Most labs will need to sterilise liquid solutions. Media, agar, saline, water and lysogeny broth to name a few, but how should you do this in an autoclave?
First, articles should be placed in the autoclave so that steam can easily penetrate them. Second, air should be evacuated so that the chamber fills with steam. Working of Autoclave: Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained. How to Validate an Autoclave: Sterilization Cycle Development. Cycle development is an important part of both the autoclave procurement and validation processes. In previous posts about validation, we explored Installation Qualification (IQ), Operation Qualification (OQ), and Performance Qualification (PQ).
An Autoclave liquid cycle is used to avoid the phenomenon known as âboil-overâ. This is when a liquid boils so violently it spills over the top of the container. This can occur if the pressure in your autoclave chamber is released too quickly. The exhaust phase of the cycle must therefore release pressure slowly to allow the liquid to cool off as the pressure decreases.
More efficient sterilising
You can use load sensed timing whereby a flexible sensor probe is fitted and submerged directly into the media or reference bottle so that the cycle is controlled directly by the temperature of the actual load, not just the chamber temperature. This ensures the sterilisation does not begin until the very centre of the load has reached the correct temperature.
Faster Sterilisation
All Astell autoclaves can sterilise fluids but to sterilise fluids quickly the addition of a cooling system and air ballast must be used. After autoclave sterilisation the chamber must cool down to a safe temperature before it can be opened. If you want to speed this process up you must be careful, using a rapid cooling system would cause a sudden change in pressure and likely cause the liquid to boil over, spoiling samples and spoiling the autoclave chamber.
There are two solutions use to overcome the problem of boil over. Option one: use assisted cooling such as an external fan, water cooling (coils) or jacket cooling. These would activate at just above 100 degrees Celsius to stop a rapid drop in chamber pressure. This is the more affordable option but does leave a delay between sterilisation completion and start of the cooling process in order to prevent boil over.
Option two: use an air ballast; compressed air is quickly injected into the chamber to replace the steam used during condensation and maintain the chamber pressure during the cooling phase. With pressure maintained this process can begin immediately following sterilisation. This can be combined with internal cooling for fast and and efficient way of cooling loads.
Method
If you have a lid for your flask place it on top and screw a half turn, make sure these are not secured as your flask will explode when the air expands as the temperature rises. If you have no lid cover your flask lightly with foil.
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Add water to the level indicator line, the water must be conductive i.e. have an ionic content therefore deionised or distilled water is not acceptable. Place your flasks into the basket and place inside the autoclave. If you are using an autoclave with load sense timing place load the sensing probe into your container. Close the lid and turn the handle to create an air tight seal. Use the control panel to set the desired cycle. The Astell autoclaves have a âFluids Cycleâ suitable for processing media or other fluids in unsealed containers.
Once the cycle is ends the display will show âcompleteâ, the door will unlock and you can rotate the handle to open the door. Please be aware that the glassware will still be hot and you will need to use heat resistant gloves to pick up your load.
For more information about sterilising liquids using autoclaves please do get in touch with one of our technical team. You can call us on 01954 233 120, email [email protected] or fill in the below contact form.
Cutaway illustration of a cylindrical-chamber autoclave
An autoclave is a pressure chamber used to carry out industrial processes requiring elevated temperature and pressure different from ambient air pressure. Autoclaves are used in medical applications to perform sterilization and in the chemical industry to cure coatings and vulcanize rubber and for hydrothermal synthesis. Industrial autoclaves are used in industrial applications, especially regarding composites.
Many autoclaves are used to sterilize equipment and supplies by subjecting them to pressurized saturated steam at 121 °C (249 °F) for around 15-20 minutes depending on the size of the load and the contents [1] The autoclave was invented by Charles Chamberland in 1884,[2] although a precursor known as the steam digester was created by Denis Papin in 1679.[3] The name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key, thus a self-locking device.[4]
Video demonstrating how autoclaves work
Uses[edit]
Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing, veterinary medicine, mycology, funerary practice, dentistry, and prosthetics fabrication. They vary in size and function depending on the media to be sterilized and are sometimes called retort in the chemical and food industries.
Typical loads include laboratory glassware, other equipment and waste, surgical instruments, and medical waste.[5][6]
A notable recent and increasingly popular application of autoclaves is the pre-disposal treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in this category largely operate under the same principles as conventional autoclaves in that they are able to neutralize potentially infectious agents by using pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressings, gloves, etc. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven.[7]
Autoclaves are also widely used to cure composites and in the vulcanization of rubber.[8] The high heat and pressure that autoclaves generate help to ensure that the best possible physical properties are repeatable. The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide.[citation needed]
Other types of autoclaves are used to grow crystals under high temperatures and pressures. Synthetic quartz crystals used in the electronics industry are grown in autoclaves. Packing of parachutes for specialist applications may be performed under vacuum in an autoclave, which allows the chutes to be warmed and inserted into their packs at the smallest volume.
Air removal[edit]
It is very important to ensure that all of the trapped air is removed from the autoclave before activation, as trapped air is a very poor medium for achieving sterility. Steam at 134 °C can achieve a desired level of sterility in three minutes, in contrast to hot air at 160 °C, which can take two hours to achieve the same sterility.[9]
Methods of air removal include:
Downward displacement (or gravity-type): As steam enters the chamber, it fills the upper areas first as it is less dense than air. This process compresses the air to the bottom, forcing it out through a drain which often contains a temperature sensor. Only when air evacuation is complete does the discharge stop. Flow is usually controlled by a steam trap or a solenoid valve, but bleed holes are sometimes used. As the steam and air mix, it is also possible to force out the mixture from locations in the chamber other than the bottom.
Steam pulsing: air dilution by using a series of steam pulses, in which the chamber is alternately pressurized and then depressurized to near atmospheric pressure.
Vacuum pumps: a vacuum pump sucks air or air/steam mixtures from the chamber. (Explained here)
Superatmospheric cycles: achieved with a vacuum pump. It starts with a vacuum followed by a steam pulse followed by a vacuum followed by a steam pulse. The number of pulses depends on the particular autoclave and cycle chosen.
Subatmospheric cycles: similar to the superatmospheric cycles, but chamber pressure never exceeds atmospheric pressure until they pressurize up to the sterilizing temperature.
In medicine[edit]
Dental equipment in an autoclave to be sterilized for 2 hours at 150 to 180 degrees Celsius
A medical autoclave is a device that uses steam to sterilize equipment and other objects. This means that all bacteria, viruses, fungi, and spores are inactivated. However, prions, such as those associated with CreutzfeldtâJakob disease, and some toxins released by certain bacteria, such as Cereulide, may not be destroyed by autoclaving at the typical 134 °C for three minutes or 121 °C for 15 minutes.[citation needed] Although a wide range of archaea species, including Geogemma barosii, can survive and even reproduce at temperatures above 121 °C, none of them are known to be infectious or otherwise pose a health risk to humans; in fact, their biochemistry is so vastly different from our own and their multiplication rate is so slow that microbiologists need not worry about them.[citation needed]
Autoclaves are found in many medical settings, laboratories, and other places that need to ensure the sterility of an object. Many procedures today employ single-use items rather than sterilizable, reusable items. This first happened with hypodermic needles, but today many surgical instruments (such as forceps, needle holders, and scalpel handles) are commonly single-use rather than reusable items (see waste autoclave). Autoclaves are of particular importance in poorer countries due to the much greater amount of equipment that is re-used. Providing stove-top or solar autoclaves to rural medical centers has been the subject of several proposed medical aid missions.[citation needed]
Because damp heat is used, heat-labile products (such as some plastics) cannot be sterilized this way or they will melt. Paper and other products that may be damaged by steam must also be sterilized another way. In all autoclaves, items should always be separated to allow the steam to penetrate the load evenly.
Autoclaving is often used to sterilize medical waste prior to disposal in the standard municipal solid waste stream. This application has become more common as an alternative to incineration due to environmental and health concerns raised because of the combustion by-products emitted by incinerators, especially from the small units which were commonly operated at individual hospitals. Incineration or a similar thermal oxidation process is still generally mandated for pathological waste and other very toxic and/or infectious medical waste.
In dentistry, autoclaves provide sterilization of dental instruments according to health technical memorandum 01-05 (HTM01-05). According to HTM01-05, instruments can be kept, once sterilized using a vacuum autoclave for up to 12 months using sealed pouches.[10]
In most of the industrialized world medical-grade autoclaves are regulated medical devices. Many medical-grade autoclaves are therefore limited to running regulator-approved cycles. Because they are optimized for continuous hospital use, they favor rectangular designs, require demanding maintenance regimens, and are costly to operate. (A properly calibrated medical-grade autoclave uses thousands of gallons of water each day, independent of task, with correspondingly high electric power consumption.)
In research[edit]
A cylindrical-chamber pass-through autoclave
Most medical-grade autoclaves are inappropriate for research tasks. General-use non-medical (often called 'research-grade') autoclaves are increasingly used in a wide range of education, research, and industrial settings (including biomedical research) where efficiency, ease-of-use, and flexibility are at a premium. Research-grade autoclaves may be configured for 'pass-through' operation. This makes it possible to maintain absolute isolation between 'clean' and potentially contaminated work areas. Pass-through research autoclaves are especially important in BSL-3 or BSL-4 facilities.
Research-grade autoclavesâwhich are not approved for use sterilizing instruments that will be directly used on humansâare primarily designed for efficiency, flexibility, and ease-of-use. They rely on efficient cylindrical pressure-chamber designs, are intended for intermittent use, and have highly customizable programmable controls.
In 2016, the Office of Sustainability at the University of California, Riverside (UCR) conducted a study of autoclave efficiency in their genomics and entomology research labs, tracking several units' power and water consumption. They found that, even when functioning within intended parameters, the medical-grade autoclaves used in their research labs were each consuming 700 gallons of water and 90 Kw/h of electricity per cycle (1,134MWh of electricity and 8.8 million gallons of water total). UCR's research-grade autoclaves performed the same tasks with equal effectiveness, but used 83% less energy and 97% less water.[11]
Quality assurance[edit]
Sterilization bags often have a 'sterilization indicator mark' that typically darkens when the bag and its contents have been adequately processed. Comparing the marks on an unprocessed bag (L) and on a bag that has been properly cycled (R) will reveal an obvious visual difference.
There are physical, chemical, and biological indicators that can be used to ensure that an autoclave reaches the correct temperature for the correct amount of time. If a non-treated or improperly treated item can be confused for a treated item, then there is the risk that they will become mixed up, which, in some areas such as surgery, is critical.
Chemical indicators on medical packaging and autoclave tape change color once the correct conditions have been met, indicating that the object inside the package, or under the tape, has been appropriately processed. Autoclave tape is only a marker that steam and heat have activated the dye. The marker on the tape does not indicate complete sterility. A more difficult challenge device, named the Bowie-Dick device after its inventors, is also used to verify a full cycle. This contains a full sheet of chemical indicator placed in the center of a stack of paper. It is designed specifically to prove that the process achieved full temperature and time required for a normal minimum cycle of 134 °C for 3.5â4 minutes.[citation needed]
To prove sterility, biological indicators are used. Biological indicators contain spores of a heat-resistant bacterium, Geobacillus stearothermophilus. If the autoclave does not reach the right temperature, the spores will germinate when incubated and their metabolism will change the color of a pH-sensitive chemical. Some physical indicators consist of an alloy designed to melt only after being subjected to a given temperature for the relevant holding time. If the alloy melts, the change will be visible.[citation needed]
Some computer-controlled autoclaves use an F0 (F-nought) value to control the sterilization cycle. F0 values are set for the number of minutes of sterilization equivalent to 121 °C (250 °F) at 100 kPa (15 psi) above atmospheric pressure for 15 minutes . Since exact temperature control is difficult, the temperature is monitored, and the sterilization time adjusted accordingly.[citation needed]
Additional images[edit]
References[edit]
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