10.12.2003
A research study has shown that disinfection is not enough! Only hot-air sterilisation at 180 ¨¬C can reliably ensure a standard-compliant sterile culture environment! How does your CO2 perform?

Download Press release, full version
CB series

An independent research has shown that spores cannot be eliminated with disinfection at 90 ¡ÆC/ humidity heat. The only standard-compliant and safe decontamination method for CO2 incubators is hot-air sterilisation at 180 ¡ÆC. Few CO2 incubators can really achieve this performance. We bring performance. The Binder CO2 incubator, CB series.

Increased safety requirements
New cell-biology fields of application such as, for instance, research on cell and tissue replacement in regenerative medicine, have radically changed the technical requirement profile of modern CO2 incubators. In addition to high-precision temperature control and the fastest CO2 control dynamics (Distler, GIT laboratory journal 7/2002 (1)), aspects such as freedom from germs and sterility of the growth environment in CO2 incubators are increasingly becoming the focus of attention. In all in-vitro applications and particularly when dealing with human cells and tissues, a sterile culture environment is an essential requirement for safe working on the basis of GLP/GMP-compliant work processes. Maximum hygiene significantly contributes to risk minimisation and increased process safety. In regenerative medicine applications, this benefits not only the scientist but, in particular, the patient who is directly affected (e.g. in the case of cartilage cell reproduction). When cultivating human cell material, similarly to the requirements for handling blood specimens, the relevant safety aspects in respect of excluding any risk of infection (Hepatitis, HIV, etc. ) must be taken into account.

From simple disinfection methods through to the standard-compliant, demonstrably safe method of 180 ¡ÆC hot air sterilisation
The CO2 incubator market now offers the appropriate solution for all culture quality and process safety (sterility of the environment) requirements: the range extends from the technically simplest units, without any device for contamination prevention or the possibility of germ elimination, through to high quality incubators which enable standard-compliant hot air sterilisation of the culture environment at 180 ¡ÆC. Technical compromise solutions can be found between the two extremes. The mode of operation of these methods - precisely characterised as disinfection methods - are based on very different principles. However, apart from their action principle, they also differ quite considerably in their effectiveness. Germ reduction through disinfection cannot qualitatively be compared with the technically complex process of 180 ¡ÆC hot air sterilisation. This is the only reliable method established in DIN 58947 (2) for safe and standard-compliant decontamination, which is also recognised internationally.

Adequate safety for the culture process through simple disinfection routines?
Popular disinfection methods and preventive methods against microbial contamination of CO2 incubators include UV radiation, permanent ambient air filtration through HEPA filters and disinfection in moist heat. In addition, a bactericidal effect is provided by metal surfaces such as e.g. copper or copper-containing metal alloys. The process safety and contamination risk in CO2 incubators correlates closely with the efficiency of the integrated decontamination method. Simple CO2 incubators without preventive protective devices accordingly also have the highest safety risk. If this results in undesirable contamination with spore-forming germs, a new incubator will have to be purchased, in extreme cases.

Strict guidelines for thermal sterilisation as per DIN 58947
Systems with integrated disinfection procedures such as UV radiation of the circulating ambient air, permanent HEPA filtration, or disinfection in moist heat, offer only very limited safety. Not all of these methods satisfy the strict guidelines for thermal sterilisation methods as defined in DIN 58947, Part 3 or as documented in Federal Health Paper 22, no. 10 (3). Mutagenic UV radiation, for instance, does not reach all areas of an incubator evenly. The risk of concealed contaminations developing in non-accessible areas can also not be excluded in incubators with HEPA filters. Causes of this are rapid ageing of the filter materials and ? due to the necessary time and cost involved ? frequently neglected filter maintenance. Disinfection at 90 ¡ÆC and high air humidity also offer only very limited safety and so, once again, a compromise solution. This method is sometimes misleadingly described on the market as 'gentle sterilisation'. This germ reduction method is based on the thermal effect of moist heat at temperatures of approx. 90 ¡ÆC over a period of several hours.

The result of an independent study: Limited action of disinfection at 90 ¡ÆC / moist heat
As an example, a neutral comparison study was performed between an incubator with 180 ¡ÆC hot air sterilisation (Binder CB, Fig. 1 and 2) and a CO2 incubator with 90 ¡ÆC decontamination routine (competitor model) in an internationally recognised microbiological test laboratory accredited to ISO/IEC 17025. The aim of this study was to provide qualitative and quantitative information about the efficiency of the two different decontamination methods under identical test conditions. The CO2 incubators were selectively, systematically and reproducibly contaminated with defined quantities of test germs in comparable areas of the incubator on ceiling, floor, inner door, and sides. The test germs used were standard vegetative germs widely used for microbiological tests in medical instruments and autoclaves, as well as spores from spore-forming germs (cf. Tab. 1). The latter were applied as spore packets, and also in opened form as a control. The CO2 incubators contaminated with test germs were subsequently subjected to the respective decontamination procedure, in accordance with the manufacturer's instructions. In the case of the Binder CB series CO2 incubator, this is a 1-hour sterilisation at 180 ¡ÆC dry heat, and in the case of the competitor model, exposure to 90 ¡ÆC / moist heat for 9 hours. The level of germ reduction was then examined for all test germs by means of smears and subsequent reproduction in appropriate culture media. Taking account of the relevant control tests and a number of test repetitions, a clear result was obtained on the mode of action of the two different methods.

No surprising findings - spores cannot be eliminated by disinfection at 90 ¡ÆC / moist heat
Evaluation of the comparison study brought a not-unexpected result. The result of the 90 ¡ÆC / moist heat disinfection was unsatisfactory. Although vegetative germ forms were inactivated, the substantially more resistant spore forms of the species Bacillus subtilis and Bacillus stearothermophilus resisted the disinfection procedure and were largely undamaged (cf. Tab. 2). In the case of the incubator with standard-compliant hot air sterilisation, all vegetative test germs and the spores were completely eliminated, as expected (cf. Tab. 2). Therefore, the goal of permanent and complete germ elimination was not achieved in the incubator with 90 ¡ÆC / most heat disinfection. This finding was reproducible in a number of test repetitions and applied to all areas tested in the incubator. This test result thus clearly contradicts the popular view of the alleged safety of this method. In particular, the findings conflict with previous pertinent documentation, which allegedly substantiates the effectiveness of the method, making reference to microbiological tests. This discrepancy probably stems from the fact that the earlier tests did not use spores, but substantially less sensitive vegetative forms.

Conclusion Germ reduction using disinfection at 90 ¡ÆC/moist heat is not qualitatively comparable with 180 ¡ÆC hot air sterilisation. This method is also unsuitable and inadequate for guaranteeing a sterile environment to the current requirements, in the case of very long exposure times over many hours. This is confirmed by the present study, performed in a certified test laboratory. The study used spores and germs which are specifically prescribed for testing medical equipment such as hot air sterilisers and autoclaves, in accordance with the mandatory standard DIN 58947. This 90 ¡ÆC/moist heat disinfection method cannot cope with the increased requirements for culture safety and sterility, particularly for applications under GLP / GMP conditions. The only demonstrably reliable and internationally recognised method for use in highly demanding cell culture under GLP / GMP conditions is thermal hot air sterilisation at 180 ¡ÆC. For critical cell culture applications with pathogenic viruses and bacteria, hot air sterilisation at 180 ¡ÆC offers unique safety. In addition, the method has the advantage of a significant time and thus cost saving of several hours, in comparison with the protracted process of disinfection at 90 ¡ÆC/moist heat. Maximum safety and process quality of cell culture in CO2 incubators also assumes a number of further factors. These are perfect tempering precision, outstanding dynamics in CO2 and temperature control, as well as the latest controller technology for optimal process control and monitoring. The Binder CB series of CO2 incubators have all of these important characteristics and provide the user with the perfect prerequisite for optimal culture results.

Bibliography:
1) P. Distler (2002), GIT laboratory journal 7, 801- 803
2) DIN 58947, Sterilisation ? Hot air sterilisers, 1990
3) Federal Health Paper (1979), 22 no. 10, 193-200