Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Developing an ecologically relevant heterogeneous biofilm model for dental-unit waterlines.

This study monitored the biodiversity of microbes cultured from a heterogeneous biofilm which had formed on the lumen of a section of dental waterline tubing over a period of 910 days. By day 2 bacterial counts on the outlet-water showed that contamination of the system had occurred. After 14 days, a biofilm comparable to that of clinical waterlines, consisting of bacteria, fungi and amoebae had formed. This showed that the proprietary silver coating applied to the luminal surface of the commercial waterline tubing failed to prevent biofilm formation. Molecular barcoding of isolated culturable microorganisms showed some degree of the diversity of taxa in the biofilm, including the opportunistic pathogen Legionella pneumophila. Whilst the system used for isolation and identification of contaminating microorganisms may underestimate the diversity of organisms in the biofilm, their similarity to those found in the clinical environment makes this a promising test-bed for future biocide testing.

Risk Assessment for the Spread of Serratia marcescens Within Dental-Unit Waterline Systems Using Vermamoeba vermiformis.

Vermamoeba vermiformis is associated with the biofilm ecology of dental-unit waterlines (DUWLs). This study investigated whether V. vermiformis is able to act as a vector for potentially pathogenic bacteria and so aid their dispersal within DUWL systems. Clinical dental water was initially examined for Legionella species by inoculating it onto Legionella selective-medium plates. The molecular identity/profile of the glassy colonies obtained indicated none of these isolates were Legionella species. During this work bacterial colonies were identified as a non-pigmented Serratia marcescens. As the water was from a clinical DUWL which had been treated with Alpron™, this prompted the question as to whether S. marcescens had developed resistance to the biocide. Exposure to Alpron™ indicated that this dental biocide was effective, under laboratory conditions, against S. marcescens at up to 1 × 10(8) colony forming units/millilitre (cfu/ml). V. vermiformis was cultured for 8 weeks on cells of S. marcescens and Escherichia coli. Subsequent electron microscopy showed that V. vermiformis grew equally well on S. marcescens and E. coli (P = 0.0001). Failure to detect the presence of S. marcescens within the encysted amoebae suggests that V. vermiformis is unlikely to act as a vector supporting the growth of this newly isolated, nosocomial bacterium.

Cryopreservation of resting cysts of the freshwater ciliate Meseres corlissi by conventional two-step methods and one-step vitrification protocols.

Trophic cells of the freshwater ciliate Meseres corlissi CCAP 1647/1 proved to be recalcitrant to all the cryopreservation methods tested; however, resting cysts of this strain were amenable to both conventional two-step cryopreservation and ultra-rapid vitrification methods. Conventional controlled rate cooling and Mr. Frosty cooling methods, employing 5 percent dimethylsulphoxide (DMSO) as a cryoprotectant were effective in preserving cysts in either liquid medium or soil suspensions. Alternative cryopreservation methods involving dehydration of soil/cyst suspensions, in the absence of any colligitative cryoprotectant, and rapid cooling over liquid nitrogen or plunge freezing were effective. The level of residual moisture was the critical factor with no survival observed in any samples with > 35 percent residual moisture, and high levels of survival (> 50 percent) in samples with < 14 percent residual moisture. Trophic cells obtained from cryopreserved cysts appeared healthy and did not differ obviously from the controls with respect to morphology, movement, division and encystment/excystment reactions. In a test culture derived from material which had been cryopreserved by the rapid cooling method a maximum growth rate of 1.32 d(-1) (at 22 degree C) was determined, a value which agrees well with earlier observations on the original strain.

Isolation of clonal cultures of endosymbiotic green algae from their ciliate hosts.

Using Paramecium bursaria as a model organism improved protocols have been developed to isolate clonal endosymbiotic algae. This involved micromanipulation of individual protists, rupturing to release endosymbionts followed by enrichment on complex media and a series of plating steps, under low light (PAR ~10µmol photons m(-2)s(-1)).

Early detection of protozoan grazers in algal biofuel cultures.

Future micro-algal biofuels will most likely be derived from open-pond production systems. These are by definition open to "invasion" by grazers, which could devastate micro-algal mass-cultures. There is an urgent requirement for methodologies capable of early detection and control of grazers in dense algal cultures. In this study a model system employing the marine alga Nannochloropsis oculata was challenged by grazers including ciliates, amoebae and a heterotrophic dinoflagellate. A FlowCAM flow-cytometer was used to detect all grazers investigated (size range <20->80 µm in length) in the presence of algae. Detection limits were <10 cells ml(-1) for both "large" and "small" model grazers, Euplotes vannus (80 × 45 µm) and an unidentified holotrichous ciliate (~18 × 8 µm) respectively. Furthermore, the system can distinguish the presence of ciliates in N. oculata cultures with biotechnologically relevant cell densities; i.e. >1.4 × 10(8) cells ml(-1) (>0.5 g l(-1) dry wt.).

Tolerance of the resting cysts of Colpoda inflata (Ciliophora, Colpodea) and Meseres corlissi (Ciliophora, Spirotrichea) to desiccation and freezing.

The survival of ciliate resting cysts, in the presence and absence of soil, was studied under two environmental stresses: desiccation and freezing. Laboratory strains of the common species Colpoda inflata and the rare species Meseres corlissi were used in these experiments, which yielded the following results: 1) Freezing of cysts in soil with a residual moisture level exceeding approximately 30% was destructive for both species. 2) Survival of Meseres corlissi cysts depended largely on the presence of soil. 3) In the absence of soil, Colpoda inflata cysts had greater tolerance to desiccation and freezing than Meseres corlissi cysts. Possible consequences for the distribution of natural populations are discussed.