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We investigated attachment processes of hydrophobic and hydrophilic particles (diameter ‫؍‬ 1 m) to mature biofilms grown on clay marbles in a sequencing batch biofilm reactor. During a treatment cycle with filtered wastewater containing different fluorescent beads, the progression of particle density in various biofilm compartments (carrier biofilm, basic biofilm layer, biofilm flocs, and sessile ciliates) was determined by flow cytometry, confocal laser scanning microscopy and automated image analysis. Particles were almost completely removed from wastewater by typical processes of particle retention: up to 58% of particles attached to clay marbles, up to 15% were associated with suspended flocs, and up to 10% were ingested by sessile ciliates. Ingestion of particles by ciliates was exceptionally high immediately after wastewater addition (1,200 particles grazer ؊1 h ؊1 ) and continued until approximately 14% of the water had been cleared by ciliate filter feeding. Most probably, ciliate bioturbation increases particle sorption to the basic biofilm. Backwashing of the reactor detached pieces of biofilm and thus released approximately 50% of the particles into rinsing water. Clay marbles in the upper part of the reactor were more efficiently abraded than in the lower part. No indications for selective attachment of the applied hydrophobic and hydrophilic beads were found. As a consequence of interception patterns, organisms at elevated biofilm structures are probably major profiteers of wastewater particles; among them, ciliates may be of major importance because of their highly active digestive food vacuoles.

A lthough some impressive applications of multicolor fluorescence in situ hybridization (M-FISH) have been demonstrated on cytogenetic preparations, there are no reports of studies that carry over these advanced multitarget techniques to histological sections of tumor tissues in molecular pathology. Despite recent advances in protocols, M-FISH with a simultaneous multicolor painting tool does not seem to be a feasible approach in histological sections, mainly because of the inherent problem of the third dimension, which leads to complex overlays of both fluorescence signals and nuclei of tumor cells. To overcome these technical limitations, we introduce here an innovative and robust method for the detection of multiple targets by interphase FISH in formalin-fixed and paraffinembedded tissue, called sequential multilocus fluorescence in situ hybridization (SML-FISH).Each single tissue section (10 m) obtained from two different paraffin blocks of formalin-fixed tissue from a surgical resection specimen of primary adenocarcinoma of distal esophagus (Union Internationale Contre le Cancer [UICC] Classification: pT1, pN0, pM0; Graduation: G2) was mounted on a glass slide. Tissue pretreatment was done by heating the slides for 20 minutes in a microwave oven (750 W); additionally, pronase E (0.05%) digestion for 10 minutes was performed. Denaturation was performed as described elsewhere (Werner et al, 1999). The following commercially available fluorescent-labeled, locus-specific (LSI), and associated centromere DNA probes (CEP) (Vysis, Inc., Downers Grove, Illinois) were sequentially applied according to manufacturers' instructions in the listed order: LSI D7S486 (c-met), CEP 7, LSI c-myc, CEP 8, LSI Cyclin D1, CEP 11, LSI Her-2/neu, CEP 17, LSI 20q13.2, and CEP 20. After hybridization of the first probe (LSI D7S486), the slides were placed in ϫ2 SSC, 0.1% NP-40 (pH 7.4) at 73°C for 2 minutes followed by embedding with an antifade solution (Vysis, Inc.) containing 4',6-diamidino-2-phenylindole (DAPI) as nuclear counterstaining. Threedimensional volume data sets for the corresponding fluorescence channel (DNA probe and nuclear counterstaining) were generated using a confocal laser scanning microscope (CLSM, Zeiss LSM 510; Carl Zeiss, Jena, Germany). For these purposes each area was scanned in high resolution mode (lens PNF 40ϫ/ 1.3 Oil; scaling X ϭ Y ϭ 0.22 m, Z ϭ 0.5 m) resulting in an image stack of DNA probes and nuclear counterstaining of each tumor area. After image aquisition, the first DNA probe (LSI D7S486) was washed out by heating the section in ϫ2 SSC at 73°C for 25 minutes, followed by denaturation at 73°C for 5 minutes in 70% formamide/ϫ2 SSC. After deletion of the DNA probe which followed after the laser scanning procedure before the next hybridization, the section was evaluated to ensure that there were no DNA probespecific fluorescent signals left. Then, the procedure (hybridization, nuclear counterstaining, image aquisition of the same tissue areas, and deletion of the probe) was repeated for each of ...

Effect of low temperature on up-flow anaerobic sludge bed (UASB) reactor performance treating raw sewage was investigated in terms of the variations in methanogenic diversity using the 16S rRNA based Fluorescence In-Situ Hybridization (FISH) technique. The diversity of microorganisms present in the anaerobic granular sludge and the structure of the granules operated at 13 degrees C have been investigated using FISH combined with CSLM (Confocal Scanning Laser Microscopy). According to FISH results, archaeal cells representing methanogens were found intensively dominant in the bottom sampling port of the UASB reactor and acetoclastic Methanosaeta was the abundant methanogen. Other methanogens such as Methanosarcina and Methanobacterium like species were also observed. The abundance of originally mesophilic Methanosaeta-related Archaea under low temperature at all sampling days revealed the microbial adaptation to psychrophilic conditions. This might be attributed to the enzymatic alterations in Methanosaeta cells originating from seed sludge, which were exposed to sub-mesophilic temperatures at start-up and then to psychrophilic conditions during gradual decreases of temperature. According to CSLM observation, even though the sludge retained in the reactor kept its granular form as a whole, the majority of the granules had a tendency to be partly broken and they lost their rigidity when raw sewage was fed following synthetic sewage. Besides, Methanosaeta related species prevailing in seed sludge have noticeably lost their long filamentous forms and deteriorated during raw sewage feeding. Members of the order Methanobacteriales constituted the major hydrogenothrophic methanogens present in the psychrophilic UASB reactor, whereas the other hydrogenothrophic methanogens--members of the order Methanococcales and Methanogenium relatives--were absent.

Thermophilic anaerobic digestion in compact systems can be an economical and ecological reasonable decentralised process technique, especially for rural areas. Thermophilic process conditions are important for a sufficient removal of pathogens. The high energy demand, however, can make such systems unfavourable in terms of energy costs. This is the case when low concentrated wastewater is treated or the system is operated at low ambient temperatures. In this paper we present experimental results of a compact thermophilic anaerobic system obtained with fluorescent in situ hybridisation (FISH) analysis and mathematical simulation. The system was operated with faecal sludge for a period of 135 days and with a model substrate consisting of forage and cellulose for a period of 60 days. The change in the microbial community due to the two different substrates treated could be well observed by the FISH analysis. The Anaerobic Digestion Model no. 1 (ADM1) was used to evaluate system performance at different temperature conditions. The model was extended to contribute to decreased methanogenic activity at lower temperatures and was used to calculate energy production. A model was developed to calculate the major parts of energy consumed by the digester itself at different temperature conditions. It was demonstrated by the simulation study that a reduction of the process temperature can lead to higher net energy yield. The simulation study additionally showed that the effect of temperature on the energy yield is higher when a substrate is treated with high protein content.