Introduction
Plant tissue culture allows propagation of disease-free plants from small explants in artificial media. Success depends almost entirely on aseptic technique — excluding bacteria, fungi, and yeast from cultures. Contamination typically appears within 3–7 days as cloudy media, fungal mats, or bacterial swarming.
Why contamination spreads so fast
Rich culture media (sucrose, amino acids, vitamins) supports microbial growth faster than plant growth. A single mold spore can overtake a petri dish overnight. Therefore every surface, tool, and airflow path must be controlled.
Step-by-step sterile workflow
- Prepare the workspace — wipe bench with 70% ethanol. Lay out only what you need. Avoid clutter that blocks laminar flow.
- Disinfect explants — common protocols use 10–20% commercial bleach (sodium hypochlorite) for 10–20 minutes, followed by 3–5 rinses in sterile distilled water. Woody species may need longer exposure or added surfactant.
- Flame tools — dip forceps and scalpels in ethanol, flame until red, then cool 10–15 seconds before touching tissue. Re-flame after touching non-sterile surfaces.
- Work inside laminar flow — keep hands downstream of explants. Minimize talking and movement to reduce dust carry-in.
- Seal cultures — parafilm or vented lids prevent desiccation while limiting entry of contaminants.
Recording and troubleshooting
Log species, explant type (leaf, node, root), disinfectant concentration, and exposure time. If cultures fail repeatedly, reduce bleach time or add ascorbic acid to reduce oxidative damage to plant cells.
Key takeaway: Sterile technique is a habit, not a one-time step. Consistency matters more than expensive equipment.