Autoclave and types of autoclave
- The temperature of saturated steam at atmospheric pressure is approximately 100°C.
- Temperature increases with pressure, e.g. at 1 bar (about 15 lb/in2 – some older autoclaves use Imperial units) it is 121°C.
- Bacteria are killed by autoclaving at this temperature for 15–20 min.
- Air has an important influence on the efficiency of autoclaving. The above relationship holds well only if no air is present.
- If about 50% of the air remains in the autoclave the temperature of the steam/air mixture will be only 112°C.
- If air is present in the autoclave load, it will also adversely affect penetration by the steam.
- All the air that surrounds and permeates the load must first be removed before sterilization can commence.
- In some autoclaves this is done by vacuum pumps.
Loads in autoclaves
- As successful autoclaving depends on the removal of all the air from the chamber and the load, the materials to be sterilized should be packed loosely.
- ‘Clean’ articles may be placed in wire baskets, but contaminated material (e.g. discarded cultures) should be in solid-bottomed containers not more than 20 cm deep.
- Large air spaces should be left around each container and none should be covered. If large volumes of fluid are to be sterilized, then the time should be lengthened to ensure that this reaches the appropriate temperature.
Types of autoclave
Only autoclaves designed for laboratory work and capable of dealing with a ‘mixed load’ should be used. ‘Porous load’ and ‘bottled fluid sterilizers’ are rarely satisfactory for laboratory work.
There are two varieties of laboratory autoclave:
- Pressure cooker types
- Gravity displacement models with automatic air and condensate discharge.
Laboratory bench autoclaves
These, as domestic pressure cookers, are still in use in many parts of the world. The more modern type has a metal chamber with a strong metal lid that can be fastened and sealed with a rubber gasket. It has an air and steam discharge tap, pressure gauge and safety valve. There is an electric immersion heater in the bottom of the chamber.
- There must be sufficient water inside the chamber.
- The autoclave is loaded and the lid is fastened down with the discharge tap open.
- The safety valve is then adjusted to the required temperature and the heat is turned on.
- When the water boils, the steam will issue from the discharge tap and carry the air from the chamber with it.
- The steam and air should be allowed to escape freely until all the air has been removed.
- This may be tested by attaching one end of a length of rubber tubing to the discharge tap and inserting the other end into a bucket or similar large container of water.
- Steam condenses in the water and the air rises as bubbles to the surface; when all of the air has been removed from the chamber, bubbling in the bucket will cease.
- When this stage has been reached, the air–steam discharge tap is closed and the rubber tubing removed.
- The steam pressure then rises in the chamber until the desired pressure and temperature are reached and steam issues from the safety valve.
- When the load has reached the required temperature, the pressure is held for 15 min. At the end of the sterilizing period, the heater is turned off and the autoclave is allowed to cool.
- The air and steam discharge tap is opened very slowly after the pressure gauge has reached zero (atmospheric pressure).
- If the tap is opened too soon, while the autoclave is still under pressure, any fluid inside (liquid media, etc.) will boil explosively and bottles containing liquids may even burst.
- The contents are allowed to cool. Depending on the nature of the materials being sterilized, the cooling (or ‘run-down’) period needed may be several hours for large bottles of agar to cool to 80°C, when they are safe to handle.
Gravity displacement autoclaves
- These may be relatively simple in their construction and operation, as shown diagrammatically in, or extremely sophisticated pieces of engineering in which air and, finally, steam are removed by vacuum pumps and in which the whole sterilization cycle may be programmed.
- The jacket surrounding the autoclave consists of an outer wall enclosing a narrow space around the chamber, which is filled with steam under pressure to keep the chamber wall warm.
- The steam enters the jacket from the mains supply, which is at high pressure, through a valve that reduces this pressure to the working level.
- The working pressure is measured on a separate pressure gauge fitted to the jacket. This jacket also has a separate drain for air and condensate to pass through.
- The steam enters the chamber from the same source that supplies steam to the jacket.
- It is introduced in such a way that it is deflected upwards and fills the chamber from the top downwards, thus forcing the air and condensate to flow out of the drain at the base of the chamber by gravity displacement.
- The drain is fitted with strainers to prevent blockage by debris. The drain discharges into a closed container (not shown in the diagram) so that there is a complete air break that prevents backflow.
- There is also a filter to ensure that aerosols are not released into the room.
- The automatic steam trap or ‘near-to-steam’ trap is designed to ensure that only saturated steam is retained inside the chamber, and that air and condensate, which are at a lower temperature than saturated steam, are automatically discharged.
- It is called a ‘near-to-steam’ trap because it opens if the temperature falls to about 2°C below that of saturated steam and closes within 2°C or nearer to the saturated steam temperature.
- The trap operates by the expansion and contraction of a metal bellows that operates a valve.
- There may be a thermometer probe in the drain but, as this registers the temperature of steam at that point and not that in the load within the chamber, this may be misleading, e.g. the temperature in the drain may reach 121°C whereas that in the load is only 50°C (Collins and Kennedy, 1999).
- In modern autoclaves flexible thermocouple probes are fitted in the chamber so that temperatures in various parts of the load may be recorded.
- In older models the thin thermocouple leads may be safely taken in through the door seals.
- There are usually interlocking devices that prevent the opening of the door before the temperature in the chamber has fallen to 80°C.
- This does not imply that the temperature in the load has also fallen to a safe level. In large, sealed bottles it may still be over 100°C, when the contents will be at a high pressure.
- Sudden cooling may cause the bottles to explode. The autoclave should not be opened until the temperature in the load has fallen to 80°C or below.
- This may take a very long time, and in some autoclaves there are locks that permit the door to be opened only fractionally to cool the load further before it is finally released.
- This is a good reason for not using these autoclaves for the preparation and melting of culture media.
Operation of a gravity displacement autoclave:
- If the autoclave is jacketed, the jacket must first be brought to the operating temperature.
- The chamber is loaded, the door is closed and the steam valve is opened, allowing steam to enter the top of the chamber.
- Air and condensate flow out through the drain at the bottom.
- When the drain thermometer reaches the required temperature a further period must be allowed for the load to reach the temperature.
- This should be determined initially and periodically for each autoclave as described below.
- Unless this is done the load is unlikely to be sterilized. The autoclave cycle is then continued for the holding time.
- When it is completed the steam valves are closed and the autoclave allowed to cool until the temperature dial reads less than 80°C.
- Not until then is the autoclave safe to open. It should first be ‘cracked’ or opened very slightly and left in that position for several minutes to allow steam to escape and the load to cool further.
The time/temperature cycles should be tested under ‘worst load’ conditions, e.g. a container filled with 5 ml screw-capped bottles.This should be placed in the centre of the chamber and, if space is available, other loaded containers may be placed around it.Thermocouple leads are placed in the middle of the load and at other places. The cycle is then started and timed.
There are three periods:
- Warming up, until the temperature in the middle of the load is 121°C.
- Sterilization, i.e. HTAT, in which the temperature in the load is maintained at 121°C for 15 min.
- Cooling down, after the steam valve has been closed and the temperature in the load has fallen to 80°C.
If the autoclave is to be operated manually these times should be noted and displayed. Automated autoclaves may be programmed.
Protection of the operator
- Serious accidents, including burns and scalds to the face and hands have occurred when autoclaves have been opened, even when the temperature gauge is read below 80°C and the door lock has allowed the door to be opened.
- Liquids in bottles may still be over 100°C and under considerable pressure. The bottles may explode on contact with air at room temperature.
- When autoclaves are being unloaded operators should wear full-face visors of the kind that cover the skin under the chin and throat.
- Also wear thermal protective gloves.