Ultrastructure of Spironucleus barkhanus N. Sp. (Diplomonadida: Hexamitidae) from Grayling Thymallus thymallus (L.) (Salmonidae) and Atlantic Salmon Salmo salar L. (Salmonidae)

ABSTRACT. A hexamitid flagellate Spironucleus barkhanus n. sp., from the lumen of the gut and gall bladder of wild grayling Thymallus thymallus , and from muscle abscesses of farmed Atlantic salmon Salmo salar from Norway, is described by light, scanning and transmission electron microscopy. The flagellate was axenically cultured in trypticase, yeast extract, iron serum medium. Live trophozoites from axenic cultures incubated at 5° C, measure 11–20 x 6–14 μm. The flagellates show a typical bi-radial symmetry. Each recurrent flagellum is almost completely surrounded by a striated lamina. In the posterior end the lamina widens, appearing heart shaped in transverse section. Accompanying each recurrent flagellum are three narrow bands of microtubules, following the longitudinal groove created by the incomplete closure of the striated lamina. The recurrent flagella emerge posterio-medially through cytostome openings halfway surrounded by crescent-shaped ridges, oriented in opposite directions in the two openings. The position and adornment of the cytostome openings, and the arrangement and number of the microtubules accompanying the recurrent flagella, distinguish Spironucleus barkhanus n. sp. from previously described species of Spironucleus .     

Spironucleus vortens (Diplomonadida) in the Ide,Leuciscus idus (L.) (Cyprinidae): a warm water hexamitid flagellate found in northern Europe

The hexamitid flagellate Spironucleus vortens, previously reported from Pterophyllum scalare from Florida, was found in the intestine of Leuciscus idus in Norway. The flagellate was cultivated and studied by scanning and transmission electron microscopy. Identification was based on a suite of ultrastructural features unique for S. vortens: compound lateral ridges, a swirled posterior end, and a distinctive microtubular cytoskeleton. Microfibrillar structures with a periodicity of 0.13 microm in the right peripheral part of the compound lateral ridges were shown to be responsible for the distinctive rope-like appearance of the peripheral ridge seen in scanning electron micrographs, and not previously reported for S. vortens. The present results show a wide geographic distribution and a wide temperature tolerance for S. vortens. The flagellate was successfully cultivated at 5 degrees C and 15 degrees C, having previously been cultivated between 2-34 degrees C. Spironucleus vortens is believed to be endemic in Norwegian waters, but an introduction hypothesis is also discussed. The similarity is striking between S. vortens and S. elegans, previously described from amphibians and fish in Europe, and the possibility of conspecificity is believed to be high.     

External morphology of the egg of the pink scavenger caterpillar,Pyroderces rileyi (Wallsingham) (Lepidoptera : Cosmopterigidae)

The egg of the pink scavenger caterpillar, Pyroderces rileyi (Walsingham), (Lepidoptera : Cosmopterigidae) was studied by scanning electron microscopy. The egg is subcylindrical to fusiform (0.43 × 0.23 mm), truncate at the anterior end and rounded at the posterior. Surface sculpturing consists of longitudinal and transverse ridges of about equal prominence. The anterior end has the form of a circular depression, bearing a rosette of short, petal-like primary cells on a slight elevation at its center. The micropylar canals open into a pit at the center of the rosette. The rosette is surrounded by a row of long, wedge-shaped secondary cells that extend outward to the rim of the depression. The depression is outlined by a prominent ridge that joins the anterior ends of the longitudinal ridges. Aeropyles occur at the anterior end along this ridge, mostly at ridge intersections, and at ridge intersections on the posterior end. The openings of the anterior aeropyles are surrounded by heavy raised collars. Those of the posterior aeropyles may or may not have collars.     

Transcriptional diversity in myogenesis.

Duodenal morphogenesis in the chick embryo has been studied to see if changes in organization of extracellular fibers are associated with villus formation. Three major processes occur during morphogenesis; longitudinal ridges form, then these ridges become zigzag in shape, and finally a double row of villi form from each zigzag ridge. Tissues of different developmental stages were progressively trypsinized to remove cellular material and were prepared for scanning microscopy to show the organization of extracellular fibers. Results show that fiber systems of increasing complexity form as the dudoenum develops, and suggest that some cellular events such as initial ridge formation precede these changes. Tissues with longitudinal ridges contain only randomly organized fibers. In tissues with zigzag ridges, oriented fibers appear along the ridges and some extend laterally from flexures of each zigzag ridge, but interridge fibers are still randomly organized. When villi form, fibers in the body of the villus are random but fibers at the base of villi and between villi are highly oriented. Transmission microscopy and collagenase treatment of partially trypsinized tissue indicate that most, if not all extracellular fibers viewed by scanning microscopy are collagenous. Therefore, since collagen fiber organization is so closely related to morphogenetic changes, we presume it plays an important role.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Quantitative and qualitative studies of the bacterial microflora of turbot, Scophthalmus maximus L., gills

Populations of aerobic heterotrophic bacteria, occurring on the gills of healthy turbot, were estimated using a dilution plate technique. From a comparison of 18 media, the highest counts, i.e. 7.0 × l0⁵ g⁻¹, were obtained after incubation at 15–25°C on a specifically formulated medium which contained low quantities of beef extract, casein, tryptone and yeast extract. These bacteria were equated with Asticacaulis sp., Hyphomicrobium sp., Janthinobacterium lividum, Prosthecomicrobium sp., Pseudomonas fluorescens and Vibrio sp. Evidence from scanning electron microscopy pointed to a general lack of intimate colonization of exposed areas of the gill. Instead, micro-organisms colonized protected niches, such as the clefts between lamellae and in secluded areas on the arches.     

Culture and Phylogenetic Characterization of Trichomitus trypanoides Duboscq & Grassè 1924, n. comb.: a Trichomonad Flagellate Isolated from the Hindgut of the Termite Reticulitermes santonensis Feytaud

ABSTRACT. A trichomonad flagellate strain R1 was isolated from the hindgut contents of the termite Reticulitermes santonensis Feytaud. The flagellate was cultivated at 28° C in anaerobic medium containing yeast extract, minerals and vitamins. The isolate fed on living bacteria. It showed the typical morphological and ultrastructural features of the trichomonads. closely resembling Trichomitus trypanoides. In order to determine its phylogenetic position the small subunit ribosomal DNA (SSU rDNA) of the flagellate was amplified in vitro using the polymerase chain reaction (PCR), cloned in a plasmid vector and sequenced. Comparison of the obtained sequence with so far available SSU rRNA/rDNA sequences showed strongest similarity (89%) to the sequence of Tritrichomonas foetus. The phylogenetic analysis with parsimony and distance matrix methods placed Trichomitus trypanoides strain R1 near by the root of the phylogenetically so far analyzed eukaryotic organisms. This confirms that termites harbour hindgut symbionts, which originate from very early evolved eukaryotes.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

The Production of a Yeast Killer Factor in the Chemostat and the Effects of Killer Yeasts in Mixed Continuous Culture with a Sensitive Strain

In batch culture in a complex medium the killer yeasts NCYC 738 and NCYC 235 gave maximal killer activity when grown in the pH ranges 4·2–4·4 and 4·6–4·8 respectively. Incubation of culture filtrates of NCYC 738 for 10 h at 25 °C or 2 h at 29 °C resulted in a 50% reduction in activity. The addition of bovine serum albumin or gelatine to a complex medium stabilized killer activity. In a defined medium the addition of yeast extract stimulated the production of killer activity. When killer yeast NCYC 738 was grown in a chemostat, killer activity was influenced by temperature, pH and the rate at which the culture was stirred. The production of killer activity was growth-linked and increased as dilution rate was raised to a maximum of 0·15 h"1. Steady state continuous cultures of the sensitive strain, NCYC 1006, were contaminated deliberately with either killer or killer-cured strains. During the first 30 h cultivation, the cell concentration of both strains increased. Subsequently the sensitive strain was displaced from the culture. When killer-cured NCYC 738 was added, the rate of displacement was proportional to the culture temperature. However, with killer NCYC 738 increase of temperature reduced the rate of displacement. When killer NCYC 235 was employed, a lowering of pH decreased the rate of displacement but had no effect when killer-cured NCYC 235 was used.     

Multiple loop purification method for selective cultivation of Pentatrichomonas hominis

It is always troublesome having protozoan cultures contaminated with other organisms in the laboratory. The method described here produces high efficiencies of purification for fast moving flagellate protozoa. A human strain Pentatrichomonas hominis was employed in the study to examine the effects of multiple loop tubes on the purification of flagellates. Trichomonads were harvested from a trypticase yeast extract iron-serum-33 (TYI-S-33) medium, adjusted to 2 X 10(5) organisms/ml, and mixed with an equal volume of 2 X 10(6) organisms/ml of bacteria. The isolation was performed at 37 degrees C in TYI-S-33 medium containing a suitable amount of antibiotics (1000 U/ml of penicillin, 1000 micrograms/ml of streptomycin, and 4 micrograms/ml of fungizone). Four days later, 10(6) organisms/ml of protozoa, free of bacteria, were observed at the other end of the single loop and the double loop tubes. About the same amount of flagellates could be found at the other end of the triple loop tube six days after incubation. The traditional U-shaped tubes were used as controls and 10(5) cells/ml of flagellates were recovered in the presence of bacteria two days after incubation. An axenic culture of P. hominis was successfully isolated from the feces of a Formosan rock-monkey, Macaca cyclopsis, by this method. Purified trichomonads were recovered from a double loop purification tube five days after incubation.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

The influence of pH and temperature on initiation of growth of Salmonella spp.

The ability of 13 strains of Salmonella , representing 12 serotypes, to grow in a tryptone-yeast extract-glucose medium, acidified with HC1 to pH values between 3.80 and 5.60 at intervals of 0.20 units, has been investigated. During incubation at 30°C, growth occurred at minimum pH values of 3.8–4.0 in 1–3 d. At 20°C, growth occurred at minimum pH values of 3.8–4.2 in 3–5 d. In tests incubated at 10°C, growth occurred at minimum pH values of 4.4–4.8 in between 10 and 19 d.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

Scanning electron microscopy of spores of the myxosporidans Chloromyxum trijugum Kudo and Chloromyxum catostomi Kudo

Spores of Chloromyxum trijugum Kudo from Lepomis macrochirus Rafinesque and Chloromyxum catostomi Kudo from Notropis dorsalis (Agassiz) were obtained from infected gall bladders, glass-bead sonicated, and examined by scanning electron microscopy. Valves of C trijugum spores each have a thick ridge running parallel to the sutural ridge. Uncapped cnidocyst pores open into the extrasutural ridges. A pyriform structure of unknown function was observed at the posterior surface in some spores. Spore valves of C catostomi are sculptured with ridged striations running in various parallel and converging patterns over the entire surface. Cnidocyst pores open into ridges adjacent to the sutural plane. Glass bead sonication was found effective in polar filament extrusion. Discharged filaments were twisted along the long axis and partially coated with mucoid globules.     

The Fine Structure of Rhynchocystis pilosa (Sporozoa, Eugregarinida)

SYNOPSIS. The trophozoite of Rhynchocystis pilosa obtained from the seminal vesicles of the earthworm Lumbricus terrestris was studied by light and electron microscopy. The trophozoite's cortical organization is particularly interesting because of its unusual evaginations and associated fibrillar structures. The pellicle is formed by 2 concentric membranes elevated into 60–70 alternating primary and secondary ridges extending posteriad. Numerous long ‘hairs’ or cytopilia originate along the primary ridges and each contains a system of fibrils originating from an underlying longitudinal myoneme. Longitudinal rows of pores lie between adjacent pollicular ridges. Three systems of fibrils lie in the cortex of the trophozoite. A longitudinal myoneme consisting of 12–18 fibrils lies below each primary pellicular ridge. Circular myonemes lie below the pellicle in a parallel array along the length of the organism. Each myoneme consists of 4–8 fibrils structurally similar to those of the longitudinal myonemes. Pairs of fine filaments also lie in the inner pellicular membrane along the apex of each ridge. The trophozoite's anterior end is modified as an attachment organelle consisting of 30–35 delicate pellicular folds which originate at the base of an anterior papilla. The folds extend approximately 15 μ posteriad where they become continuous with the primary pellicular ridges. The nucleus lies in the cytoplasm near the posterior level of the attachment organelle and is surrounded by a double membrane perforated by numerous pores. The cytoplasm contains numerous small vesicles which may be found in dense aggregations. These aggregations often occur in proximity to Golgi complexes and certain membrane-bound bodies. Mitochondria are abundant in the cytoplasm as are large, ovoid paraglycogen bodies. Occasionally layers of granular membranes are arranged parallel to the surface of the paraglycogen bodies but also occur thruout the cytoplasm.     

Growth of Particle-Bearing and Particle-Free Paramecium aurelia in Axenic Culture*

SYNOPSIS. Several strains of particle-bearing and particle-free Paramecium aurelia have been cultivated in an axenic medium composed of proteose peptone, trypticase, yeast nucleic acid, MgSO⁴.7H²O, TEM-4T (diacetyl tartaric acid esters of tallow monoglycerides), stigmasterol and a mixture of vitamins. The “yeast fraction,” an indispensable component of previous media used for the cultivation of these ciliates has been replaced by a mixture of trypticase, yeast nucleic acid and TEM-4T. Particle-bearing animals of stock 299 lambda, 138 mu, and 139 pi maintain their particles when cultivated in the medium, whereas particle-bearing animals of stock 51 kappa, 225 kappa and 114 signia do not. With the exception of stock 92 (syngen 3) the medium appears to be selective in its ability to support the growth of animals of the even- but not odd-numbered syngens of P. aurelia. Maintenance of the particles was dependent only to a small degree upon environmental conditions brought about by changes in pH and temperature. Division of the particles was found to be comparable with the division of the protozoan. Methods for the growth, maintenance and mass cultivation of particle-bearing P. aurelia are given in detail.     

Laboratory growth of larval lampreys (Lampetra (Entosphenus) tridentata Richardson) at different food concentrations and animal densities

Lampreys are important research animals. This study investigates some of the Parameters important for culturing the Suspension feeding larvae: food concentration, temperature and crowding. Large larvae ( Lampetra ( Entosphenus ) tridentata Richardson) were used, weigh-ing from l·5 to 3·0 g (wet). Two food types were employed: suspended yeast cells ( Saccharo-myces cerevisiae , 0–20 mg1 – (dry), or, in a few tests, a fine particulate fish food, Liquifry® (Interpet LTD, 0–13 mg l^-1). At both 14 and 4°C, yeast could sustain weight increases comparable to those in nature: >6% month^-1 for up to 6 months, the duration of the study. In a single lest, a vitamin Supplement failed to improve growth on yeast. Growth-was fastest at 14°C (+41% month^-1, max. weight increase), although also substantial at 4°C (+11% month^-1, max). Growth could not be sustained at 20°C, due perhaps to difficulty in removing products of food decay from the aquaria. Food level being constant, growth rate varied inversely with animal density. It is suggested that larval lampreys release a growth-inhibiting substance into the sand which they inhabit. Overall, the best growth was obtained at 14° C, with <0·05g of animal (wet weight) – aquarium water and average daily yeast concentrations between 4 and 13 mg –. Liquifry was associated with lowered growth rates when present continually above 4 mg (dry weight) – (14° C), although growth did occur at lower concentrations.     

A thermotolerant and high acetic acid-producing bacterium Acetobacter sp. I14–2

A thermotolerant bacterium with high production of acetic acid was isolated from spoiled banana in Taiwan. The isolate, I14–2 ,was considered to be an Acetobacter sp. according to phenotypic and chemotaxonomic characteristics. Optimal cultural conditions for Acetobacter sp. I14–2 to produce acetic acid were studied under cultivation in a medium containing 2 mg l⁻¹ acetic acid and 5% ethanol at 30 °C. Acetic acid productivity by Acetobacter sp. I14–2 was almost two and three times the amount produced by Acet. aceti IFO3283 and Acetobacter sp. CCRC 12326, respectively. The isolate retained 22% residual acetic acid-producing activity after 3 d incubation in a medium containing 8% ethanol, and produced acetic acid in a medium containing 10 g l⁻¹ acetic acid. This bacterium is thermotolerant and retained 97% and 68% of acetic acid-producing activity after 3 d incubation at 35 °C and 37 °C, respectively, compared with that when incubated at 30 °C.