Protocols
Sterilization and aseptic technique
Most contamination in tissue culture is introduced during transfers, not from the media itself. This protocol covers autoclave cycles, bleach surface sterilization, and laminar flow setup — with notes on how to document each as a repeatable SOP in xPlant.
Why aseptic technique matters
Tissue culture media is rich in sugars and salts that bacteria, fungi, and yeast thrive in. A single break in technique — touching an explant with an unflamed tool, opening a vessel in still air, or plating onto warm media — can contaminate an entire batch. Consistent, documented technique is the most effective contamination control available.
Safety note: Bleach and ethanol produce hazardous fumes at high concentrations. Work with good ventilation. Never mix bleach with other cleaners. Flame ethanol away from open bleach solutions.
Autoclave sterilization
Autoclaving is used for media, glassware, and heat-stable instruments. The standard cycle for tissue culture media is 121 °C at 15 psi for 15–20 minutes.
- 1Fill the reservoir to the minimum-fill line with distilled or deionised water.
- 2Load items loosely — overcrowding blocks steam penetration. Leave caps slightly loose on bottles.
- 3Select the correct cycle: 121 °C / 15 psi for 15–20 min (liquid) or 30–40 min (porous/wrapped loads).
- 4Allow pressure to drop to zero before opening. Remove liquids with heat-resistant gloves.
- 5Allow media to cool to ~50 °C before pouring or adding heat-sensitive additives (e.g. PGRs).
Log it in xPlant:Create an SOP called “Media autoclave — liquid cycle” with each step above. When you autoclave a batch, add an SOP log entry noting the cycle date, run duration, and any deviations (e.g., extended run for larger volumes).
Bleach surface sterilization
Used to decontaminate explant surfaces before culture initiation. Bleach concentration and exposure time vary by species — delicate leaf tissue may need a shorter duration or lower concentration than woody stems.
- 1Prepare a 10–20% commercial bleach solution (1–2% sodium hypochlorite final) with 1–2 drops of Tween-20 per 100 mL.
- 2Immerse explant material for 10–20 minutes, agitating gently. Adjust time for species sensitivity.
- 3Rinse 3× with sterile distilled water inside the laminar flow hood.
- 4Allow excess surface moisture to evaporate on sterile filter paper before plating.
Species variation:Start conservative (10% bleach, 10 min) and record outcomes. Adjust per SOP based on contamination and browning results. xPlant's SOP notes field is a good place to capture species-specific adjustments.
Laminar flow hood operation
A laminar flow hood protects work from ambient contaminants by pushing HEPA-filtered air in a continuous, unidirectional stream. It does not protect the operator — it is not a biosafety cabinet. Always work in the laminar flow stream, never against it.
- 1Turn on the blower at least 20–30 minutes before work begins.
- 2Wipe all interior surfaces with 70% ethanol. Work from back to front, top to bottom.
- 3Flame all tools (scalpels, forceps) in 70% ethanol and allow to cool before contact with tissue.
- 4Work at least 15 cm inside the hood opening. Never block airflow between your work and the filter.
- 5Minimise movement in front of the hood — turbulence disrupts laminar flow.
- 6Leave tools inside the hood for the entire session; re-flaming after any contamination event.
Documenting this as an SOP
Sterilization is typically broken into at least two SOPs: one for media prep (autoclave) and one for transfer work (hood setup + surface sterilization). Keeping them separate lets you log them independently and assign each to the correct stage type.
- →"Media autoclave — liquid cycle" — attach to all media-prep stages
- →"Explant surface sterilization — bleach" — attach to initiation stages; note the species in the log
- →"Laminar flow setup" — a short checklist SOP you run at the start of every transfer session