Protocols
Media preparation protocol
Consistent media is the foundation of reproducible tissue culture. This protocol walks through weighing, dissolving, pH adjustment, dispensing, and autoclaving — with a sample Murashige and Skoog (MS) formulation and notes on recording each batch as an xPlant media recipe.
These are reference steps only — not a substitute for validated lab-specific procedures. Verify formulations against authoritative sources and your own experimental data before use.
Reference: MS basal medium (1 L)
Murashige and Skoog (1962) is the most widely used basal medium in tissue culture. The following table is a quick reference — use your lab's validated recipe or a quality premix where available.
| Component | Amount | Note |
|---|---|---|
| Macro salts (MS premix) | 4.4 g | Or weigh individual salts |
| Sucrose | 30 g | Standard; reduce for some species |
| Myo-inositol | 100 mg | Often included in premix |
| Thiamine HCl | 0.1–1 mg | Often included in premix |
| Agar | 7–8 g | Omit for liquid culture |
| Distilled water | To 1 L | |
| pH | 5.7–5.8 | Adjust before autoclaving |
Preparation steps
- 1
Gather materials
Analytical balance, distilled or deionised water, volumetric flask (1 L), magnetic stirrer + hotplate, pH meter, KOH or HCl for pH adjustment, agar (if solidifying), media salts and PGRs.
- 2
Dissolve salts
Add ~800 mL of distilled water to the flask. Weigh each macro- and micronutrient salt and dissolve in order of concentration (macros first). Stir gently; some salts may need gentle heat to dissolve fully.
- 3
Add sugar and vitamins
Add sucrose (typically 30 g/L for MS). Add vitamins (thiamine, glycine, myoinositol) if not included in a premix. Stir to dissolve.
- 4
Add plant growth regulators
Add PGRs (auxins, cytokinins) last before pH adjustment. Many PGRs are prepared as stock solutions (1000× or 10 000×) dissolved in KOH or ethanol — add the calculated volume from stock.
- 5
Adjust pH
Calibrate your pH meter. Adjust to pH 5.7–5.8 (or protocol-specific target) using 1 M KOH to raise or 1 M HCl to lower. pH will shift slightly on autoclaving — stop at 5.8 if autoclaving a solidified medium.
- 6
Add agar (solid media)
Bring volume to 1 L. Add agar (typically 7–8 g/L for standard solid media). Stir on hotplate until agar is fully suspended — it will not dissolve until autoclaved.
- 7
Dispense into vessels
Pour ~20–25 mL per culture tube or jar before autoclaving. Leave caps loose or use autoclave-safe vented caps. Do not overfill — agar expands slightly and needs headspace.
- 8
Autoclave
121 °C, 15 psi, 20 minutes (liquid cycle). Allow to cool to ~50–55 °C before pouring plates or adding heat-sensitive additives. Solidified media in vessels can cool in a rack.
- 9
Cool and store
Allow to solidify at room temperature. Inspect vessels for unusual colour, particulates, or contamination before use. Store at 4 °C for up to 4 weeks or at room temperature for up to 2 weeks (sealed, dark).
Logging batches in xPlant
xPlant media recipes are designed to track the formulation context, not to enforce exact measurements. Use them to record what went into a batch and attach the recipe to the stages that used it.
- →Create a media recipe named by formulation and purpose — e.g. "MS 0.1 BAP — Multiplication" or "WPM 0.5 IBA — Rooting".
- →Add ingredients with amounts and units. xPlant stores this as reference context, not a formulation calculator.
- →When you prepare a batch, attach the recipe to the relevant stages. If you deviated (adjusted pH differently, swapped a salt source), note it in the stage transfer log.
- →Review which recipes are used most often via the media recipe list — it helps identify which formulations to standardise first.
Common preparation issues
Agar won’t gel or gels unevenly
Likely cause: pH too low before autoclaving, or agar not fully suspended before dispensing.
Try: Target pH 5.8 before autoclaving. Stir agar thoroughly while hot. Check agar source — some suppliers have lot-to-lot quality variation.
Media turns brown after autoclaving
Likely cause: Over-autoclaving, temperature too high, or high sugar concentration.
Try: Check autoclave calibration. Reduce autoclave time for small volumes. Sucrose undergoes Maillard-type reactions at high heat — some browning is normal in small quantities.
Media contaminates within days
Likely cause: Loose vessel caps, handling before media cooled, or contaminated water source.
Try: Use autoclave-safe vented caps or foil. Handle vessels in laminar flow after autoclaving. Verify water source purity (use fresh distilled or 18 MΩ deionised).