Culture media

1. Water

• Laboratory media are prepared with distilled water of conductivity no greater than 2 µS/cm, or with deionised water of purity grade I–II. Tap and artesian water are acceptable only in production formulations (for example, the modified Zarrouk medium), where the hardness is taken into account by reducing the CaCl₂ dose.
• Freshly distilled water should be left to stand for 1–2 days before preparing oligotrophic media (Chu-10, ASM-1, WC): this removes dissolved CO₂ and stabilises the pH.
• Natural seawater is collected well offshore, filtered through 0.45 µm, held in the dark for 2–4 weeks ("ageing") and filtered again through 0.22 µm. Ageing is necessary for the mineralisation of dissolved organic matter.

2. Order in which salts are dissolved

• Universal rule: salts are added one at a time into the most dilute solution possible, each weighed amount being fully dissolved before the next is added. Concentrated stock solutions are never mixed directly with one another.
• Calcium salts (CaCl₂, Ca(NO₃)₂) are added LAST — after the phosphates and carbonates, with vigorous stirring, in a volume of at least 80 % of the final one. Departing from this order produces a white turbidity of Ca₃(PO₄)₂ and CaCO₃ that irreversibly removes phosphorus and calcium from the medium.
• Iron salts are added only together with a chelator (EDTA, citrate, NTA), having first been mixed in a separate portion of water and held for 20–30 min until complexation is complete — until a stable yellow colour appears with no precipitate.
• Silicates (Na₂SiO₃·9H₂O) are added at the very end and only to a medium already adjusted to its working pH: in an acidic medium, amorphous SiO₂ precipitates and is unavailable to diatoms.
• Sodium bicarbonate is added last, or prepared as a separate solution — especially when calcium is also present.

3. Stock (concentrated) solutions

• Most media are more conveniently prepared from ×100 … ×1000 stock solutions than from dry weighings: a 6 mg weighing cannot be made accurately, whereas measuring 1 mL of a 6 g/L solution is straightforward.
• Rule of separate storage: phosphates, calcium salts, carbonates and iron solutions are kept in SEPARATE bottles. Combining them in one stock will invariably produce a precipitate.
• Shelf life: macronutrient salts 6–12 months at 4 °C; iron solutions 3–6 months (replace as soon as a brown suspension appears); vitamins 6 months at −20 °C.
• Labelling is mandatory: solution code, date of preparation, expiry date and the name of the person who prepared it. Solutions are kept in dark glass; plastic is acceptable for trace elements but not for EDTA solutions in long-term storage.

4. pH adjustment

• The pH is adjusted AFTER all components have been added but BEFORE sterilisation, using dilute 1 M NaOH (KOH) or 1 M HCl. Concentrated acids and alkalis cause local precipitation at the point of addition.
• Autoclaving shifts the pH: bicarbonate-containing media (Zarrouk, SOT) rise by 0.5–1.0 units as NaHCO₃ decomposes and CO₂ is lost; media with organic buffers usually fall by 0.1–0.3 units. The target value is set with this shift in mind.
• Buffer systems: phosphate buffer (BBM), Tris 10–20 mM (TAP, WC, Provasoli), HEPES 10 mM (BG-11 modifications), and the carbonate–bicarbonate system (BG-11, Zarrouk). Tris and HEPES are not autoclaved together with sugars.

5. Sterilisation

• Autoclaving: 121 °C at 1 atm gauge pressure, 15–20 min for volumes up to 1 L; 25–30 min for 2–5 L. Exceeding these times caramelises the agar and decomposes bicarbonate.
• Sterile filtration (0.22 µm membrane) IS MANDATORY for: vitamins, glucose and other organic substrates, bicarbonate solutions, hypersaline media (Johnson medium), and seawater containing silicate.
• Separate sterilisation: incompatible components (bicarbonate + calcium; phosphates + calcium; sugars + amino acids) are autoclaved in separate vessels and combined aseptically only after they have cooled completely to room temperature.
• Pasteurisation (80 °C for 30 min, three times at 24 h intervals) is a gentler alternative for seawater that preserves the carbonate equilibrium.

6. Agar-solidified media

• Agar concentration: 10–12 g/L for plates, 6–8 g/L for semi-solid media, 15 g/L for agar slants in tubes. Use bacteriological or purified agar; technical-grade agar contains inhibitory impurities.
• Agar is autoclaved SEPARATELY in twice the volume of water, the medium is made up at double strength, and the two are combined at 45–50 °C. Autoclaving agar together with phosphates and calcium gives cloudy plates and reduces plating efficiency.
• For cyanobacteria, 0.3 g/L Na₂S₂O₃·5H₂O is added to the agar: thiosulphate neutralises the growth-inhibiting products of agar's thermal decomposition.
• Plates are dried in a laminar flow cabinet for 20–30 min lid-down and stored at 4 °C in the dark for no more than 3–4 weeks.

7. Inoculation and culture conditions

• Inoculum volume is 5–10 % of the medium volume, giving a starting density of 10⁵–10⁶ cells/mL. Too small an inoculum prolongs the lag phase; too large a one exhausts the nutrients quickly.
• Irradiance: 20–50 µmol photons·m⁻²·s⁻¹ for maintaining collections, 100–200 for batch cultures, up to 500 for inducing carotenogenesis. Photoperiod 12:12 or 16:8; continuous light for many cyanobacteria.
• Temperature: 20–25 °C for most freshwater forms, 28–35 °C for Arthrospira and Chlorella sorokiniana, 45–55 °C for thermophilic cyanobacteria, 15–20 °C for polar and cold-water strains.
• Collection cultures are subcultured every 4–8 weeks. For long-term storage, use cryopreservation (10 % DMSO or 5 % methanol, −196 °C) or immobilisation in agar under a layer of mineral oil.

8. Common errors and how they present

• White turbidity immediately on mixing — precipitation of calcium phosphate or carbonate: the order of salt addition was wrong, or concentrates were mixed.
• Brown flocs and a rusty deposit on the bottom — iron hydrolysis: too little chelator, an expired Fe solution, or too high a pH at the moment of addition.
• No growth in an apparently normal medium — vitamins destroyed during autoclaving, no carbon source in a closed vessel without sparging, or residual nitrate in a supposedly nitrogen-free medium.
• Browning and lysis of the culture after 3–5 days — the buffering capacity is exhausted and the pH has risen above 10 through photosynthetic CO₂ consumption.
• Chlorosis (yellowing) of the culture — nitrogen or iron starvation; under nitrogen starvation, cyanobacteria additionally degrade their phycobilisomes.