In addition, the indicator phenol red was added to all wells of the Taxa Profile™ A and C microtiter plates to optimize detection. The blank value was measured for each biochemical reaction on the same plate and subtracted from measured values. In order to assess inter-assay variability five independent experiments per strain were conducted. For evaluation of the newly developed Brucella specific 96-well microtiter plate three trials

per strain were run independently. Intra-assay variability was assessed with the reference strains testing all substances twice within the same experiment. Since the blank values measured on extra plates proved to be constant a fixed mean value of each substrate was subtracted from the measured data. Data acquisition and analysis Turbidity and colour change were measured photometrically using a Multiskan Ascent® photometer LY2606368 in vivo (Labsystems,

Helsinki, Finland) at a wave length of 405 nm, 540 nm and 620 nm according to manufacturer’s recommendations. Optimal OD cut-off values were empirically adapted from the preliminary test results of the 384-wells Taxa Profile™ microtiter plates. Stable and discriminatory markers were selected to design a 96-well Micronaut™ plate (Figure 2) to identify bacteria of the genus Brucella and to classify their species and biovar. Dendrograms were deduced from Erastin nmr the biotyping data using SPSS version 12.0.2 (SPSS Inc., Chicago, IL, USA). First of all, three different character data sets were defined following

the metabolic activity tested (Taxa Profile™ A (“”amino acids”"), C (“”carbohydrates”"), and E (“”other enzymatic reactions”")). Each character was considered as equal within the particular data set. Both the raw OD data and the binary coded data based on the empirically set cut-off were analyzed using the Pearson coefficient and the categorical coefficient, respectively. Hierarchical cluster analysis was performed by the Ward’s linkage algorithm, and a dendrogram was generated. If necessary, analysis was repeated within each cluster for further discrimination. Secondly, a separate data analysis Interleukin-3 receptor of the 23 Brucella reference strains representing the currently known species and biovars was performed including all biochemical reactions of the Taxa Profile™ system or exclusively the TPCA-1 clinical trial substrates selected for the newly developed plate. Finally, the whole collective of 113 strains tested with the Brucella specific Micronaut™ microtiter plate was analyzed to prove the diagnostic system. An identification table presenting quantitative and qualitative metabolic activity was created [Additional file 7] and the specificity of the test system to differentiate Brucella species and biovars was calculated (Table 1). Acknowledgements The project was partially supported by research funds of the Bundeswehr Medical Service. We are grateful to Dr.