This is an execution manual — the master holds the "why"
This document is the procedure for the modern world: what to do, in order, start to finish. It assumes you can buy pre-mixed MS medium, bleach, alcohol, agar, and hormones, and power a pressure cooker and lights. For the science behind each step — contamination biology, the hormone ratio, every media formula and dosing number, diagnostics, and the full FAQ — see the master reference. Keep it open beside this.
Links work when all three files sit in the same folder.
This track assumes you can buy pre-mixed MS medium, bleach, alcohol, agar, and hormones, and can power a pressure cooker and lights. It's the fastest, most repeatable path, and the way to learn the craft. Work through it top to bottom. Difficulty rises with woodiness — begin with an easy herbaceous plant.
Assemble the kit
Buy or gather, once:
- Pressure cooker / canner (reaches 121 °C — your sterilizer), a stove or hotplate.
- Still-air box (clear tote on its side) to start; a DIY HEPA flow hood later.
- Glass jars with vented lids (micropore tape or polyfill-plugged hole); baby-food/canning jars are ideal.
- Scalpel + blades, fine forceps, and an alcohol lamp or bead sterilizer.
- 70% isopropyl/ethanol in a spray bottle; household bleach; a drop-bottle of surfactant (dish soap/Tween).
- Pre-mixed MS basal salts + vitamins powder; sucrose (table sugar); agar (or Phytagel); hormones BAP and IBA (add NAA optionally).
- Distilled/RO water; a 0.01 g scale; pH meter or strips; nitrile gloves + mask.
- Grow shelf with LED/fluorescent light on a timer, ~22–25 °C.
Set up two zones
A transfer zone (the still-air box or hood, in a closed low-traffic room) and a growing zone (the lit shelf). Wipe the box interior with 70% alcohol; if it's a still-air box, close it and let the air settle 10–20 minutes before opening any jar inside. Keep the room draft-free.
Mix and sterilize the medium
Multiplication medium — 1 liter
- Dissolve MS powder (~4.4 g/L) + 30 g sucrose in ~900 mL distilled water.
- Add hormones from stock: BAP 1.0 mg/L (+ NAA 0.1 mg/L optional). (Make a 1 mg/mL BAP stock: 100 mg in a drop of dilute HCl, water to 100 mL → add 1 mL per liter.)
- Top to 1 L; adjust pH 5.6–5.8 with dilute acid/base.
- Add 7 g agar; heat, stirring, until clear.
- Pour ~1.5–2 cm into jars; cap loosely.
- Pressure-sterilize 121 °C / 15–20 min. Cool undisturbed in the clean area; rest jars 2–5 days and discard any that cloud.
Also make a rooting medium the same way but with ½× MS, 15–20 g sugar, IBA 0.5 mg/L (or no hormone). And autoclave several jars of plain distilled water for rinsing.
Prepare the mother plant (Stage 0)
Two to six weeks ahead: bring the source plant under cover, water only at the base to keep foliage dry, and prune to force clean new growth. Optionally spray a preventive fungicide. Harvest from the youngest vigorous shoots.
Take and sterilize the explant
Surface sterilization
- Cut shoot tips / nodal segments slightly oversized; wash under running water (drop of soap for fuzzy tissue, rinse off).
- 70% alcohol, 30–60 s only.
- ~0.5–1.0% bleach (10–20% of household stock) + a drop of surfactant, 10–20 min, agitating. Milder/shorter for soft tissue.
- In the box, rinse in sterile water 3–4×, ≥1 min each.
- On a sterile surface with a flamed scalpel, trim off bleach-burned edges, size the explant, place cut-side to the gel, right way up.
For woody/browning species, dip in vitamin-C/lemon water, add activated charcoal to the medium, and plan to transfer to fresh medium after 24–48 h.
Initiate (Stage 1)
Seal, label (species / tissue / medium / date), place on the lit shelf at ~23 °C. Watch daily for 1–4 weeks. Cloudy/slimy/fuzzy = contamination → remove and sterilize-kill immediately, away from clean jars. Swelling and greening = success. Expect to lose 20–50% early; keep notes on what preceded each loss.
Multiply (Stage 2)
Move clean growth to multiplication medium. Every 4–6 weeks, in the box: lift the shoot cluster onto a sterile surface, divide with a flamed blade into pieces each bearing a shoot/bud, and place each on fresh medium. Re-flame the blade between cuts. One explant becomes hundreds over cycles. Avoid over-multiplying through callus (somaclonal variation) — re-start from the mother periodically.
Root (Stage 3)
When shoots reach ~1–2 cm and you have enough, move to rooting medium (IBA or none). Roots appear in days–weeks. Or root ex vitro: dip shoot bases in IBA and root them directly in the weaning tray — often better, and it merges Stages 3 and 4.
Wean to soil (Stage 4)
Hardening off — the second great killer
- Remove plantlet; gently wash all gel off the roots (leftover sugar rots).
- Pot into sterile, fast-draining mix (perlite/vermiculite/coir/pumice blend).
- Enclose at high humidity under a dome/tote — recreate the jar.
- Vent progressively over 2–4 weeks so it builds cuticle, stomata, tough roots.
- Bright indirect light, no direct sun, no fertilizer at first; ease both in.
- Graduate to nursery pot → harden outdoors → plant out.
Refine, store, and scale
Keep a notebook on every jar (sterilization times, hormone levels, outcome) — iteration is the skill. For long-term holding, put backup cultures in cool/dim slow-growth storage (subculture every 6–12 months), and for rare lines add ELISA/PCR-indexed clean stock and, if you have liquid nitrogen, cryopreservation. Keep working cultures, slow-growth backups, and seed/synthetic-seed archives as redundant layers.
Buy the kit → settle the air → mix & pressure-sterilize MS → clean the mother plant → alcohol-then-bleach-then-rinse the explant → initiate → multiply every 4–6 weeks → root (in jar or ex vitro) → wean slowly → log everything. Start herbaceous, graduate to woody, index and bank what matters.
Build a DIY Laminar Flow Hood
A still-air box gets you started; this is what you graduate to for higher throughput and lower losses.
- HEPA filter — a true H13/H14 panel (furnace, cleanroom, or salvaged medical). Its face area sets your working width. The one part you can't fake — a fan without real HEPA just blows spores at your work.
- Blower — a squirrel-cage/centrifugal blower sized to push gently and evenly through the filter's pressure drop. You want a laminar 0.3–0.5 m/s face velocity, not a gale. Undersize beats oversize.
- Plenum box — a sealed plywood/acrylic chamber between blower and filter that pressurizes evenly so air exits the whole filter face uniformly. Seal every seam; leaks ruin laminarity.
- Pre-filter — a cheap furnace filter on the intake extends HEPA life.
- Orientation — filter face vertical, blowing horizontally toward you, sweeping spores off the front edge. Work close to the filter face, upstream of your hands.
Test it: hold a smoking stick at the filter face — smoke should travel in smooth parallel lines, not tumble. Tumbling = leaks or wrong blower. Never run an alcohol lamp in the airstream (flame blows sideways). A hood protects the work, not you.
Common Modern Mistakes
Read this before you start — most first-timer failures are here.
- Trusting 99% alcohol. It's worse than 70% — dilute it. Pure alcohol seals cells before killing them.
- Under-timing the autoclave. Timing starts at full pressure, not power-on; big volumes need longer. A short cycle leaves spores.
- Fast hands in the still-air box. Quick motion re-suspends settled dust. The box only works with slow movement and a settle wait.
- Skipping the settle wait because the hood/box "looks clean."
- Over-multiplying. Chasing numbers through callus and high cytokinin breeds hyperhydricity and off-types.
- Cytokinin too high "to be safe." Drives hyperhydricity and ethylene damage — use the minimum that works.
- Weaning too fast. Beautiful jars, dead trays. The commonest heartbreak.
- Not resting media before use — cloudy jars from prep contamination get used on precious explants instead of discarded.
- Reusing one "sterile" water jar across explants, cross-contaminating.
- Ignoring the notebook — repeating the same failure because nothing was recorded.
Fix two things and most failures vanish: genuine heat sterilization (real 121 °C — never a single boil) and still, settled, clean air with slow hands. Nearly every failure traces to one of those two. For symptom-by-symptom diagnosis of everything else — hyperhydricity, shoot-tip necrosis, browning, recalcitrance — see the master's deep diagnostics.