4). The residues on the filter were subsequently used for the microscopic verification of purification success. All samples purified by the six procedures were stored at 4°C no longer than 12 h until further processing. Verification of purification procedures One important criterion for a purification BYL719 datasheet method is a minimized loss of cells. Unfortunately, cell densities of untreated biogas reactor samples could not be calculated by particle AZD5153 cost counting due to interfering particles and cell aggregates. Hence, pure cultures of E. coli were used for determination of cell losses during the purification procedures. Cell counts were determined in triplicates by Coulter
Counter (Multisizer™ 3 Coulter Counter®, Beckman Coulter, Germany). Each triplicate was measured three times and the standard deviation of the nine measurements was calculated. Measurements were carried out with a 50 μm capillary, and the measurement volume was 50 μl. To determine the particle number and size within the electrolyte solution (‘background control’), the electrolyte was measured without addition of any microorganisms. For the verification of the purification click here success in
terms of cell aggregates disbandment and detachment of microorganisms from particles, the washed pellets, the supernatants, and the residues on the filter were visually evaluated by fluorescence microscopy. For microscopic analyses 10 μl of residues on the filter, pellet samples, and supernatants each diluted 1:500 in sterile water were coated on separate wells
of a 10-well-slide in triplicates. After drying the samples at 40°C the antifading reagent Citifluor A1 (PLANO GmbH, Wetzlar, Florfenicol Germany) was added to coat each well and 0.2 μl of a 20 μg ml-1 stock solution of 4’,6-diamidino-2-phenylindole (DAPI) were carefully injected into the Citifluor A1 drop. The size of cell aggregates was determined by microscopic field analyses using an ocular micrometer at 630× magnification. Five randomly chosen microscopic fields from each sample were analyzed in terms of the sizes of cell aggregates, the presence of organic and inorganic particles, and their microbiological growth. One microscopic field comprised the total area of 144 μm2 and was divided into 10 × 10 sub-fields of 5.76 μm2 each. All microscopic analyses were conducted with a Nikon Optiphot-2 microscope (Nikon, Duesseldorf, Germany) fitted with a DAPI AMCA filter tube or with an Olympus BX51 fluorescence microscope (Olympus GmbH, Hamburg, Germany) fitted with a U-MWU2 filter module. Fluorescence in situ hybridization (FISH) FISH was carried out with domain specific probes EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′)  and ARCH915 (5′-GTGCTCCCCCGCCAATTCCT-3′)  for the detection of bacteria and archaea, respectively. For the detection of undesired cross hybridization with non-target microorganisms the nonsense probe NonEUB338 (5′-ACTCCTACGGGAGGCAGC-3′)  was used.