Aseptic technique

Awareness level only. This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.

Why it matters

Aseptic technique is the set of habits that keeps unwanted microbes out of your work, and keeps your work's microbes where they belong. A single contaminating bacterium or fungal spore can ruin a cell culture that took weeks to grow, give a false positive in a clinical culture, or contaminate a batch in a biomanufacturing facility. Good technique also protects you and others. Every biological technician, medical laboratory technologist and biochemist is expected to perform it automatically.

Sterile versus aseptic

• Sterile means free of all living microorganisms. Items are made sterile by autoclaving (steam under pressure), filtration, irradiation or similar.
• Aseptic technique means working in a way that keeps sterile items sterile and prevents contamination during handling.

You cannot make your bench sterile, but you can avoid contaminating sterile media, tips and cultures while you work.

Where you work

• Biosafety cabinet (BSC). Cell culture and work with many microorganisms is done in a Class II BSC, which draws air through HEPA filters to protect the work, you and the room. Do not block the front or rear grilles, work at least several centimetres inside the cabinet, and move slowly so you do not disrupt the air curtain. Bunsen burners are generally not used in a BSC: the heat disrupts airflow and can damage the filter.
• Open bench with a flame. Many microbiology teaching labs work on the bench beside a Bunsen burner, which creates an updraft, and flame loops and tube mouths. Follow what your lab's SOP specifies.

The biosafety level of the agent (BSL-1 to BSL-4) decides which containment and practices are required. See the CDC/NIH guide Biosafety in Microbiological and Biomedical Laboratories (BMBL): https://www.cdc.gov/labs/bmbl/ (Checked on 2026-10-23.)

The core habits

1. Prepare. Plan the steps, gather everything you need, and label tubes and plates before you start, so the cabinet is not crowded and open containers wait as little as possible.
2. Hands and PPE. Wash hands, put on a clean lab coat and gloves as your SOP requires. Spray gloves with 70 percent ethanol and let them dry.
3. Disinfect the work surface before and after, usually with 70 percent ethanol (which works best with some water present and needs contact time) or another approved disinfectant.
4. Disinfect items going into the cabinet by wiping or spraying bottles and racks.
5. Open containers only when needed and close them promptly. Hold caps in your hand or place them face down on a disinfected surface, as your SOP specifies. Never touch the inside of a cap, the neck of a bottle or a sterile tip.
6. Never pass hands or arms over open containers. Particles fall from you onto whatever is below.
7. Use each sterile pipette or tip once. If it touches anything non-sterile, discard it and take a new one.
8. Work in a logical flow, from clean to dirty, and keep waste at the side.
9. Clean up. Discard waste into the correct biohazard containers, disinfect the surface again, remove gloves and wash hands.

Worked example: an aseptic transfer of medium

In a BSC, a technician moves 10 mL of sterile medium into a labelled sterile flask:

1. Wipes the medium bottle and flask with 70 percent ethanol and places them in the cabinet.
2. Attaches a sterile 10 mL serological pipette to the pipette controller without touching the tip.
3. Opens the bottle, keeps the cap in the other hand, draws 10 mL, recaps the bottle.
4. Opens the flask, dispenses down the inside wall without touching the neck, recaps the flask.
5. Discards the pipette into the waste container without touching anything else on the way.

Every step keeps sterile surfaces from touching non-sterile ones and keeps containers open as briefly as possible.

Checking your technique

• Negative controls: incubate a tube or plate of medium you opened but did not inoculate. Growth means your technique or materials introduced contamination.
• Settle plates and environmental monitoring are used in cleanrooms and biomanufacturing.
• In cell culture, watch for cloudy medium, sudden colour changes and floating particles.

Common mistakes

• Talking, coughing or reaching over open containers.
• Overcrowding the cabinet and blocking its grilles.
• Touching the tip of a pipette to the outside of a bottle neck and carrying on.
• Leaving bottles open while you fetch something.
• Skipping the negative control.

Quick recap

• Sterile is a state; aseptic is a set of working habits.
• Work in a BSC (no flame) or beside a flame, as your SOP says.
• Disinfect, open briefly, never touch sterile surfaces, never reach over open containers, single-use tips.
• Prove your technique with negative controls.

Aseptic work with real cultures is learned only in your teaching lab or workplace, after biosafety training and under supervision.

Try it

Open the Do tab to rehearse the motions of an aseptic transfer using only clean, empty containers and tap water. No microbes, media or cultures are involved.