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Colloids and Surfaces B: Biointerfaces 20 (2001) 37 – 50

Microbiological methods for testing disinfectant efficiency on Pseudomonas biofilm
G. Wirtanen *, S. Salo, I.M. Helander, T. Mattila-Sandholm
VTT Biotechnology and Food Research, PO Box 1500, Tietotie 2, FIN-02044 VTT Espoo, Finland Received 22 November 1999; accepted 10 May 2000

Abstract Biofilms of theGram-negative bacteria Pseudomonas aeruginosa and Pseudomonas fragi were grown on stainless steel surfaces (AISI 304, 2B) for 4 days in slime broth. These biofilms were treated with four commercial disinfectants. The disinfectants were alcohol-based, tenside-based, peroxide-based and chlorine-based products, covering most disinfectant types used in the food industry. The effects of thedisinfectants on the bacterial cells were first investigated in suspension using the permeabilisation test, which is based on fluorescence assessment of hydrophobic 1-N-phenylnaphtylamine (NPN). The surfaces covered with disinfectant-treated biofilms were investigated using conventional cultivation, impedimetry and epifluorescence microscopy in combination with image analysis of preparations stained with theDNA-stain acridine orange and with the metabolic indicator system CTC-DAPI. The results showed that the tenside-based and peroxide-based disinfectants permeabilised the cells in suspension. The overall biofilm results showed that of the agents tested, the peroxide-based and chlorine-based disinfectants acted most effectively on cells in biofilms. © 2001 Elsevier Science B.V. All rights reserved.Keywords: Biofilm; Pseudomonas sp.; Disinfection; CTC-DAPI; Impedance; Microscopy; Permeability

1. Introduction The key to effective cleaning of a food plant is understanding the type and nature of the substances to be cleaned including the microbes to be removed from the surfaces. Once microbes grow into a firm biofilm, cleaning and disinfection become much more difficult [1,2]. Furthermore,disinfectants do not satisfactorily penetrate the
* Corresponding author. Tel.: + 358-9-4565222; fax: +3589-4552103. E-mail address: (G. Wirtanen).

polysaccharide and glycoprotein matrix left on the surfaces after inadequate cleaning, and thus they do not destroy all living cells in biofilms [3,4]. Tests of a detection method based on microelectrodes and scanning confocal lasermicroscopy have shown that the agent used for disinfection of biofilms did not penetrate the cell clusters entirely [5,6]. Criteria to be met to indicate cleanliness in a food plant are physical, chemical and microbiological [7,8]. No visible soil, foreign matter or slime, or undesirable chemical residues and microbes should remain on the equipment surfaces

0927-7765/01/$ - see front matter © 2001Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 0 ) 0 0 1 7 3 - 9


G. Wirtanen et al. / Colloids and Surfaces B: Biointerfaces 20 (2001) 37–50

[8]. Factors affecting the elimination of those substances are chemical and mechanical effects, holding time and temperature [9 – 11]. Studies have also revealed that bacteria on surfaces isolated from milking lines are moreresistant to cleaning-in-place disinfectants than those in solutions [12]. Efficient disinfection is required in food plants where wet surfaces provide favourable conditions for microbial growth [13 – 16]. Disinfectants used in the food processing industry include oxidising agents, e.g. hypochlorite, hydrogen peroxide, ozone and peracetic acid; denaturating agents, e.g. alcohol-based products;non-oxidising and surface tension diminishing agents; and enzyme-based compounds [11,17]. The disinfectants must be effective, safe and rinsable, as well as easy to use, and they should not affect the sensory qualities of the product [9,11,18]. Although disinfectants are developed to destroy microbes [19], microbes have been found in disinfectant solutions. Pseudomonas sp. have been found in...
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