Effects of enrichment on protist abundances and bacterial composition in simple microbial communities

We experimentally investigated effects of nutrient enrichment and trophic structure in a microbial food web consisting of mixed bacteria, two bacterivorous ciliates ( Tetrahymena sp. and Colpidium sp.) and an omnivorous ciliate ( Blepharisma sp.) feeding on both trophic levels. We assembled all possible food webs including one or more of the ciliate species and cross-classified them with four levels of enrichment of the bacterial medium. The qualitative outcome of food web interactions was independent of enrichment and always the same: Tetrahymena strongly depressed or excluded Colpidium , and Blepharisma strongly depressed or excluded both bacterivores. Consequently, in all sub-webs only the dominant ciliate species responded positively to enrichment. The total density of bacteria increased with enrichment irrespective of food web composition. In contrast, the response of single-celled bacteria to enrichment depended on food web composition and was only weakly positive in most food webs with the omnivore. Enrichment had a positive effect on the relative success of (presumably more defended) bacterial aggregates. The outcome of interspecific interactions among ciliates could not be predicted from monoculture experiments and deviated from earlier experiments in which each bacterivore coexisted separately with the omnivore. As a potential explanation we suggest that changes in experimental protocol reduced spatial heterogeneity and increased attack rates. A simple, dynamical model shows that increased attack rates can indeed greatly decrease the upper limit and range of enrichment over which intermediate consumers can coexist with omnivores.     

Protists with different feeding modes change biofilm morphology

The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders), Spumella , and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder Dexiostoma campylum did not alter biofilm volume and porosity but stimulated the formation of larger microcolonies compared with ungrazed biofilms. In contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the biofilm surface, leading to smaller microcolonies and lower maximal and basal layer thickness compared with ungrazed biofilms. Microcolony formation was not stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped bacteria from the outer surface leading to mushroom-shaped microcolonies. In the presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times lower compared with ungrazed biofilms. However, the biofilm porosity and the ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times higher, respectively. Thus, exchange of nutrients and gases between the biofilm and its surrounding fluid should also be improved in deeper biofilm layers, hence accelerating microbial growth.     

Packaging of live Legionella pneumophila into pellets expelled by Tetrahymena spp. does not require bacterial replication and depends on a Dot/Icm-mediated survival mechanism

The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulse-chase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication. Pellets of virulent L. pneumophila may contribute to the transmission of Legionnaires' disease, an issue currently under investigation.     

Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective mechanism, developmental stage and protozoan feeding mode

In a previous study we identified microcolony formation and inhibitor production as the major protective mechanisms of Pseudomonas aeruginosa biofilms against flagellate grazing. Here we compared the efficacy of these two key protective mechanisms by exposing biofilms of the non-toxic alginate overproducing strain PDO300 and the wild-type toxic strain PAO1 to a range of feeding types commonly found in the succession of protozoans associated with natural biofilms. Alginate-mediated microcolony formation conferred effective protection for strain PDO300 against the suspension feeding flagellate Bodo saltans and, as reported earlier, the surface feeding flagellate Rhynchomonas nasuta, both of which are considered as early biofilm colonizers. However, microcolonies of mature PDO300 biofilms were highly susceptible to late biofilm colonizers, the surface-feeding amoeba Acanthamoeba polyphaga and the planktonic ciliate Tetrahymena sp., resulting in a significant reduction of biofilm biomass. Mature biofilms of strain PAO1 inhibited growth of flagellates and A. polyphaga while the grazing activity of Tetrahymena sp. remained unaffected. Our findings suggest that inhibitor production of mature P. aeruginosa biofilms is effective against a wider range of biofilm-feeding predators while microcolony-mediated protection is only beneficial in the early stages of biofilm formation.     

Peristance of bacteria in the presence of viable,nonencysting, bacterivorous ciliates

Laboratory studies of the interactions between a bacterial population and a population of bacterivorous ciliates consistently show that the bacteria are able to persist in the presence of viable ciliates. Reproduction of the bacteria, presumably at the expense of substrates produced by death and lysis of the ciliates and/or by their metabolic activity, has been suggested to be a factor involved in the observed bacterial persistence. Rates and extents of growth ofEscherichia coli in broths of mixed cultures of this bacterium and the ciliateTetrahymena pyriformis were determined in order to provide some data necessary to assess the importance of the suggested factor. In addition, an attempt was made to suppress bacterial growth on produced substrates so that feeding of the ciliates could be studied free of this complication. However, the procedure tested—addition of the antibiotic chloramphenicol (CM) at a concentration of 150g/ml—led to other complications that made it impossible to obtain the desired information about feeding.     

Role of quorum sensing and antimicrobial component production by Serratia plymuthica in formation of biofilms, including mixed biofilms with Escherichia coli

We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.     

Ciliates are the Dominant Grazers on Pico- and Nanoplankton in a Shallow,Naturally Highly Eutrophic Lake

Abundance and biomass of the microbial loop members [bacteria, heterotrophic nanoflagellates (HNF), and ciliates] were seasonally measured in the naturally eutrophic and shallow (2.8 mean depth) Lake Võrtsjärv, which has a large open surface area (average 270 km²) and highly turbid water (Secchi depth <1 m). Grazing rates (filter feeding rates) on 0.5-, 3-, and 6-μm-diameter particles were measured to estimate pico- and nanoplankton grazing (filter feeding) by micro- and metazooplankton. Among grazers, HNF had a low abundance (<50 cells mL^?1) and, due to their low specific filtering rates, they only grazed a minor fraction of the bacterioplankton (≤4.2% of total grazing). Ciliates were relatively abundant (≤158 cells mL^?1) and, considering their high specific feeding rates, were able to graze more than 100% of the bacterial biomass production in the open part of the lake, whereas the average daily grazing accounted for 9.3% of the bacterial standing stock. Ciliates were potentially important grazers of nanoplanktonic organisms (on average, approximately 20% of the standing stock of 3-μm-size particles was grazed daily). Metazooplankton grazed a minor part of the bacterioplankton, accounting for only 0.1% of standing stock of bacteria. Grazing on nanoplankton (3–6 μm) by metazooplankton was higher (0.4% of standing stock). The hypothesis is proposed that ciliates dominate due to a lack of top–down regulation by predators, and HNF have a low abundance due to strong grazing pressure by ciliates.     

Kinetics of growth of the ciliate Tetrahymena pyriformis on Escherichia coli

The growth of the ciliate Tetrahymena pyriformis on non-growing Escherichia coli has been studied by following the time courses of population densities and protozoan mean cell volume in batch cultures. Viable, non-encysted protozoa always stopped feeding before the bacterial density was reduced to zero and non-feeding ciliates tended to swim faster than feeding ciliates. In addition, the number of bacteria and other particles of bacterial size consumed in the formation of one new ciliate, when averaged over the lag and reproductive phases of a culture, declined toward a limiting value of about 1.6 x 10(4) particles per ciliate as the initial density of such particles was increased.     

Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non‐photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.     

Enhanced survival of Salmonella enterica in vesicles released by a soilborne Tetrahymena species

Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of Salmonella enterica serovar Thompson with a soilborne Tetrahymena sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of Tetrahymena at several ratios. During incubation with serovar Thompson, Tetrahymena cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on Listeria monocytogenes cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to Tetrahymena cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (F = 28.3, P < 0.001) and unstarved (F = 14.09, P < 0.005) Tetrahymena cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health.     

Factors affecting preference responses of the freshwater ciliate Uronema nigricans to bacterial prey

ABSTRACT. To enhance our understanding of the factors affecting feeding selectivity of bacterivorous protists in aquatic systems, we examined the preference responses of the freshwater ciliate Uronema nigricans towards three bacterial prey taxa, Pseudomonas luteola, Serratia rubidaea , and Aeromonas hydrophila . Potential factors influencing the predator–prey contact rate included the previous feeding history of the ciliate and physiological state of bacteria. Preference indexes were obtained from multiple-choice mazes in which ciliates moved preferentially towards alternative bacteria or the prey species on which they had been feeding. Uronema nigricans showed differential attraction towards the offered prey types, and these preferences varied as a function of the ciliate feeding history: U. nigricans growing on P. luteola showed lower preference responses towards the offered bacteria than U. nigricans growing on S. rubidaea . The bacteria in stationary phase elicited a higher degree of attraction than bacteria in exponential phase, probably due to a higher concentration of carbohydrates in the former. Therefore, this protist will preferentially swim towards bacteria in stationary growth phase, although the degree of this response will be affected by the recent feeding history of the ciliate.     

Role of anaerobic ciliates in planktonic food webs: abundance, feeding, and impact on bacteria in the field

We studied the dynamics of two populations of anaerobic ciliates, Plagiopyla sp. and Metopus sp., and of their potential prey, heterotrophic and phototrophic purple bacteria, in Lake Cisó throughout a 1-year cycle. The abundance of both ciliates was very low (less than 2 individuals per ml). During mixing, Plagiopyla ciliates exhibited high clearance rates (about 100 nl ciliate h), its integrated abundance increased with a net doubling time of 47 days, and its potential doubling times, as calculated from the number of bacteria consumed, ranged between 5 and 8 days. During stratification, the activity of Plagiopyla ciliates was reduced and the population decreased; this was related to the higher amounts of sulfide present. The impact of predation by the Plagiopyla population on bacterioplankton was found to be insignificant, less than 0.1% of bacterial biomass consumed per day. Thus, anaerobic ciliates cannot control the bacterioplankton in Lake Cisó because of both the low abundance over the period studied and the low feeding rates during certain periods. A review of available field studies suggests that this conclusion can be extrapolated to most other anoxic systems.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Using a green fluorescent protein gene-labeled p-nitrophenol-degrading Moraxella strain to examine the protective effect of alginate encapsulation against protozoan grazing

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.     

A Fossilized Microcenosis In Triassic Amber

ABSTRACT Detailed data on bacterial and protistan microfossils are presented from a 0.003 mm³ piece of Triassic amber (Schlierseerit, Upper Triassic period, 220-230 million years old). This microcenosis, which actually existed as such within a very small, probably semiaquatic habitat, included the remains of about two bacteria species, four fungi ( Palaeodikaryomyces baueri, Pithomyces -like conidia, capillitium-like hyphae, yeast cells) two euglenoids, two chlamydomonas ( Chlamydomonas sp., Chloromonas sp.), two coccal green microalgae ( Chlorella sp., Choricystis -like cells), one zooflagellate, three testate amoebae ( Centropyxis aculeata var. oblonga-like, Cyclopyxis eurystoma -like, Hyalosphenia baueri n. sp.), seven ciliates ( Pseudoplaryophrya nana -like, Mykophagophrys terricola -like, Cyrtolophosis mucicola -like, Paracondylostoma sp., Bryometopus triquetrus -like. Tetrahymena rostrata -like, Paramecium triassicum n. sp.) the microfossils correspond to or diverge from extant species only slightly.     

Effects of temperature, sulfide, and food abundance on growth and feeding of anaerobic ciliates

The trophic role of ciliates in anaerobic food webs has not been assessed experimentally. In order to obtain basic information necessary to interpret field situations, we studied the effects of temperature, sulfide concentration, and food abundance on the growth and feeding activities of two anaerobic ciliates, Plagiopyla nasuta and Metopus es. The growth rate of P. nasuta increased with temperature from 8 to 18 degrees C (Q(10) = 2.0) and remained constant in the range between 18 and 24 degrees C (0.22 day). Sulfide concentrations of between 0 and 1 mM did not affect the feeding activities, but concentrations greater than 2 mM were inhibitory. The functional response of P. nasuta feeding on fluorescently labeled heterotrophic and phototrophic bacteria was investigated. In both cases, the parameters of the functional response were almost identical when expressed in terms of biovolume: the maximal uptake rate (U(m)) was 1,800 mum ciliate h and the half-saturation constant for ingestion (k) was 1.5 x 10 mum ml. The functional response of M. es feeding on heterotrophic bacteria was found to be similar to that of P. nasuta. These ciliates needed high bacterial abundances in order to maintain their growth (k of about 4 x 10 bacteria ml), implying that they will frequently be food limited in planktonic environments. Both the maximal uptake rates and the maximal clearance rates were comparable to those of aerobic ciliates. By combining the growth and feeding data, we estimated gross growth efficiencies of 12 and 13% for P. nasuta and M. es, respectively. These results indicate that the feeding rates of anaerobic ciliates are similar to those of aerobic ciliates. Their slower growth must, therefore, be due to the lower gross growth efficiency (likely due to anaerobic metabolism).     

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).     

Modeling the Individual Growth of Tetrahymena Sp. and its Population Consequences

ABSTRACT. Three types of mathematical growth models are presented to describe the individual growth of the ciliate Tetrahymena sp. feeding on the bacterium Pseudomonas fluorescens . Both organisms were isolated from a domestic waste-water treatment plant. Growth of individual ciliates and the consequences for the whole population are considered. Experimental data, obtained by following the individual ciliate during its lifespan from cell division to cell division, are used for parameter estimations. Differences between growth models for individuals turn out to have little effect on the specific population growth rate and the mean cell volume. In case of exponential growth of individuals the unstructured and structured population models are equivalent, even in time-variant environments. This knowledge can be applied in the stability analysis of food chains or forced systems. The results obtained facilitates quantification of protozoa biomass as a function of bacterial biomass in chemostats. More specifically, it highlights the dynamic behaviour of bacteria and protozoa in waste-water treatment plants.     

Aqueous two-phase system-derived biofilms for bacterial interaction studies

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a β-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.