Colorimetric assay for biofilms in wet processing conditions

Abstract

Controlling bacterial biofilms is necessary for food safety and industrial processing in clean room environments. Our goal was to develop a method to quantitatively measure biofilm produced by pathogens under wet poultry production and processing conditions. Stainless steel and glass coupons were incubated in aqueous media containing reduced nutrients and exposed to Listeria monocytogenes under static temperature and humidity conditions. Samples were measured separately by biofilm assay and viable cell density, and then confirmed by spectrophotometry and microscopy. The biofilm assay resulted in different t groupings from the cell density. The mean from the biofilm assay was 0.50, and the error% was 0.595. The mean of the log10 density (cfu/cm2) was 5.90, and the standard deviation ranged from 0.127 to 0.438 on 24 coupons. The typical sequence of biofilm development, followed by microscopy of biofilm grown on glass coupons, exhibited a change from dispersed single cells to an all-over pattern of clumps with few dispersed cells. L. monocytogenes formed biofilms on all of the substrata tested. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiment and reached equilibrium between 6 and 24 h. The cell density log10/ml was 8.01, 8.03, 7.69, and 6.66, respectively; and the standard deviation ranged from 0.156 to 0.394. The data will be used to grow stable biofilms of Listeria spp. collected from the food processing environment for further study. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions. The methods tested are applicable to other bacteria and substrata.

Critical review on biofilm methods

“…gravitation. Appropriate washing steps can remove sedimented non-attached cells limiting or eliminating this problem; (iii) lack of reproducibility (Arnold, 2008;Peeters et al, 2008); (iv) nonspecific nature of CV that does not allow species differentiation in poly-microbial communities; (v) absence of a standardized protocol, despite its widespread use, resulting in a broad variety of staining protocols (Stepanovic et al, 2007) that make comparison of results between studies difficult. Colorimetric methods have also been used to assess cellular physiology in biofilms.…”

An Alternative Approach to Investigate Biofilm in Medical Devices: A Feasibility Study

“…A staining method for microscopic observation of biofilms, as originally described by J. W. Arnold [ 16 ], has the advantage to rapidly monitor biofilm evolution onto physical supports. However, it does not provide a quantitative assessment of stained surface.…”

“…Crystal violet staining of biofilm populated biomaterial. One of the most common methods to assess biofilm formation current relies on a 96 well microtiter plate assay, which usually involves a colorimetric detection of the dye removed from the previously stained biofilm [ 16 ]. Crystal violet is a basic protein dye that marks the surface negatively charged molecules (i.e., peptidoglycan) and extracellular matrix of polysaccharides [ 17 ].…”

Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials

“…This includes the fluorochromes DAPI [20,25,26], SYTO 60 [28] and SYTO 9 [22]. These nucleic acid stains provide greater specificity and higher sensitivity as compared to other methods, such as ATP, DNA, crystal violet staining, viable cell count and AODC [4,6,7,12,29,33,38,40]. For example, simple quantification by microscopy is difficult due to bacterial aggregation and slime formation [18].…”