The sterilization process in an autoclave (steam sterilizer) can be very difficult. If, for example, liquids or solids (instruments, glassware, filters, textiles) are sterilized for later use in the laboratory, the sterilization process must ensure a reproducible, sterile product at all times. Products that are sterilized for use in the laboratory cannot be tested for sterility, as they are contaminated by the test and can therefore no longer be used in the laboratory.
The optimal autoclave for you?
The validation of steam sterilization processes is an increasingly important topic in order to ensure verifiably reproducible results. Furthermore, safety aspects must be taken into account in steam sterilization in general, but especially in the sterilization of liquids. As a rule, sterilization is carried out at a temperature of 121 ⁰C. This corresponds to a steam pressure of approx. 2 bar. These high temperatures and the associated pressure can pose a considerable potential risk to the user if the steam sterilization process is incorrectly designed or carried out.
Sterilizing liquids is one of the most demanding tasks in the laboratory. The sterilization processes can sometimes take a very long time, bottles must be open or at least ventilated, some of the liquid boils away, liquids can boil over and bottles can even burst. Another question that needs to be asked is whether the liquids in the bottles even reach the desired sterilization temperature (e.g. 121 ⁰C) and when they can be safely removed from the autoclave once the sterilization process is complete.
If you look at a sterilization process for liquids, it consists of three phases:
Figure 1 illustrates the individual phases graphically.
The blue line represents the temperature in the pressure vessel of the autoclave, the red line the temperature in the liquid. It can be clearly seen that the desired temperature of 121 ⁰C is reached very quickly in the autoclave 's pressure vessel, while the liquids in the bottles take considerably longer to reach the sterilization temperature. The thermal energy of the steam is transferred to the bottles during the heating time by condensation of the steam. This condensation process and the associated heat transfer takes some time, which explains the time difference between simply heating the pressure vessel and heating the liquid itself. The time required to reach the same temperatures in the autoclave pressure vessel and in the liquids is referred to as the equalization time.
Many autoclaves used in laboratories today are still not equipped with a temperature measurement in a reference vessel. The exact temperature of the liquid to be sterilized is therefore not recorded and cannot be used to control the sterilization process. These autoclaves start the sterilization time when the desired temperature is reached in the autoclave's pressure vessel. The equalization time required for the liquids to reach the desired temperature is not taken into account. The liquids therefore never reach the sterilization temperature of 121 ⁰C, for example, and the biological effectiveness of the sterilization process is therefore no longer guaranteed. Depending on the resistance of the microorganisms to be inactivated, they are only partially inactivated or not inactivated at all.
Figure 1 - Sterilization process / phases
Temperature measurement in a reference vessel
By measuring the temperature in a reference vessel using a temperature sensor, the exact temperature of the liquid to be sterilized can be determined and also used to control the sterilization process. The sterilization time only starts when the desired sterilization temperature has been reached in the liquid. The reference vessel is filled with water for this purpose. It is important that the size and fill level of the reference vessel correspond to those of the largest vessel filled with the liquid to be sterilized.
The temperature sensor for measurement in a reference vessel is therefore required to ensure that the sterilization temperature is reached in the liquid. However, it is also required to ensure a safe removal temperature after sterilization. In an autoclave, liquids are heated well above the normal boiling point (100 ⁰C). The heat introduced into the liquid, combined with the associated overpressure, can pose considerable risks to the operator of an autoclave. For example, boiling delay can occur, which means that the liquid starts to boil spontaneously when the autoclave is opened. This spontaneous boiling creates a pressure wave of steam and hot liquid that shoots out of the vessels, similar to a geyser. 1 liter of water produces 1000 liters of steam!
Due to this considerable hazard potential, steam sterilizers used for sterilizing liquids are subject to corresponding regulations. DIN EN 61010-2-040 requires that steam sterilizers for the sterilization of liquids must be equipped with safety devices that prevent the autoclave from opening until the liquids have cooled down to a safe withdrawal temperature for the user. The standard defines a safe withdrawal temperature as 20K below the boiling point of water at atmospheric pressure. This corresponds to a safe withdrawal temperature of 80 ⁰C. Modern autoclaves are equipped with a temperature and pressure-dependent door lock. This prevents the autoclave from opening as long as the pressure vessel is pressurized and as long as the temperature measured in the liquid is above the required 80⁰C.
Cooling the liquids to the safe removal temperature can take a very long time. A commonly used size for autoclaves in laboratories is an autoclave with a pressure vessel capacity of approx. 150 liters. If such an autoclave is fully loaded with bottles containing the liquid to be sterilized, an entire sterilization cycle can take up to 10 hours. This means that not even one sterilization process can be completed in one working day. It is therefore advisable to equip the autoclave with a re-cooling system, which significantly reduces the total batch time and eliminates further hazards and disadvantages when sterilizing liquids.
Rapid re-cooling - maximizing productivity and safety
A basic distinction must be made between two types of cooling systems available for autoclaves.
Cooling byevaporation is probably the most commonly used type of cooling in an autoclave. These can be, for example
All of the above types of cooling have serious disadvantages in the sterilization of liquids and, if the sterilization process is not carried out correctly, can pose a considerable potential risk, as this type of cooling requires the liquid to be cooled to boil.
When purchasing an autoclave, it is therefore advisable to define exactly which applications it will be used for and how it should be equipped in terms of productivity and safety.
Radiation cooling
Cooling by radiation (rapid recooling with support pressure) has considerable advantages over cooling by evaporation. With rapid recooling with support pressure, the entire surface of the pressure vessel is cooled with cold water using external cooling coils. Before cooling is activated after the sterilization phase, the steam in the pressure vessel is replaced by sterile-filtered compressed air. The compressed air reliably prevents the liquid from boiling during the cooling phase. The heat is transferred from the liquid to the cold walls of the pressure vessel by radiation, thus cooling the liquid.
Rapid recooling with support pressure allows a considerable increase in productivity, as the process times are significantly reduced compared to self-cooling. While self-cooling still requires up to 10 hours for an entire autoclaving process, the recooling time can be reduced by up to 60% with rapid recooling with support pressure, depending on the load quantity. Furthermore, all the risks and disadvantages described for evaporation cooling (boiling delay, loss of liquid, boiling over, no cooling for tightly closed bottles) are reliably eliminated, as the liquid no longer boils. With this type of cooling, the bottles can be filled to the maximum fill level (productivity gain of 50 to 70%) and even tightly closed bottles can be used. It is not necessary to open or prime the bottles.
Further optimize process times
Modern autoclaves offer the option of further optimizing the cooling of liquids in modules. This further increases productivity, but also has an impact on the quality of the liquids to be sterilized. Many liquids contain ingredients that are not very heat-stable. Although the liquids should be sterilized, the time during which the liquids are exposed to heat should be kept as short as possible so as not to negatively affect heat-labile ingredients.
Module 1 - Centrifugal fan
During the cooling phase, the radial fan generates an air flow in the pressure vessel of the autoclave. This air flow forces the heat from the bottles to the walls of the pressure vessel, which are cooled by the rapid recooling with support pressure. This process can reduce the recooling time by up to 70% compared to self-cooling.
Figure 4 - Cooling with support pressure and radial fan
Module 2 - Ultracooler
The Ultracooler is an additional, water-cooled heat exchanger that is integrated directly into the autoclave's pressure vessel. This allows the heat to be extracted from the bottles directly where it is located: in the pressure vessel. Thanks to the significantly improved heat transfer, the recooling time can be reduced by up to 90% compared to self-cooling.
Note: As the radial fan and ultracooler are installed inside the pressure vessel, care must be taken to ensure that they do not reduce the available usable space of the autoclave.
When sterilizing solids (e.g. instruments, empty glassware, pipette tips in boxes, filters and textiles) and when destroying waste in destruction bags, care must be taken to ensure that a steam atmosphere is created exactly where it is needed. Namely on and in the product to be sterilized. Many autoclaves do not reliably remove air from the autoclave and the product. Where air remains in the autoclave and in the product, there is no sterilizing effect, as only steam transports the necessary heat energy that reliably inactivates microorganisms.
Ineffective venting
Figures 5 and 6 show ineffective venting using the example of a box with pipette tips and a destruction bag. If the autoclave is simply heated up, air is displaced from it and a steam atmosphere is created in the autoclave's pressure vessel, while air remains in the product to be sterilized. Remaining air in the product prevents the steam from penetrating where its heat energy is needed to achieve a sterilizing effect.
Air at the same temperature as steam (e.g. 121 ⁰C) contains many times less heat energy. For products that cannot be sterilized in a steam atmosphere, there are hot air sterilizers, but these sterilize at higher temperatures (180 to 250 ⁰C) and for a much longer time (up to several hours sterilization time). The sterilizing effect of air at temperatures typically used in steam sterilizers of 121 ⁰C to 134 °C and a sterilization time of 3 to 20 minutes is therefore practically non-existent.
Figure 5 - Ineffective deaeration for solids
Effective venting
A fractionated pre-vacuum must be used for the complete and reproducible removal of air from the autoclave and the product to be sterilized. The autoclave is equipped with a vacuum system for this purpose. During the heating phase, vacuum cycles are used to actively remove the air, followed by bursts of steam. As a rule, a triple fractionated pre-vacuum is used, but more fractions may be necessary depending on the product.
Drying for solids - Superdry
For drying, solids such as instruments or empty glassware are usually placed in a drying cabinet after the sterilization process. Modern autoclaves allow solids to be dried directly after the sterilization process. Sterilization and drying in one process. Further handling of the sterilization items with the risk of recontamination is not necessary.
The sterilization of hazardous biological substances is a particular challenge. During the heating phase, the air in the autoclave is replaced by steam. For this purpose, the air is displaced from the autoclave and released into the room in which the autoclave is installed.
TRBA 100 - Technical Rules for Biological Agents - requires that process exhaust air from an autoclave in laboratories must be treated from safety level S2, as this exhaust air could be contaminated by microorganisms from the product to be sterilized. A suitable process must be used for this. For autoclaves, this is usually filtration. For this purpose, the autoclave is equipped with an exhaust air filter. All air displaced from the autoclave is passed through the filter and microorganisms are retained in it. The filter is sterilized "in-line" during the sterilization process in order to inactivate the microorganisms retained in it. TRBA 100 only deals with the exhaust air discharged from the autoclave, but not with the condensate produced. During the sterilization process, steam condenses on the product and thus turns back into water (condensate). This water can also potentially be contaminated by microorganisms. Therefore, the condensate must remain in the autoclave during the sterilization process and must also be sterilized "in-line" before it is discharged into the drain after successful sterilization.
During qualification, it is checked whether a device is suitable for its intended use and whether a process, e.g. a sterilization process, can always be carried out with the same (reproducible) result, a sterile product, taking into account the product to be sterilized.
In principle, the qualification process is divided into three main parts:
The aim of qualification and validation is to provide documented proof that a device is suitable for its intended use.
The sterilizing effect of a steam sterilization process is verified in the OQ (empty chamber) and PQ (with product) using external measuring equipment for temperature and pressure and with the aid of bioindicators based on Bacillus stearothermophilus. While external measuring devices for temperature and pressure provide proof that the autoclave control system displays and documents reliable values and performs the sterilization process within the defined tolerances, bioindicators provide proof of biological effectiveness. For the placement of the bioindicators, it is necessary to determine in which areas of the items to be sterilized it is most difficult to achieve biological effectiveness. It is precisely in these areas that bioindicators must be placed in order to cover the "worst case", so to speak. All steps of an IQ, OQ and PQ must be documented in detail. In any case, close coordination between the user and the manufacturer is necessary when carrying out an IQ, OQ and PQ.
This article was published in the GIT Labor-Fachzeitschrift, 7/2016, p. 14-18.
Find the right autoclave for your sterilization processes: