Development of appropriate fast methods for cleaning service enterprises to analyze cleanliness and hygiene in cleanrooms
IGF 60 EN
Cleanrooms are closed system rooms in which the particle content of the air is controlled according to the specific clean room class. Such rooms are used in specific manufacturing processes, e.g. in the aseptic preparation of pharmaceuticals, foods and in hospitals. These controlled areas are subject to strict cleanliness and hygiene requirements. A continuous and comprehensive microbial monitoring of clean rooms is of high importance and represents a fundamental keystone within the quality management system. Currently, the assessment of the number and type of microorganisms on surfaces is performed using contact plates or the swab method. A major disadvantage of such classical microbiological methods is the incubation time and the analysis of samples by external laboratories that usually takes at least two days. Thus, corrections and adjustments to the cleaning and disinfection procedures can be made only after a considerable delay.
The aim of the project was the development of a suitable rapid method for the evaluation of the hygiene of cleaned and disinfected surfaces in cleanrooms.
An efficient method for the sampling of microorganisms was established by means of a sampling matrix based on a special clean room approved wipe made out of polyester/cellulose, which was moistened with 0.85 % NaCl solution. For the sampling of plane as well as geometrically complex surfaces, a sampling pen with an elastic tip was developed that holds the sampling matrix.
The elution of microorganisms from the sampling matrix was carried out by “stomaching”. For subsequent quantification of microorganisms, an analytical method based on flow cytometry was developed. For this purpose, at first a 15 minute “one-step” staining with two suitable fluorescent dyes was established, which allowed the discrimination of live and dead cells. Following, the fluorescent cells were detected and quantified using a flow cytometer.
The developed rapid method provided reproducible and reliable results when applied to surfaces such as stainless steel, glass or plastics. In addition, representatives of different groups of microorganisms (e.g., Gram-positive and Gram-negative bacteria and yeasts) and bacterial spores could be quantified.
Any residues on surfaces such as blood, silicone, wool grease, surfactants and disinfectants showed no negative influence on the developed methods as demonstrated in practice-relevant concentrations.
The entire process, i.e., surface sampling, elution of microorganisms, cell staining and flow cytometric analysis, requires less than 20 min. This allows a simple, fast and cost-effective assessment of cleaning and disinfection procedures in cleanrooms and enables a simple documentation of results. For the service companies entrusted with clean room cleaning and for clean room operators the developed method offers a valuable mean for internal self-control and quality assurance.
The IGF-project 60 EN of the research association Europäische Forschungsgemeinschaft Reinigungs- und Hygienetechnologie e.V., Campus Fichtenhain 11, D-47807 Krefeld, was supported via the AiF within the funding programme „Industrielle Gemeinschaftsforschung und –entwicklung (IGF)“ by the Federal Ministry of Economic Affairs and Technology (BMWi) due to a decision of the German Parliament.
The research report is available on request from FRT.