Are you serious about sterility?

In applications as diverse as dairy, brewing and molecular microbiology, drinking water analysis and pharmaceutical research, clinical waste disposal and sewage treatment, in fact anywhere where living cells are cultured or could interfere with experimental work, sterility is the key word in good laboratory practice.

By sterility we mean a complete absence of viable microbiological organisms, viruses and bacterial spores, that means 100 percent compliance not just 95 percentile, a situation which is not easy to achieve and even harder to validate. The sort of loads that need to be sterilised vary depending on the activities in the laboratory, but would, typically, include experimental and analytical glassware, instruments, packaging, culture media and chemical solutions. In addition, under duty of care legislation, any waste material that is potentially bio-hazardous has to be sterilised prior to disposal.

While there are many chemicals, both inorganic and organic, which will kill micro-organisms, they are not totally effective and may leave undesirable residues. Ultraviolet radiation is an effective biocide, modifying the DNA in the cell to prevent it replicating, but it will only achieve logarithmic removal not 100 per cent compliance.

So if sterility is an absolute requirement, today's laboratory scientists turn, as did their predecessors, to heat. Like most of us, bugs tend to become more active as the temperature rises but, at around 80°C (pasteurisation temperature) most of them die. At above 120°C you can guarantee that there are no living micro-organisms. The most widely used piece of heat sterilising equipment is the laboratory autoclave.

Autoclaves

Laboratory autoclaves use steam to provide the required sterilisation temperature. Although this sounds a fairly straightforward operation, the application is not easy.

Firstly, to achieve a temperature of 121°C or more requires steam to be pressurised to at least 1.05 barG (2.05 bar (Abs)).

This means that the autoclave which contains it is classified as a pressure vessel and must be designed to a strict engineering standard: BS5500, ASME VIII or similar.

Secondly, when the autoclave is first loaded it is full of air that has to be displaced by the steam, and this requires a venting or vacuum extraction system.

Thirdly, the sterilisation temperature has to be reached in all parts of the autoclave and its load. Finally, as steam cools down it condenses, which means that uncontrolled steam evacuation and cooling after sterilisation can leave the load wet.

Industrial steam boilers are constructed from carbon steel but the cyclical nature of autoclave operation, and the exposure to atmospheric air, would cause severe corrosion of carbon steel so stainless steel is invariably used for autoclaves. While low cost 304 stainless steel is just about adequate, for a long trouble-free life there is no substitute for 316L, the material used universally in the pharmaceutical manufacturing industry.

Aside from the sterilisation process itself, the most important criterion in autoclave operation is safety. Pressurised steam is a scalding hazard, and it is important that equipment meets the safety standards set out by OSH. This ensures that the steaming cycle cannot start until the temperature has fallen to a safe level. It's not just steam that can be a hazard with fluid cycles. If the door were to be opened while the autoclave is too hot, the result would be a sudden drop in temperature which could cause liquids to boil over and glass bottles to be cracked by thermal shock.

Automatic

The time and amount of steam needed for the load to reach sterilisation temperature will vary with the thermal capacity of the load. A couple of litre bottles of culture media will need more steam than a dozen smaller containers, and heating will take longer.

The time needed to achieve sterilisation once the temperature has been attained will depend on the type of microbial organisms present. Bacteria and fungi are rapidly killed by heat, but viruses and particularly bacterial spores are very persistent and may need quite long sterilisation times. The development and wide availability of reliable microprocessors means that most autoclaves are user-programmable, allowing the sterilisation temperature and time and the rate of cooling to be set to suit the requirements of the load.

The temperature of steam is a function of its pressure, but simply achieving pressure in an autoclave does not necessarily mean that the corresponding temperature has been reached. The presence of pockets of air in the load can dilute the steam, so the partial pressure of steam can be significantly lower than the autoclave pressure.

To ensure that sterilisation is effective, it is better to measure and control temperature rather than pressure. A free steaming stage (at atmospheric pressure) is useful for applications where initial air removal may be difficult, such as pre-disposal 'make safe' processing of discard loads, where there are often pockets of trapped air. With particularly difficult loads a pulsed pre-vacuum system will provide the most efficient air removal. Water or assisted cooling sequences are also widely available for rapid cool down giving shorter sterilisation cycles.

In laboratory units the sterilising steam is usually generated in the autoclave chamber itself by means of electric immersion heaters. On small units water may be added manually but, generally, it is much more convenient to buy an autoclave with an automatic filling system which can be plumbed into the water supply - most autoclaves run on ordinary mains water but softened water is obviously preferable.

Validation

Achieving sterilisation of the load is only part of the story. Just as critical is compliance with Quality Assurance procedures that require validation that sterilisation has been achieved. This is usually carried out by monitoring the conditions inside the load during the sterilisation procedure either by observation and logging or by automatic recording.

Astell Scientific points out that it is false economy to buy a cheap autoclave. With several loads a day a typical autoclave will perform around 10,000 sterilisation cycles in a 10 year period, so reliability is essential. Not that 10 years is old for an autoclave; there are many examples of 20 year old autoclaves still in daily use. As a NAMAS accredited autoclave validation laboratory, Astell provides a multipoint validation service for autoclaves and a wide range of other thermal equipment.

The Astell range of laboratory autoclaves offers capacities from 23 up to 500 L as well as larger units for certain specialised applications. All units are fully automatic, using a micro-computer controller, which is user programmable, with delayed start and timed free-steaming facilities, and even has an RS232 interface for connection to an external PC. An integral data printer provides a hard copy record of the sterilisation sequence for QA purposes. Astell's autoclaves are controlled to a temperature setpoint that the user can adjust in 1°C steps between 100-138°C with sterilisation time variable from 1-99 min at 1 min increments.