Aseptic technique and autoclaving in the laboratory

Aseptic technique is a set of working habits that stop unwanted microorganisms getting into a sample, a culture or a sterile solution. Autoclaving is the most common way labs sterilise equipment, media and waste, using steam under pressure. The two go together: an autoclave makes things sterile, and aseptic technique keeps them that way while you work. This guide covers both, in the context of microbiology and cell culture.

Terms are defined in the glossary.

Sterile, aseptic and clean

These words are often mixed up:

• Sterile means free of all living microorganisms, including spores. It is an absolute state: something is sterile or it isn't.
• Aseptic describes a way of working that keeps sterile things sterile, and stops contamination getting in.
• Disinfected means most microorganisms have been killed or removed, but not necessarily spores. Wiping a bench with 70% ethanol disinfects it. It doesn't sterilise it.
• Clean means free of visible dirt and residues. Cleaning comes before disinfection or sterilisation, because dirt protects microbes.

Core habits of aseptic technique

The details vary between labs, but the principles are the same.

Prepare the space

• Work in the right place. Cell culture is done in a microbiological safety cabinet, which supplies filtered air over the work surface. Much bacterial work is done on an open bench near a Bunsen burner, whose rising hot air carries airborne particles away from the work.
• Disinfect the surface before and after work, usually with 70% ethanol. It works better than pure ethanol, because the water helps it get into cells.
• Clear the space of anything you don't need.
• Plan the work so everything is within reach and you aren't reaching over open containers.

Handle materials carefully

• Open containers for as little time as possible, and hold lids facing down or keep them in your hand rather than putting them face up on the bench.
• Don't pass hands or arms over open vessels.
• Use sterile, single-use consumables such as pipette tips, tubes and loops, or sterilise reusable ones. In microbiology, wire loops are flamed until red hot before and after use.
• Work with small portions. Take what you need from a stock into a separate tube rather than dipping into the stock repeatedly.
• Label everything with the contents, date and initials.

Protect yourself and the work

• Wear a lab coat and gloves, and spray gloves with 70% ethanol before working in a cabinet.
• Tie back long hair, and don't talk, cough or sneeze over open work.
• Check for contamination. Cloudy media, unexpected colonies or a change in colour show something has got in.

Good microbiological practice protects the person as well as the work. The WHO manual treats it as the basis of all laboratory biosafety (WHO).

How an autoclave works

An autoclave is a sealed chamber that heats items with steam under pressure. At normal pressure, water boils at 100 °C. Raising the pressure lets steam reach higher temperatures, and moist heat at those temperatures kills microorganisms, including spores, much faster than dry heat.

A typical cycle:

1. Air removal. Air is pushed out of the chamber, either by steam flowing through or by a vacuum pump. Trapped air keeps steam from reaching the load and leaves cold spots.
2. Heating. Steam fills the chamber until the load reaches the set temperature.
3. Holding. The temperature is held for a set time. Common settings are 121 °C for at least 15 minutes, or 134 °C for about 3 minutes for suitable loads. The time counts from when the whole load is at temperature, not the chamber.
4. Exhaust and cooling. Pressure is released, and liquids are allowed to cool slowly so they don't boil over.

What can go in an autoclave

Usually suitable: glassware, metal instruments, culture media and water in loosely capped containers, polypropylene plastics, and biological waste in autoclave bags.

Not suitable:

• Flammable, volatile or corrosive chemicals, including solvents and bleach.
• Sealed containers, which can burst. Loosen caps first.
• Plastics that melt, such as polystyrene. Check the plastic's markings.
• Heat-sensitive solutions, such as many proteins, peptides, vitamins and antibiotics. These are filter-sterilised instead (see below).
• Some volumetric glassware, because repeated heating can change its calibration. See pipettes and volumetric glassware.

Checking that it worked

• Indicator tape changes colour with heat. It shows the pack went through a cycle, not that it was sterilised.
• Chemical indicators inside the load react to time, temperature and steam together.
• Biological indicators contain spores of a heat-resistant bacterium, Geobacillus stearothermophilus. After the cycle they are incubated. If nothing grows, the cycle killed the spores.
• Cycle records from the autoclave's own printout or data log show the temperature and time actually reached.

Autoclave safety

Autoclaves combine high pressure, steam and hot liquids. Only trained staff should run them, and they should never be opened until the pressure is released and the load has cooled. In the UK, an autoclave is a pressure system, so the Pressure Systems Safety Regulations 2000 apply. The owner needs a written scheme of examination, drawn up by a competent person, and the autoclave must be examined in line with it (legislation.gov.uk; HSE). For the wider law on hazardous substances at work, see COSHH and safety data sheets.

Filter sterilisation

Solutions that heat would damage are sterilised by passing them through a membrane filter with pores of 0.2 or 0.22 µm, which hold back bacteria. The filtered solution is collected in a sterile container, working aseptically.

Filtration doesn't remove viruses or the smallest particles, and it doesn't remove dissolved substances. Some molecules, including some peptides, can stick to certain membrane materials, so labs choose a low-binding membrane where that matters. See bacteriostatic water vs sterile water for how sterile water is made.

Related guides

For safe use of centrifuges in the same lab, see centrifuges. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.