Bdellovibrio bacteriovorus Parasitizing Rhizobium in Western Australia

S ummary . Bdellovibrio bacteriovorus strains which parasitize Rhizobium meliloti, R. trifolii, Agrobacterium tumefaciens and A. radiobacter have been found in Western Australian soils. Rhizobium lupini was not lysed by any strain. Some Bdellovibrio cells pass through 0·22 μm membrane filters, whilst the 0·45 μm filters allow several saprophytic soil bacteria to pass. The strength of the medium is shown to be of great importance for the detection or counting of Bdellovibrio cells. The Bdellovibrio strains are capable of destroying large numbers of rhizobia in the laboratory.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm⁻² glucose for E. coli and up to 2·7 × 10⁻⁹ mol cm⁻² iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h⁻¹ in a biofilm and μ = 0·96 h⁻¹ in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h⁻¹ and in planktonic culture 0·78 h⁻¹. This method allows the limitation of the size of a biofilm population to a chosen value.     

An assessment of the Sartorius MD8 microbiological air sampler

Tests described in this paper show that gelatine membrane filters used in the MD8 microbiological air sampling system collected monodispersed aerosols between 0·7 and 1·0 μm containing viable Bacillus subtilis var. niger spores, with an efficiency of 99·9995%. Gelatine membrane filters linked to the MD8 control pump system were as effective as the well established Casella slit-to-agar device for collecting some viable bacteria, nebulized under controlled experimental conditions and naturally occurring airborne micro-organisms in a pharmaceutical plant. By using a long flexible hose connection to the control pump, the head could be positioned where sampling was required in locations remote from the pump exhaust, making it suitable for microbiological monitoring in critical locations such as laminar flow stations and isolators.     

Difference in blood and water diffusion distance in gill lamellae of diploid and triploid tench Tinca tinca (L.)

Image analysis of sagittal sections of gill lamellae of diploid and triploid tench Tinca tinca revealed the blood and water diffusion distance in diploids (2·07 μm) to be significantly higher than that of their triploid siblings (1·46 μm; P < 0·01). Lamellae of diploids compared to triploids were found to be significantly shorter (105·84 v. 132·11 μm) and thicker (18·47 v. 14·21 μm; all at P < 0·05) than those of their triploid siblings but with similar mean sectional areas (1965·44 v. 1910·86 μm²).     

A Note on the Diagnosis of Ratoon Stunting Disease of Sugarcane by Negative-Stain Electron Microscopy of the Associated Bacterium

After negatively staining with 1% (w/v) sodium phosphotungstate (pH 6·5) or 1% (w/v) ammonium molybdate (pH 6·5), the cell wall, cytoplasmic membrane and mesosomes of the RSD-associated bacterium obtained from the fibrovascular fluid of infected sugarcane were usually clearly displayed. The cells measured 0·19–0·39 μm (av. 0·27 μm) in width and 0·6–3·4 μm in length. Few mesosomes were visible and the cells were approx. 40% wider (0·27–0·52 μm, av. 0·38 μm) when stained with 1% (w/v) uranyl acetate (pH 3·0–4·2). Freezing and thawing the suspension before negative staining with sodium phosphotungstate did not greatly affect the size of the cells or resolution of the mesosomes. Glycine (0·25 M) as the suspending medium, fixation in 2% (w/w) glutaraldehyde, or placing wet instead of dry specimen grids in the electron microscope resulted in wider cells usually lacking mesosomes.     

Myxobolus dahomeyensis infection in ovaries of Tilapia species from Benin (West Africa)

The prevalence of Myxobolus dahomeyensis in ovaries of Tilapia zillii was 31·6% and 18·3% in Sarotherodon melanotheron melanotheron . The parasite induced destruction of the oocytes and host castration. Its ovoid spore was 6·5–12·0 × 6·0–8·0 μm and the polar capsules averaged 3·6 × 2·2 μm ( n = 30).     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Morphology and fine structure of the heart of the burbot, a cold stenothermal fish

The ventricle of the burbot Lota lota heart comprised 0·148 ± 0·006% of the body mass which is nearly two-fold heavier than the relative ventricular mass ( M _V) of other similarly sized teleosts. The shape of the ventricle is pyramidal and the wall is exclusively composed of spongious muscle without a distinct compact layer. The atrium forms 0·017 ± 0·002% of the body mass. Length, width, sarcolemmal surface area and volume of enzymatically isolated myocytes from burbot ventricle were 147·2 ± 10·2 μm, 6·3 ± 0·4 μm, 2440·8 · 251·5 μm² and 2356·8 ± 316·6 μm³, respectively. The myofibrils were peripherally located and their volume density was remarkably high: 65 ± 2 and 68 ± 3% in ventricle and atrium, respectively ( P >0·05). Although not particularly conspicuous, some nonjunctional and junctional sarcoplasmic reticulum (SR) was present in both atrial and ventricular myocytes. The SR formed peripheral couplings with the sarcolemma and the junctional clefts were frequently occupied by foot processes. These findings suggest that cold-adaptation is achieved by cardiac enlargement, high volume density of myofibrils and well-developed peripheral couplings in the SR in the heart of stenothermal burbot.     

Ultrastructural observations of euspermatozoa and paraspermatozoa in a copulatory cottoid fish Blepsias cirrhosus

Euspermatozoa and paraspermatozoa of a copulatory (internal insemination with external sperm transfer) cottoid fish Blepsias cirrhosus were observed ultrastructurally. Euspermatozoa of B. cirrhosus consisted of an acrosome‐less, thin, disk‐like sperm head (1·6-2·0 μm in length and 1·3-1·6 μm in width), a long middle piece, and a long flagellum ( c . 30 μm). Aberrant spermatids, which were rich in cytoplasm and possessed two nuclei, occurred in testicular lobules. They were also observed in semen and were round (5·0-5·3 μm in diameter) and biflagellate, suggesting that they are released along with euspermatozoa at ejaculation. The nuclei of aberrant spermatids developed into masses of highly electron‐dense globules. Judging from their form, nuclear condition, and connection with normal spermatids by intercellular bridges during spermiogenesis, aberrant spermatids of B. cirrhosus are considered hyperpyrenic paraspermatozoa formed by incomplete cytokinesis at the second meiotic division.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0·45 μm membrane filters which were then placed upon appropriate media and incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37°C in 2·5% CO₂ for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia , and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

UPTAKE OF CALCIUM BY SUBCELLULAR FRACTIONS ISOLATED FROM OUABAIN-TREATED CEREBRAL TISSUES

Abstract— Slices of cerebral cortex were incubated in medium containing 0·75 or 2·8 mM ⁴⁵CaCl₂, in the presence or absence of 0·01–0·1 m m -ouabain. Ouabain induced accumulation of calcium by slices to a maximum of 4 μmoles/g of tissue/hr (0·75 m m -CaCl₂ in the medium) and to 8 μmoles/g of tissue/hr (2·8 m m -CaCl₂ in the medium). Accumulation of Ca²⁺ occurred more slowly than loss of K⁺ from the slices and more closely resembled the pattern of Na⁺ uptake.
Mitochondrial fractions isolated from ouabain-treated slices contained significantly more calcium than controls. Inclusion of EDTA in the homogenization medium resulted in decreased amounts of particulate-bound calcium.
The effect of ouabain on accumulation of calcium is discussed with regard to possible relationships to processes of active and passive transport.     

Viable ultramicrocells in drinking water

Aims:  To examine the diversity of cultivable 0·2 micron filtrate biofilm forming bacteria from drinking water systems.
Methods and Results:  Potable chlorinated drinking water hosts phylogenetically diverse ultramicrocells (UMC) (0·2 and 0·1  μ m filterable). UMC (starved or dwarf bacteria) were isolated by cultivation on minimal medium from a flow system wall model with polyvinyl chloride (PVC) pipes. All cultivated cells (25 different isolates) did not maintain their ultra-size after passages on rich media. Cultured UMC were identified by their 16S ribosomal DNA sequences. The results showed that they were closely related to uncultured and cultured members of the Proteobacteria, Actinobacteria and Firmicutes. The isolates of phylum Actinobacteria included representatives of a diverse set of Actinobacterial families: Micrococcaceae, Microbacteriaceae, Dermabacteraceae, Nocardiaceae and Nocardioidaceae.
Conclusions:  This study is the first to show an abundance of cultivable UMC of various phyla in drinking water system, including a high frequency of bacteria known to be involved in opportunistic infections, such as Stenotrophomonas maltophilia, Microbacterium sp., Pandoraea sp. and Afipia strains.
Significance and Impact of the Study:  Chlorinated tap water filtrate (0·2 and 0·1  μ m) still harbours opportunistic micro-organisms that can pose some health threat.     

Effects of bottle size in determinations of primary productivity by phytoplankton

Determinations of primary productivity were made by the radiocarbon method at regular intervals over a year in samples, taken from the eutrophic waters of the Menai Strait, North Wales, and contained in bottles of capacities from 135 ml up to 2290 ml. Fixation per unit volume in particulate matter retained by 0 · 45 m pore size membrane filters was independent of bottle size over most of the year. There was a consistent divergence, however, during the period of rapid phytoplankton growth in the spring, when small bottles gave up to three times higher values than large ones. Possible effects of bottle size on primary productivity determinations are discussed but the cause of this particular result is not apparent.     

The density and dimensions of exchange vessels in Octopus pallidus

The microvasculature of the brachial muscle and neuropil tissues of Octopus pallidus was investigated quantitatively with the aid of electron microscopy. Morphometrically the microvascular beds of the two tissue types proved very similar. The average lumenal diameter of the exchange vessels was approximately 8·0 μm, and the average minimum vessel wall (or pericyte) thickness and associated basement membrane thickness were about 0·1 and 0·08 μm, respectively. Forty-four or 45 vessels were encountered per mm². The results were incorporated into the Krogh-Erlang equation. The analysis indicated that the Octopus tissues studied are likely to quickly suffer oxygen deprivation once the metabolic rate moves above the resting level. The results are compared with the vertebrate system where vessels are essentially similar in lumenal diameter and wall thickness, but where the microvascular density is an order of magnitude, or more, higher.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade OSS, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 μm. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 μm) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.     

A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.