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Sterilization procedures for 2 incubators">CO2 incubators

Knegendorf, Leonarda; Brill, Holgerb; Steinmann, Joergc; Pahl, Steffenb; Steinmann, Eiked; Brill, Florian H.H.b

Reviews in Medical Microbiology: October 2018 - Volume 29 - Issue 4 - p 147–152
doi: 10.1097/MRM.0000000000000145

Cell culture methods are utilized ubiquitously in science and pharmaceutical and biotech industries. Sterility is one crucial factor for maintenance of cells and creation of valid data from experiments. As cells are usually cultured in 2 incubators">CO2 incubators, those are one bottleneck in terms of sterility in a cell culture lab. This review gives an overview on the different sterilization procedures for 2 incubators">CO2 incubators on the market with emphasize to considerations from a practical point of view. It compares sterilization by dry heat, steam, gas and ultraviolet radiation in terms of validity and practicability in accordance with international standards and regulations and transfers literature consensus about these methods to 2 incubators">CO2 incubators. As conclusion, the authors give recommendations for a sterile working environment in line with good cell culture practice.

aInstitute for Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover

bDr Brill + Partner GmbH, Institute for Hygiene and Microbiology, Bremen

cInstitute of Clinical Hygiene, Medical Microbiology and Infectiology, Paracelsus Medical University, Klinikum Nürnberg, Nuremberg

dDepartment for Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany.

Correspondence to Dr Florian H.H. Brill, Dr Brill + Partner GmbH, Institute for Hygiene and Microbiology, Stiegstück 34, DE-22339 Hamburg, Germany. E-mail:

Received 12 July, 2018

Accepted 27 July, 2018

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To address the needs of customers in science and industry regarding cell culture equipment, various systems are marketed by a number of manufacturers, each recommending specific disinfection and sterilization procedures for proper and safe use of the equipment. Cell cultures and the alongside used incubators are often highly susceptible to contamination, which requires the establishment of professional use instructions and high standards in applied hygiene. Therefore, decontamination procedures must be routinely applied in need of prevent the equipment being contaminated by various microorganisms, including vegetative cells of bacteria, bacterial spores, molds and their spores or viruses. Furthermore, specific measures effective against prions, DNA and/or RNA, bacteriophages and mycoplasma species may be necessary as well. This review provides a comparison of various sterilization procedures and evaluates them in terms of hygiene.

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Contamination and microbial transmission

There are several possible pathways for contamination of the interior of 2 incubators">CO2 incubators. Primary sources of contamination are the ambient air, the attending staff, lab equipment for cell culture needs and the material being incubated inside the device. Figure 1 illustrates examples of these potential pathways. Special importance relates to interruption of the pathways by which microorganisms could possibly be transmitted and contaminate the incubator. Furthermore, it is highly recommended to perform validated cleaning, disinfection and sterilization procedures as instructed by the manufacturer. Nevertheless, adapted processes might need to be implemented to meet the individual hygiene requirements of each laboratory. Disinfection and sterilization represent two terms that continuously keep being confused. Although sterilization aims at achieving a microorganism-free environment, disinfection routines are performed to eliminate pathogenic microorganisms only. The most important parameters are presented together for comparison in Table 1.

Fig. 1

Fig. 1

Table 1

Table 1

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Definition and requirements of sterilization procedures

According to the 8th edition of the European Pharmacopoeia (Ph. Eur. 8.0) sterility is defined as ‘absence of viable microorganisms’ [1]. The entire development of sterilization standards and procedures has been characterized by an inherent conflict between the imperative of creating this idealized absolute condition of completely microorganism-free conditions and its practical feasibility. Robert Koch's work in the late 19th century had already expressly established the requirement for sterilization of killing bacteria spores, which led logically from the use of spore-containing soil as a performance criterion for sterilization procedures to the development of standardized bio indicators for monitoring sterilization [2]. Especially in light of the continued and even increasing importance of diseases that are transmissible through viruses or other nonbacterial pathogens, any definition of sterility limiting the term to ‘microorganisms capable of reproduction’ is now obsolete. Wallhäußer proposed a very comprehensive definition: ‘The term “sterile” means an absence of biological material that has the ability to reproduce themselves, such as microorganisms (e.g. bacteria and fungi), or to transfer genetic material through phages, viruses, plasmids, prions or infectious nucleic acids’ [3].

Even though it is known that transferal of genetic material plays no role in prion pathogenesis [4], this definition still very accurately captures the stringent criteria that must be applied to sterilization procedures. Accordingly, in this review we define sterilization, in contradiction to disinfection, as process including measures against nonviable forms of microorganisms and contamination threats incapable of reproduction in general. This definition, however, gives rise to a series of problems regarding the practical application of sterilization procedures, problems that may concern the safety concept associated with sterilization.

The European Pharmacopoeia defines the requirements for medical products that are to be marked as ‘sterile’ as a ‘theoretical value of no more than one living microorganism (being) present in 1 × 106 sterilized units of the end product’ [1]. These requirements are also referenced by DIN EN 556 [5]. Furthermore, the effective sterilization principle should be characterized according to ISO 14937 [6].

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Challenges associated with sterilization procedures

The mechanisms by which all sterilization methods produce their effects are based on irreversible structural changes in biochemical components with resulting effects on the integrity, metabolism or reproduction capability of microorganisms. However, the necessary intensive physical and chemical effects cannot be limited to the desired antimicrobial effects, therefore ultimately resulting in possible adverse effects on both the material characteristics and the functionality of the sterilization load. As there is no universally applicable sterilization procedure, in practice assessment and application of appropriate and possibly adapted processes for each type of sterilization load are required. However, as the result of a sterilization process cannot be confirmed in practice by a test, the application of a suitable and validated procedure is a prerequisite to ensure proper sterilization [1,7].

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Key features of sterilization procedures

It can be assumed that marketed 2 incubators">CO2 incubators have been qualified, therefore fulfilling all needed requirements for being sterilized. However, the sterilization procedure needs to be evaluated by validation and revalidation processes under the terms of quality assurance protocols. Special procedures are to be applied to that end because the result of applying the sterilization procedure cannot be evaluated through subsequent checks or tests at the incubator itself [8].

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Dry heat

For sterilization purposes by dry heat, exclusively temperature and time need to be considered as parameters, including their specification, observation and documentation alongside the applied sterilization process [9]. Use of this method is very widespread owing to the relatively modest technical demands that its application presents. Its limits are found in the long equalization and exposure times as well as the required thermostability of the materials [10]. Elevated temperatures are applied mainly by hot air heated through a heating register. Heat transfer can be enhanced by mechanical means of air movement (blower) [11]. In this regard, heat conduction represents a critical parameter, as transfer of heat from the air to a solid body that is intended to be sterilized is influenced by the physical properties of the sterilization load (thermal resistance, heat capacity and thermal conductivity).

The effect of dry heat on microorganisms is supposed resulting from a complex process, in which denaturing of proteins is accompanied by irreversible damages to cell membranes and DNA. Based on the high-temperature resistance of bacterial spores, these microorganisms are most often the limiting factor to a successful sterilization process [12]. The main influencing factor on this can be considered moisture, either of the spores itself [13], the carrier of the spores [14], or the humidity of the sterilization system [15]. Exposure time and temperature of dry heat are easily determined in case that the microbial contamination, that is the number of microorganisms (N 0) and their resistance to a reference-temperature (D 160 value for 160 °C and z value as temperature resistance coefficient), is known. The D-value is described as temperature–time relationship at which the number of surviving organisms decreases by a power of ten within a several exposure time [12]. Increased humidity of spores results in an increased D-value. The necessary minimum exposure time F is calculated for the preselected temperature T i using the following equations:

In case the contamination source is unknown, however, the presence of a large quantity of highly resistant microorganisms needs to be expected, therefore requiring the setting of parameters with an arbitrary margin of safety. In the past, these parameters were published authoritatively in the national pharmacopoeias. However, published heat resistance data strongly varied when comparing these national guidelines. According to the Ph. Eur. 8.0, at every point within the usable space and sterilization load, a temperature of 160 °C and an exposure time of 120 min are required for effective sterilization [1]. Perkins and Wallhäußer, however, provide more detailed recommendations on sufficient time–temperature relationships for dry heat sterilization [3,16]. Table 2 provides an overview of recommended time–temperature relationships for sufficient dry heat sterilization from several sources.

Table 2

Table 2

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Steam sterilization (in an autoclave) is a routinely applied procedure and the probably most often applied reference method for sterilization purposes [1,18]. With this method contained saturated steam inside a pressure chamber is utilized for microorganism kill.

We see the crucial advantages of steam sterilization as follows:

  1. More than 100 years of experience and countless scientific studies have brought the procedure to a high level in terms of both technology and safety.
  2. As a sterilizing agent, steam is toxicologically harmless, as is the condensate produced during the process.
  3. The sterilization procedure is economical and, aside from its energy consumption, environmentally compatible.

The physical properties of steam render it especially suitable as a sterilizing agent. When water is heated, the temperature increases continuously to its boiling point, from which on all of the absorbed heat is used for the conversion from water into steam without further increasing the water temperature. Only after full conversion of the water into steam a further increase in steam temperature will occur. Steam vaporizing above a free water surface in equilibrium with the water is referred to as saturated steam, which is one of the fundamental conditions for steam sterilization processes to be fully effective [7].

In accordance to the Ph. Eur. 8.0 and DIN EN 556 a holding temperature of at least 121 °C for 15 min is required for sterilization procedures utilizing saturated steam. However, further temperature–time relationships are permitted if adequate effectiveness is ensured. For example, in DIN EN 285 [23] the alternatively valid temperature–time relationships of 10 min/126 °C and 3 min/134 °C are stated. For calculation of equivalent temperature–time combinations, the F 0 concept, as contained in the Ph. Eur. 8.0, can be utilized. It applies if the standard temperature cannot be used in case of temperature-sensitive sterilization loads, or when parts of the heating and cooling phases are to be factored into the total lethality of the procedure. However, the F 0 concept is based on very simplified assumptions and has been criticized in regard to both its practicability and the level of certainty of sterilization it affords [24,25].

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Because of its existing residue issues, problems associated with its practical performance and the risk it poses to staff, ethylene oxide gas sterilization will not be discussed in the context of this review. Furthermore, for low-temperature steam formaldehyde sterilization and sterilization by glutaraldehyde identical concerns apply, although use of both methods is widely accepted in Anglo-American regions.

As a replacement for both ethylene oxide gas sterilization and low-temperature steam formaldehyde sterilization, both methods utilizing extremely toxic agents, hydrogen peroxide gas plasma sterilization was developed to provide a method applying less toxic substances, as decontamination exclusively creates O2 radicals and no toxic by-products [3,26]. Systems operating on this basis are very complex owing to the fact that a vacuum has to be created, all surfaces need to be saturated with a sufficiently high concentration of H2O2, and all materials require being resistant to the agent. Because of their toxicity to tissue cultures, it is absolutely necessary to flush out the oxygen radicals that are produced during hydrogen peroxide application. Effectiveness of sterilization by hydrogen peroxide can further be enhanced by ultraviolet (UV) light application [27,28]. Despite its status as a yet-to-be standardized sterilization procedure, it found broad practical application.

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Ultraviolet radiation

UV radiation is characterized by a broad spectrum of wavelengths with antimicrobial effects and provides an alternative to heat-based sterilization procedures. However, the sterilizing action of UV radiation is primarily mediated via the nonionizing UV-C and UV-B wavelength range between 190 and 320 nm with an optimum effect at 254 nm [29]. Treatment with UV radiation, in contrast to procedures applying ionizing radiation, is seldom recommended for sterilization purposes. UV radiation has a very shallow penetration depth, therefore being inappropriate for sterilization of other than plain and nonporous surfaces. Furthermore, UV lamps weaken over time, making it necessary to replace them regularly to ensure proper sterilization [30,31]. However, approaches exist to fully exploit the antimicrobial potential of nonionizing UV radiation. For example, omnidirectional homogeneous UV radiation, in which the sterilization load is UV radiated simultaneously from all sides to reduce the formation of shadows, allows effective treatment of structured objects [32]. Reductions of greater than 6–7 logs in bacterial spores have been observed on various materials applying this method [3,33].

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Conclusion and discussion

Preventive measures

It is advisable to take preventive measures to avoid contamination of cell or tissue cultures [34] and of special importance in regard of good cell culture practice [35,36].

The authors recommend the following procedure:

  1. Clean the collection and sampling areas.
  2. Sanitize the laboratory environment.
  3. Prepare the biological safety enclosure.
  4. Sterilize all cell culture materials such as reagents and nutrient media.
  5. Include antibiotics and/or antimycotics.
  6. Maintain the ‘clean end’.
  7. Extract the tissue quickly.
  8. Use aseptic work techniques.
  9. Introduce the cells into the culture quickly.
  10. Monitor the cultures.
  11. Sterile filter all gas mixtures being introduced into the incubator.

Products of approximately 35 manufacturers of 2 incubators">CO2 incubators are on the market worldwide. The disinfection or sterilization methods being used for incubators are mainly based on dry or damp heat application, UV radiation, the antimicrobial effect of hydrogen peroxide, or a combination of the above. In case chemical cleaning and disinfection agents are used, operating personnel should take care that such products do not intoxicate the culture system, as they often contain substances that are harmful to cultured cells and tissues, ultimately adding a chemical hazard. Taking the aforementioned disadvantages into account, thermal procedures should be preferred over chemical treatment.

In comparison of these methods in terms of their effect on undesirable biological materials, such as microorganisms or nucleic acids, dry heat sterilization must be regarded as the most reliable sterilization method for 2 incubators">CO2 incubators. The success of sterilization measures and the reliability of the methods depend exclusively on the exposure and temperature parameters. At temperatures of 180 °C and an exposure time of 1 h, it can be assumed that all agents capable of damaging the culture, including bacterial spores and nucleic acids, are eliminated.

Other widely applied procedures, such as hydrogen peroxide sterilization or UV light radiation, require a careful handling and nevertheless do not guarantee a successful kill of microorganisms. Neither hydrogen peroxide nor UV radiation might sufficiently penetrate existing cavities of sterilization load or, for example, dust particles, in which microorganisms might be residing. Incubators combining both procedures are on the market as well. However, these incubators are quite complicated in design and consequently difficult to deal with in practical use. We may therefore conclude that, for 2 incubators">CO2 incubators, sterilization with dry heat appears to be most reliable when sufficiently high temperatures and sufficiently long exposure times are chosen.

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Conflicts of interest

There are no conflicts of interest.

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