Operación de lógicas de automatismo

The mean, standard deviation, standard error of mean (SE), and coefficient of variance (%CV) was routinely calculated for all experimental data. This was used to assess both inter-assay (biological replicates) and intra-assay (technical replicates) variability. Typically five biological replicates of each assay were completed. The N value for each assay is indicated in the figure legends.

2.10.1 Statistical analysis

Statistical analysis was carried out using GraphPad Prism software (GraphPad software). Significance was assessed by calculating Students‟ t-test, Tukey, and Bonferroni post-test probabilities. A P value below 0.05 was considered significant. Linear regression was used in the cell density measurements presented in Section 3.2.5.

2.10.2 Dot plot analysis

The cje0556 and cje1441 genes were compared using the dotpath software, available on the EMBOSS website (http://emboss.bioinformatics.nl accessed 18 December 2014) and following the software instructions.

Helen Louise Brown TTC staining of the biofilm

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3 Development of a novel staining method for assessment

of C. jejuni biofilm formation in the presence of organic

materials.

This chapter is based on the paper: Brown H L, van Vliet A H M, Betts R P, Reuter M (2013) Tetrazolium reduction allows assessment of biofilm formation by Campylobacter jejuni in a food matrix model. Journal of Applied Microbiology, 115, p1212-21

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Introduction

3.1

Levels of biofilm formation are usually assessed by dye-based staining techniques, which broadly fall into two categories: non-specific dyes and dyes targeting specific molecules within the biofilm, such as fluorescent dyes, which can be visualised using confocal laser scanning microscopy (Lawrence and Neu, 1999). Such specific dyes are useful for detailed and dynamic imaging of biofilms (Baird et al., 2012), but are costly and have labour-intensive and time consuming methodologies, making them an inappropriate tool when rapid visualisation of biofilms is required. Conversely non-specific dyes, although rapid, may overestimate the quantity of viable biofilm cells present, due to their non-specific binding of the matrix or surface components.

The most commonly used method to detect and quantify biofilms is staining with crystal violet (Chavant et al., 2007). This dye is non-specific, as it binds to all surface molecules of a negative charge, which can be found on either the bacteria or ECM (Extremina et al., 2011, Pan et al., 2010). Although crystal violet is frequently used to detect and quantify biofilm formation, some authors have criticized crystal violet for its relatively high inter-assay variability, particularly when compared to other imaging methods (Li et al., 2003).

Tetrazolium salts are the basis of several redox sensitive dyes, and have been used previously to study cell growth and biofilm formation in various bacterial models (Tengerdy et al., 1967, Schaule et al., 1993), but not with C. jejuni biofilms. In this chapter it is demonstrated that crystal violet gives high levels of non-specific (false-positive) staining in the presence of organic food matrices. As an alternative to crystal violet the reduction of the metabolic stain TTC to insoluble, red crystals of 1,3,5-triphenylformazan (TFP) (Bakor and Fahselt, 2005, Berends et al., 2010) was tested, before optimisation for use with C. jejuni biofilms.

Helen Louise Brown TTC staining of the biofilm

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Results

3.2

3.2.1 Crystal violet is able to non-specifically stain chicken juice

Crystal violet staining is a frequently used method for detection and quantification of

biofilm formation, and has been used previously with C. jejuni biofilms (Joshua et

al., 2006, Reeser et al., 2007, Reuter et al., 2010). However, initial experiments with

crystal violet, chicken juice (for details of the chicken juice method see 2.2.2.6.1 and

(Figure 3-1). Tubes incubated with chicken juice but no C. jejuni cells gave high

levels of staining, visualized as both a diffuse stain below the air/liquid interface and

a strongly stained ring at the air-liquid interface (Figure 3-1b). This staining is

independent of the presence or absence of C. jejuni. Similar results were obtained

when replacing the chicken juice with skimmed milk solution (Figure 3-1c), another

food-relevant matrix that has previously been used (Chmielewski and Frank, 2003).

Figure 3-1 Representative images of CV stained chicken juice and skimmed milk

Images show crystal violet staining of test tubes incubated for 48 hours with Brucella medium only (A), 5% chicken juice (B) or 5% skimmed milk (C). No C. jejuni is added to these test tubes so staining shown reflects the ability of crystal violet to non-specifically the attached particulates.

This non-specific staining necessitated the development of an alternative method for measuring biofilm levels of C. jejuni. As shown in Figure 3-2, to allow TTC staining the standard biofilm assay was modified. In both the crystal violet and TTC methods, C. jejuni NCTC 11168 biofilms were cultivated using static culture (primary culture). In addition the TTC method required replacement of primary culture growth medium with fresh, TTC- supplemented media (secondary culture).

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Figure 3-2 Diagrammatic representation of the staining assay methodologies showing incubation length and assay steps.

The workflow of both the TTC and crystal violet assays are shown to highlight both the methodological and time-span differences between the two methods.

During the secondary incubation, viable cells within the attached population were able to reduce the TTC, because the addition of the fresh medium allowed metabolic activity to increase, leading to conversion of TTC and the formation of a visible red formazan ring. Supplementation of the medium used during the primary incubation with TTC led to a diffuse red dye, which could not be collected and quantified, hence a secondary incubation step was added to the biofilm method, allowing quantification of the biofilm only. For the secondary incubation, only Brucella medium was tested as the C. jejuni cultures were known to be metabolically active and form biofilms within this medium. Since this medium is also widely used by this group and other groups working in the field, the results could be compared more easily to existing studies. The formazan crystals could be quantified by dissolving the bound dye and measuring absorbance at a wavelength of 500 nm (Figure 3-3). This formazan conversion allowed distinction between bacterial populations and attached organic material, a distinction which could not be achieved by use of non-specific dyes such as crystal violet (Figure 3-3).

Helen Louise Brown TTC staining of the biofilm

Page 97 of 294 Figure 3-3 TTC does not non-specifically stain 5% chicken juice

Representative images of test tubes which have been incubated for 48 hours to allow biofilm formation before staining with either crystal violet (first and third columns) or TTC (second or last columns). The upper panel highlights that TTC is unable to convert to red formazan crystals without the presence of viable C. jejuni, whereas crystal violet confound biofilm quantification by staining both attached cellular populations and chicken juice (* denotes the approximate level of the meniscus following a 48 hour incubation). Chicken juice was selected for use here since BSA, another protein rich commonly used medium supplement, inhibited biofilm formation by C. jejuni NCTC 11168 as so could not be used in experiments where C. jejuni biofilm formation was required.

3.2.2 The reduction of TTC by C. jejuni is more effective under microaerobic