## 1 Introduction  
Microorganisms are present in all ecosystems and due to their rapid responses to physical and chemical changes, they can be used as bioindicators of environmental quality. The Community-Level Physiological Profiling (CLPP) is a rapid and relatively inexpensive technique to relate microbial functional diversity over space and time to changes in the environment [1; 2; 3; 4]. Biology\({}^{\text{\textcircled{R}}}\) EcoPlates\({}^{\text{TM}}\) (Biology Inc., Hayward, CA, USA) were developed to analyse the functional diversity of bacterial communities by means of measuring their ability to oxidize carbon substrates. An EcoPlate is a 96-well microplate that contains 31 common carbon sources from altogether five compound groups--that is, carbohydrates, carboxylic and ketonic acids, amines and amides, amino acids and polymers--plus a blank well as a control, all these replicated thrice to control variation in inoculum densities. Each EcoPlate is filled with a dilution of one soil suspension, thus representing one soil sample. The utilization rates of carbon compounds in the wells are quantified spectrophotometrically by following the reduction of water-soluble colourless triphenyl tetrazolium chloride to purple triphenyl formazan. For the measurements of optical density (OD), two filters are used: (a) 590 nm (absorbance peak of tetrazolium) to evaluate colour development plus turbidity values and (b) 750 nm to measure turbidity values only. The turbidity of dilutions is due to clay and humic particles in soil colloidal suspension. Every bacterial community has a characteristic reaction pattern with different OD values for different carbon compounds, called a'metabolic fingerprint' [2; 5]. While the inoculated bacterial density significantly affects the rate of colour development in the wells, on the other side the choice of a wrong inoculum can compromise the results of this techniques [5; 6]. For this reason, it should be necessary to accurately choose the inoculum densities for different soil samples using a plate count culture-based method. Moreover, a correct definition of blank is essential for calculating the related diversity indices. Biology\({}^{\text{\textcircled{R}}}\) EcoPlates\({}^{\text{TM}}\) have been successfully adopted in our laboratories for studying bacterial soil communities from different soil environments, polluted soils and soils subjected to various agronomic treatments [6; 7; 8; 9]. Unfortunately, the studies regarding soil as a matrix for bacterial communities often include a Biology\({}^{\text{\textcircled{R}}}\) analysis but the reagents and apparatus for soil preparation, dilution and incubation time used, standard deviations and number of replicates and precise formulas for calculations are often not reported, so it becomes difficult for other researchers to understand how to correctly use this method. On this basis and taking into account our previous research on soils, we here propose for the first time a standardized and accurate step-by-step method for estimating the functional diversity of a soil bacterial community by Biology\({}^{\text{\textcircled{R}}}\) EcoPlates\({}^{\text{TM}}\) assay. We tried to adopt a clear terminology and parameters, explaining them in detail, in order to facilitate the calculation of the most relevant and used Biology\({}^{\text{\textcircled{R}}}\)-related indices of microbial functional diversity. Even with its limitations [10], the Biology\({}^{\text{\textcircled{R}}}\) EcoPlates\({}^{\text{TM}}\) method remains a quick and relatively simple and inexpensive technique for comparing microbial communities. The standardization of this technique could be essential for indicating differences in CLPPs between samples within a single experiment, for comparing different experiments, soil types and management systems or simply soils sampled at different times.