Contributors: Tank
Following the 17 July no-growth result, designed a set of M9 supplement-gradient plates to find the minimum supplementation needed to rescue growth. All conditions built on a 50 mL base: 12.5 mL 4X M9 salts, 1.0 mL 20% glucose, 31.5 mL agar-water, plus chloramphenicol (58.8 µl of 34 mg/mL stock ≈ 40 µg/mL final).
Group A — casamino acids gradient (fixed 1 mM thiamine): CA0 (0%), CA1 (0.05%), CA2 (0.1%), CA3 (0.2%), CA4 (0.4%) casamino acids, to identify the minimum concentration that rescues growth.
Group B — thiamine gradient (fixed 0.1% casamino acids, which also covers leucine): TH0 (0 mM), TH1 (0.1 mM), TH2 (0.25 mM), TH3 (0.5 mM) thiamine, to identify the minimum thiamine requirement.
Group C — leucine alone (fixed 1 mM thiamine, no casamino acids): Leu1 (0.4 mM), Leu2 (0.8 mM) leucine, to test whether leucine alone (without full casamino acids) supports growth.
[Note: the exact per-condition stock volumes are recorded in the source protocol sheet but were lost to formatting in the digitised copy — the condition labels and target concentrations above are recoverable and correct; volumes should be re-confirmed against the original before repeating.]
Stock solutions used: 4X M9 salts, ~4.76% agar-water base, 20% w/v glucose, 100 mM thiamine HCl (filter-sterilised, foil-wrapped, −20 °C), casamino acids (dissolve only, do not autoclave), 10% w/v MgSO₄·7H₂O, 80 mM CaCl₂ (anhydrous), chloramphenicol (34 mg/mL working stock).
Note: MgSO₄ stock was prepared from the heptahydrate (MgSO₄·7H₂O); a monohydrate version also exists in storage with a different molecular weight — do not substitute by mass alone.
Results: not yet transcribed from the plate-reader tank at time of writing.