Tutorial: aseptic technique and explant sterilisation

Culture medium is full of sugar, which makes it a feast for bacteria and fungi. A single spore that lands on it will outgrow your plant tissue in days. Aseptic technique is simply the set of habits that stops that happening.

Setting up the laminar flow hood

1. 1Switch the hood on 15–20 minutes before work so the airflow settles.
2. 2Wipe every surface with 70% ethanol, working from back to front.
3. 3Spray and wipe anything you bring in: bottles, racks, the outside of vessels.
4. 4Keep the work area uncluttered. Don't block the air coming from the filter at the back.
5. 5Sterilise tools between cuts, using a bead steriliser or by dipping in ethanol and flaming, and let them cool before touching tissue.

Sterilising an explant surface

Exact times depend on how tough the tissue is, so treat these as starting points and test on a few pieces first.

1. 1Wash the material under running tap water, with a drop of detergent, for 10–30 minutes.
2. 2In the hood, dip it in 70% ethanol for 30–60 seconds.
3. 3Move it to sodium hypochlorite (about 1–2% available chlorine) with a drop of Tween 20 for 10–15 minutes, swirling now and then.
4. 4Rinse three times in sterile distilled water to remove all the bleach.
5. 5Trim away bleached edges and place the explant on medium.

Habits that matter

• Work with your hands and arms, not your face, inside the hood. Don't talk over open vessels.
• Never pass your hand over an open container.
• Wear clean lab coats and gloves, and tie long hair back.
• Check cultures every few days and remove contaminated ones quickly, closed, so they don't spread spores.