 1.430 | 0.190 |
| 7 | 2.470 | 0.090 | 1.830 | 0.190 |
| 8 | 2.520 | 0.090 | 1.850 | 0.180 |
Both negative controls (2, 4) stayed flat near baseline throughout, as expected. Culture 3 (full supplement) grew robustly, tracking below but roughly parallel to the LB positive control.
[Note: cultures 5 and 6 (no-thiamine, no-FeSO₄) grew comparably to or better than culture 3 across the same time course — exact values were corrupted in the source file’s table formatting during digitisation and aren’t reliably recoverable, but the qualitative result (no drop in growth) is clear and is consistent with the 13 August entry’s conclusion that thiamine and FeSO₄ are non-essential.]
Culture 7 (no casamino acids) was lost — the bottle was smashed while taking an OD reading, so this condition has no data from this run.
Cultures 3 and 5 showed visible white pellets forming around hour 4, consistent with CaCl₂ precipitating out on pipetting into the culture.