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Since more than half of the faecal nitrogen (N) originates from microbial N, the objective of the study was to develop a method for quantitatively detecting microbial biomass and portion of living microorganisms in dairy cattle faeces, including bacteria, fungi and archaea. Three techniques were tested: (1) the chloroform fumigation extraction (CFE) method, (2) detection of the fungal cell-membrane component ergosterol and (3) analysis of the cell wall components fungal glucosamine and bacterial muramic acid. In a second step, an N deficient (ND) and an N balanced (NB) diets were compared with respect to the impacts on faecal C and N fractions, microbial indices and digestibility. The mean values of microbial biomass C and N concentrations averaged around 37 and 4.9 mg g(-1) DM, respectively. Ergosterol, together with fungal glucosamine and bacterial muramic acid, revealed a 25% fungal C in relation to the total microbial C content in dairy cattle faeces. Changes in ruminal N supply showed significant effects on faecal composition. Faecal concentrations of NDF, hemicelluloses and undigested dietary N and the total C/N ratio were significantly higher in ND treatment compared to the NB treatment. N deficiency was reflected also by a higher microbial biomass C/N ratio. It was concluded that the assessment of microbial indices provides valuable information with respect to diet effects on faecal composition and the successive decomposition. Further studies should be conducted to explore the potentials for minimising nutrient losses from faeces.