Development of improved sampling methods for the evaluation of cleaning and disinfecting measures within self control systems in hygienically demanding areas (with IUTA/DIL)
IGF 276 ZN
The success of cleaning and disinfecting measures needs to be controlled in healthcare sector and food industry on diverse surfaces. In these hygienically demanding areas hygiene standards and monitoring requirements were increased constantly in recent years. Therefore, fast and easy self-control methods are required to control the bioburden of surfaces within monitoring systems.
However, surfaces with a complex geometry (e.g. convex or concave surfaces or corners) cannot effectively be sampled with standard microbial wiping methods or contact slides. In addition, standard methods like ATP-monitoring are not capable to detect small numbers of bacteria and can’t distinguish between live and dead cells. Further more ATP occurs in all types of cell making a differentiation of bacteria and eukaryotic cells impossible.
The aim of this research project was therefore the development of an improved sampling method using a gelatine matrix in combination with flow cytometry analysis as a tool for an easy, self-practicable and inexpensive assessment of cleaning and disinfecting measures. Flow cytometry is a standard technique in biology for counting, analysing and sorting of particles in relation to their size, inner complexity and fluorescence. In the flow cell of a flow cytometer a liquid stream carries and aligns stained cells that they pass in single file through a laser beam for sensing.
For sampling a gelatine matrix was developed to completely remove bacteria from surfaces. A surface with complex geometry (e.g. milk pipe thread or perforated metal plates) can for example be sampled with this liquid matrix. After hardening of gelatine the microorganisms are included in the gel matrix and can be detached residue-free from the surface of interest. To analyse the gel matrix with the flow cytometer the gelatine need to be liquefied by an enzymatic treatment.
The cells are stained with two different nucleic acid binding dyes to be able to distinguish between living and dead bacteria when performing the flow cytometry analysis. The developed method takes less than 90 minutes inclusive preparation of gelatine, enzymatic treatment, staining and analysis with the flow cytometer.
The IGF-project 276 ZN 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