Quantification of lipids and protein in thin biofilms by fluorescence staining

Abstract

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised.

Keywords:

• fluorescent staining
• biofilms
• NanoOrange
• Nile Red
• CBQCA
• FITC

Acknowledgements

The authors are grateful to Anne Eg Lauritsen from Novadan ApS., Denmark, for kindly providing cleaning agents for interference tests and technical expertise on CIP procedures. Daniel Otzen, Aalborg University, is acknowledged for providing insulin for protein specificity tests. Funding for this study was obtained by The Danish Ministry of Food, Agriculture and Fisheries' project “Hygiene in Danish Seafood Industries” to JLN.