The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2-5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10(3).