SubscribeAre you getting hot under the collar about sterility??
In applications as diverse as brewing and molecular biology, drinking water analysis and pharmaceutical research, clinical waste disposal and sewage treatment, sterility is the key word in good laboratory practice.
Sterility is a complete, 100% absence of viable microbiological organisms, viruses and bacterial spores, which is by no means easy to achieve and even harder to validate. The sort of loads that need to be sterilized vary depending on the activities in the laboratory, but could include experimental and analytical glassware, instruments, packaging, culture media and chemical solutions. In addition, under duty of care legislation, any material, which is potentially bio-hazardous, must be sterilized prior to disposal.
While there are many chemicals, both organic and inorganic, which will kill micro-organisms they are not totally effective and may have undesirable residues. Ultraviolet irradiation is also an effective biocide, modifying the DNA in the cell to prevent it replicating. However, UV irradiation will only produce a logarithmic reduction in cells, not an absolute eradication. Therefore, if sterility is a requirement, today’s laboratory scientists turn, as did their ancestors, to heat. Like most of us, bugs tend to become more active as the temperature rises, but at around 80°C, most of them die. At above 120°C, you can guarantee that there will be no living organisms. Autoclaves are the most widely used means of heat sterilization in the laboratory.
Autoclaves
Laboratory autoclaves use steam to provide the required sterilization temperature. Although this sounds like a very straightforward operation, the application is not so easy. Firstly, to achieve a temperature of 121° C or more requires steam to be pressurised to at least 1.05BarG, which is 2.05bar (Abs). This in turn means that the autoclave must be categorized as a pressure vessel and must be designed in accordance with a strict engineering standard, such as BS 5500, ASME VII or similar. Secondly, when the autoclave is loaded it is full of air, which has to be displaced by steam, which requires a venting or vacuum extraction system. Thirdly, the sterilization 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 sterilization 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 both cause severe corrosion so stainless steel is invariably used for autoclaves. Whilst low cost 304 grade stainless steel is just about adequate, there is no substitute for 316 grade stainless steel, if you want an autoclave with a long, trouble-free life.
Aside from the sterilization process itself, the most important criterion in autoclave operation is safety. Pressurised steam is a scalding hazard and it is important that equipment meets safety standards set out in HSE.PM73. This standard stipulates that the steaming cycle cannot begin until the autoclave is properly closed and that the autoclave cannot be opened until the temperature has dropped to a safe level. However, it is not just steam that is a hazard in fluid cycles. If the autoclave were to be opened while it was still hot, this could cause a sudden drop in temperature which could cause liquids to boil over and glass bottles to be cracked by thermal shock.
Automation
The time and amount of steam needed for the load to reach sterilization temperature will vary with the thermal capacity of the load. A couple of 1L media bottles of culture media will need more steam than a dozen smaller containers and the heating will take longer. The time needed to achieve sterilization once the temperature has been attained will depend on the type of micro-organisms present. Bacteria and fungi are rapidly killed by heat, but viruses and especially bacterial spores are very persistent and may need long sterilization times. The development and availability of microprocessors means that most autoclaves are user-programmable, allowing the sterilization temperature, the sterilization time and the rate of cool down to be specified, depending on the load. The temperature is all-important in the sterilization process. The temperature of any gas 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 that its partial pressure can be significantly lower than the overall pressure in the autoclave. To ensure that sterilization is effective, it is better to measure and control the temperature in the autoclave, rather than the pressure. A free-steaming phase, at atmospheric pressure is useful in applications where there is a good chance that air pockets are in the load. 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 sterilization cycles.
In laboratory units, the sterilizing steam is usually generated in the autoclave chamber itself by means of electric immersion heaters. On smaller units, water is sometimes added manually, but generally it is more convenient to have an automatic autoclave which can be plumbed in. softened water is preferable, although mains water is generally fine.
Achieving sterilization of the load is only part of the story. Just as critical is compliance with quality assurance procedures which require validation that sterilization has been achieved. This is usually carried out by monitoring the conditions inside the load during the sterilization process, either by observation and logging or by automatic recording.
The Astell range offers standard models from 23L to 500L, with larger units for certain specialized applications. All the units are fully automatic, using Astell’s Secure micro-computer controller which is user-programmable with delayed start and free-steam facilities. It has an RS-232 interface for connection to an external PC. An integral data printer provides a hard copy record of the sterilization sequence for QA records. The Astell range of autoclaves are controlled to a temperature set point which the user can adjust in 1° C between 100°C and 138°C with sterilization time variable between 1 and 99 minutes in 1 minute increments. Another feature of Astell autoclaves is the Holdwarm facility, which maintains the autoclave at about 55°C for up to 18 hours after the sterilization cycle is completed. This is particularly useful for overnight sterilization of nutrient media, which can then be held at its pouring temperature until required for use the next morning.