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PubMed2001

A scanning technique to measure regional cerebral blood flow and oxyhemoglobin level.

OBJECTIVE: The application of a laser scanning technique to measure regional cerebral blood flow (CBF) and tissue hemoglobin oxygenation (HbO2) using the rat closed cranial window preparation is described. METHODS: Twenty-nine male Wistar rats were used to consecutively measure local CBF by laser Doppler flowmetry and tissue HbO2 by a microspectrophotometric method at multiple corresponding cortical locations. The scanning technique used a computer-controlled micromanipulator. Data from three experimental models are presented: the whisker stimulation model, the ischemia-reperfusion model, and the sinus-vein thrombosis model. Sequential changes in local CBF and HbO2 data before, during, and after stimulation, ischemia, and sinus thrombosis were examined. Data from predefined locations within the same region were correlated with the topographical location and then arranged in a three-dimensional image. RESULTS: In the whisker stimulation model, we found a disproportionate increase in CBF (32 +/- 12%) as compared with that of HbO2 (9 +/- 4%) during stimulation. In the ischemia-reperfusion model, the three-dimensional image showed heterogeneous low CBF (depending on the area) and homogeneous HbO2 at a reduced level during ischemia and postischemic hyperperfusion. However, the range of oxygenation was normal after reperfusion. In the sinus-vein thrombosis model, drainage of the unsaturated blood via the collateral pathways was noted. CONCLUSION: The laser scanning technique is useful for visualizing sequential changes in hemodynamic-metabolic interactions of cortical brain tissue. This technique can reveal phenomena not detected by traditional monitoring procedures.

Animals↗
PubMed2002

Donor-derived circulating endothelial cells after kidney transplantation.

BACKGROUND: In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of kidney allograft recipients and are often associated with acute rejection and active infections with human cytomegalovirus. In the present study we hypothesized that cEC after kidney transplantation are of donor origin, thus reflecting transplantation-related damage to the allograft endothelium. METHODS: Using hydraulic micromanipulation equipment, we isolated single cEC (n=153) from the peripheral blood of nine kidney allograft recipients at various time points after transplantation. We demonstrated the origin of these cells (donor or recipient) by typing their HLA-DRB alleles by single-cell, genomic, nested polymerase chain reaction. RESULTS: The majority (71.8%) of cEC were of donor origin and could be detected up to 141 days after onset of acute rejection episodes. Although less frequent (28.2%), recipient-type cEC were detected in the same time course as donor-type cEC. CONCLUSION: We conclude that posttransplantational injury to the allograft endothelium is reflected by the presence of donor-derived cEC in the blood.

Acute Disease↗
PubMed1997

Obtaining clone-specific primer and probe for the immunoglobulin heavy-chain gene from paraffin-embedded tissue of B-cell lymphoma: technical considerations.

The complementarity determining region (CDR) III of the immunoglobulin heavy-chain (IgH) gene is a tumor-specific marker for B-cell malignancies that has been widely exploited for the monitoring of minimal residual disease in B-precursor acute lymphocytic leukemia. There are a number of technical problems in applying the same technology for B-cell non-Hodgkin's lymphoma (B-NHL). Several procedures have been useful in overcoming these unique problems encountered in obtaining the tumor-specific sequence of the IgH-CDRIII in B-NHL, including the use of denaturing gradient gel electrophoresis or micromanipulation of tissue sections in separating the tumor-specific CDRIII products from those of contaminating normal B-lymphocytes. Minor modifications of a commercial kit greatly improve the purity of the polymerase chain reaction (PCR) products for sequencing. Modifications of the 5'-ends of the VH and IH primers, coupled with the cycle sequencing technique, make it possible to obtain unambiguous sequences on direct sequencing of short PCR products. Computer informatics and programs that facilitate the design of tumor-specific primers and probes from CDRIII sequences are described.

DNA Primers↗
PubMed1997

Efficiency of single-cell polymerase chain reaction from stained histologic slides and integrity of DNA in archival tissue.

Molecular analysis of isolated single cells is a powerful tool for studying heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Here, we investigate the applicability of molecular techniques at a single-cell level by using routinely processed archival tissue. An ultraviolet laser in conjunction with a computer-controlled micromanipulator and a microscope were used for the contamination-free isolation of single tumor cells from stained sections of diffuse-type gastric cancer. A total of 1,328 single cells and 654 clusters of 10-30 cells each, taken from specimens of 14 patients, were analyzed for parts of the E-cadherin gene by the polymerase chain reaction (PCR). With increasing length in base pairs (bp) of the amplified fragments, the efficiency of single-cell PCR as measured by the rate of detectable amplification products declined from approximately 25% (156, 213, and 228 bp) to 14% (246 bp) and 11% (264 and 296 bp). For groups of 10-30 cells, a similar effect was seen at a higher level at 33% (246 bp), 31% (264 bp), and 26% (296 bp), respectively. To our knowledge, this is the first report that has studied the outcome of single-cell PCR on a large systematic scale. The average degree of DNA disintegration in paraffin-embedded, stained tissues was estimated to be approximately 100 bp when the aforementioned data were used in a mathematical model. This study provides evidence that in order to obtain reasonable sensitivity with single-cell PCR, short fragments, preferably < 200 bp long, should be used. Furthermore, whenever applicable, pooling of cells of interest may be another favorable option.

DNA↗
PubMed2000

Clonal proliferation of B lymphocytes in the germinal centers of human reactive lymph nodes: possibility of overdiagnosis of B cell clonal proliferation.

Clonal expansion of the germinal center B cells of human reactive lymph nodes was analyzed. By micromanipulation, 28 germinal centers were microdissected from three nonneoplastic lymph nodes that had been fixed with formalin. Immunoglobulin heavy chain variable (V) region gene rearrangement was examined by seminested polymerase chain reaction (PCR) using two sets of primers (FR2-J and FR3A-J). An oligoclonal development (one to five clones) was found in each germinal center. Depending on the primer used, four or five (16%) of the germinal centers showed a single rearrangement band. The average number of B-cell clones in each germinal center was approximately 2.5. Next, the authors analyzed 50 endoscopic biopsy specimens from 6 patients with non-mucosa-associated lymphoid tissue (MALT) type gastric lymphoma, 25 patients with chronic gastritis, and 19 patients with nonspecific colitis. In addition to the samples from the 6 patients with malignant lymphoma, 8 of 44 biopsy samples (18.2%) from patients diagnosed as having chronic gastritis or nonspecific colitis showed one or two amplified bands. These results indicate that PCR analysis of immunoglobulin heavy chain V region gene rearrangement in small biopsy specimens could be misleading, causing overdiagnosis of reactive lymphoid tissue as B-cell clonal proliferation.

Adenocarcinoma↗
PubMed2005

Recording of neural activity from goat cervical facet joint capsule using custom-designed miniature electrodes.

STUDY DESIGN: To establish a methodology for the neurophysiologic study of mechanoreceptors in the cervical facet joint capsule. OBJECTIVES: To test a custom designed miniature dual bipolar electrode for recording the neural activity in cervical dorsal roots. To determine if the neural activity from different receptors in the capsule can be differentiated using this methodology. SUMMARY OF BACKGROUND DATA: Injury to cervical facet joint capsules has been regarded as an important source of whiplash pain, but no neurophysiologic study has been performed to demonstrate or characterize sensory nerve function in the capsule. METHODS: Nineteen goats weighing 34 to 55 kg were used under general anesthesia. A C4-C6 laminectomy was performed to expose the C6 nerve root. Custom designed miniature dual bipolar electrodes were used to record neural activity in the left C6 branches. Electrical and mechanical stimuli were used to evoke receptor activity in the dorsal aspect of the C5/6 capsule. Conduction velocities (CVs) of evoked units were determined by electrical stimulation and dual-bipolar-electrode recording methods. The units were classified based on their CVs. The waveform of each classified unit was saved as a template for later single unit discharge search among multiunit discharges during the stretch of the capsule. The C5/6 facet joint with capsule was pulled by a computer-controlled actuator instrumented with a load cell at a rate of 0.5 mm per second. The evoked neural activity and load were recorded, digitized, and analyzed to determine CV, discharge rate, and response to the stretch. RESULTS: Miniature bipolar electrodes recorded the neural activity in both channels, with single unit CVs being measured. There was no discernible motion between the electrode and dorsal root when the capsule was pulled. Both local compression and stretch on capsule evoked multiunit discharges. A-beta, A-delta, and C-fiber units were found among these multiunit discharges. The rate of single unit and multiunit discharges increased during capsule stretch in the physiologic range and afterdischarges occurred beyond the physiologic range. CONCLUSIONS: The novel miniature electrodes not requiring a micromanipulator made it feasible and reliable to record neural activity from short cervical spinal roots. Waveforms of different units could be identified, making it possible to study sensory functions of the facet joint capsule. A-beta, A-delta, and C-fiber units were found responding to mechanical stimuli, indicating that facet joint capsule has functional proprioceptors and nociceptors.

Action Potentials↗
PubMed2003

Intrarenal blood flow distribution in hyperdynamic septic shock: Effect of norepinephrine.

OBJECTIVES: To measure changes in medullary and cortical renal blood flow during experimental hyperdynamic sepsis and the effect of subsequent norepinephrine infusion on such flows. DESIGN Experimental animal study. SETTING: Animal laboratory of university-affiliated physiology institute. SUBJECTS Eighteen anesthetized merino sheep. INTERVENTIONS: A transit-time flow probe was placed around the left renal artery. Laser Doppler flow probes were inserted in the left renal medulla and cortex by micromanipulation to measure changes in regional intrarenal blood flow. MEASUREMENTS AND MAIN RESULTS: Systemic pressures, cardiac output, renal, and intrarenal blood flows were measured continuously. A bolus of Escherichia coli (7.5 x 10(9) colony forming units) was given intravenously to induce hyperdynamic sepsis. After the onset of hyperdynamic sepsis, all animals were randomly allocated to either norepinephrine (0.4 microg.kg-1.min-1 for 30 mins) or observation for 30 mins in random order. E. coli injection induced a significant decrease in mean arterial pressure (102.2 +/- 15.2 mm Hg to 74.3 +/- 16.1 mm Hg, p <.05) and an increase in mean cardiac output (4.60 +/- 1.62 L/min to 5.93 +/- 1.18 L/min, p <.05). However, renal blood flow did not change significantly (326.4 +/- 139.4 mL/min to 293.1 +/- 117.5 mL/min, not significant) despite a 30% increase in renal conductance (3.27 +/- 1.52 to 4.13 +/- 2.01 mL.min-1.mm Hg-1, p <.05). Cortical blood flow decreased by 15% (not significant) and medullary flow by 5% (not significant) during sepsis, but individual changes were unpredictable. On the other hand, norepinephrine infusion caused a significant improvement in mean arterial pressure (74.3 +/- 16.1 to 105.7 +/- 17.7 mm Hg, p <.05) and a further increase in cardiac output (5.93 +/- 1.18 to 7.13 +/- 1.52 L/min, p <.05). Mean renal blood flow also increased (293.1 +/- 117.5 to 384.5 +/- 168.1 mL/min, p <.05) despite decreased renal conductance (4.13 +/- 2.01 to 3.73 +/- 1.91 mL.min-1.mm Hg-1, p <.05). Infusion of norepinephrine significantly increased medullary blood flow by 35% compared with baseline (p <.05) and by 54% compared with untreated sepsis (p <.05), whereas the increases in cortical blood flow (16 and 53%, respectively) were not significant. CONCLUSIONS: Hyperdynamic sepsis caused renal vasodilation but had limited effects on regional intrarenal blood flow. Norepinephrine infusion (0.4 microg.kg-1.min-1) during sepsis significantly increased global and medullary renal blood flow and restored renal vascular tone toward but not above normal.

Animals↗
PubMed2004

Safe and reliable sound threshold measures with direct vibration of the ossicular chain.

OBJECTIVES: The aim of the study was to evaluate the safety and feasibility of piezoelectric malleus vibration audiometer (MVA), which presents micromechanical vibrations to the umbo membranae tympani. STUDY DESIGN: Phase I study performed in a tertiary referral center (University Hospital). METHODS: The coupling rod of the MVA was moved slowly through the outer ear canal toward the eardrum with a micromanipulator. Coupling was completed when the rod tip touched the umbo membranae tympani. Basic audiologic measures of sound threshold obtained with direct stimulation of the malleus are presented. We used MANOVA (multivariate repeated measures ANOVA) to investigate the repeatability of MVA thresholds from one day to the other and when decoupling and retracting the coupling rod 2 mm off the umbo. We also selected the MANOVA to test for unwanted bone-conduction threshold shifts after MVA application. We assessed normality of the data by quantile-quantile plots of the residuals. RESULTS: Twenty-eight male and 10 female subjects with normal hearing, 22.2 to 34.6 years old (median age, 27.2 yr) underwent an examination. Thirty-six subjects underwent MVA, because 2 of the 38 subjects who volunteered for the study have not undergone the procedure due to the external auditory canal anatomy preventing application of the MVA. The results show that it is possible to safely and reliably measure thresholds of direct vibration of the ossicular chain. Using pure tone audiograms, no pure tone bone- and/or air-conduction threshold shifts occurred after the procedure. None of the subjects reported any other ear-related symptoms such as vertigo, tinnitus, or dizziness. Geometric mean vibratory displacements at threshold ranged from 0.55 nm at 250 Hz to 0.03 nm at 6 kHz. MANOVA demonstrated a repeatability of MVA thresholds. CONCLUSION: Malleus vibration audiometry will not allow exact linkage of actual implantable hearing aid. But the present study demonstrates that MVA can provide an audiometric tool for assessing ossicular function and integrity prior to implantation of an electronic hearing amplifier.

Adult↗
PubMed2005

Characterization of the elastic properties of the nuclear envelope.

Underlying the nuclear envelope (NE) of most eukaryotic cells is the nuclear lamina, a meshwork consisting largely of coiled-coil nuclear intermediate filament proteins that play a critical role in nuclear organization and gene expression, and are vital for the structural stability of the NE/nucleus. By confocal microscopy and micromanipulation of the NE in living cells and isolated nuclei, we show that the NE undergoes deformations without large-scale rupture and maintains structural stability when exposed to mechanical stress. In conjunction with image analysis, we have developed theory for a two-dimensional elastic material to quantify NE elastic behaviour. We show that the NE is elastic and exhibits characteristics of a continuous two-dimensional solid, including connections between lamins and the embedded nuclear pore complexes. Correlating models of NE lateral organization to the experimental findings indicates a heterogeneous lateral distribution of NE components on a mesoscopic scale.

Elasticity↗
PubMed1977

Chromosome distribution: experiments on cell hybrids and in vitro.

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.

Animals↗
PubMed1997

Amaricoccus gen. nov., a gram-negative coccus occurring in regular packages or tetrads, isolated from activated sludge biomass, and descriptions of Amaricoccus veronensis sp. nov., Amaricoccus tamworthensis sp. nov., Amaricoccus macauensis sp. nov., and Amaricoccus kaplicensis sp. nov.

Three isolates of gram-negative bacteria, strains Ben 102T, Ben 103T, and Ben 104T, were obtained in pure culture by micromanipulation from activated sludge biomass from wastewater treatment plants in Italy, Australia, and Macau, respectively. These isolates all had a distinctive morphology; the cells were cocci that usually were arranged in tetrads. Based on this criterion, they resembled other bacteria from activated sludge previously called "G" bacteria. On the basis of phenotypic characteristics and the results of 16S ribosomal DNA sequence analyses, the three isolates were very similar to each other, but were sufficiently different from their closest phylogenetic relatives (namely, the genera Rhodobacter, Rhodovulum, and Paracoccus in the alpha subdivision of the Proteobacteria) to be placed in a new genus, Amaricoccus gen. nov. Each of the three isolates represents a new species of the genus Amaricoccus; strains Ben 102T, Ben 103T, and Ben 104T are named Amaricoccus veronensis, Amaricoccus tamworthensis, and Amaricoccus macauensis, respectively. An isolate designated Ben 101T, which was isolated independently by Cech and Hartman in Kaplice, Czech Republic, was also characterized and belongs to the same genus. We propose that the isolate of Cech and Hartman should be placed in another new species, Amaricoccus kaplicensis.

Carbohydrate Metabolism↗
PubMed1999

Tessaracoccus bendigoensis gen. nov., sp. nov., a gram-positive coccus occurring in regular packages or tetrads, isolated from activated sludge biomass.

An isolate of a Gram-positive bacterium, designated strain Ben 106T, was obtained in pure culture by micromanipulation of a biomass sample obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci or clusters of cocci arranged in regular tetrads and was morphologically similar to the dominant organism observed in the biomass. This morphology resembled that of some Gram-positive and -negative bacteria and the so-called 'G-bacteria' commonly seen in activated sludge samples. Strain Ben 106T is a non-motile, facultative anaerobe. It is oxidase-negative, catalase-positive and is capable of reducing nitrate. This organism can grow between 20 and 37 degrees C, with an optimum temperature of 25 degrees C. The pH range for growth is between 6.0 and 9.0, with an optimum pH of 7.5. The isolate stained positively for intracellular polyphosphate granules. The diagnostic diamino acid of the peptidoglycan is LL-diaminopimelic acid (LL-A2pm) with a glycine moiety at position 1 of the peptide subunit, which characterizes the presence of a rare peptidoglycan (type A3-gamma'). Two menaquinones, MK-9(H4) and MK-7(H4), are present and the main cellular fatty acid is 12-methyltetradecanoic acid. The G + C content is 74 mol%. From phenotypic characteristics and 16S rDNA sequence analysis, the isolate differed sufficiently from its closest phylogenetic relatives, namely Propionibacterium propionicum, Propioniferax innocua, Friedmanniella antarctica, Luteococcus japonicus and Microlunatus phosphovorus in the A1 subdivision of the Gram-positive bacteria (i.e. Firmicutes with a high G + C content), suborder Propionibacterineae, to be placed in a new genus, Tessaracoccus, as Tessaracoccus bendigoensis gen. nov., sp. nov. The type strain is Ben 106T (= ACM 5119T).

Bacterial Typing Techniques↗
PubMed1999

Friedmanniella spumicola sp. nov. and Friedmanniella capsulata sp. nov. from activated sludge foam: gram-positive cocci that grow in aggregates of repeating groups of cocci.

Two Gram-positive, non-motile, non-spore-forming, strictly aerobic, pigmented cocci, strains Ben 107T and Ben 108T, growing in aggregates were isolated from activated sludge samples by micromanipulation. Both possessed the rare type A3 gamma' peptidoglycan. Major menaquinones of strain Ben 107T were MK-9(H4) and MK-7(H2), and the main cellular fatty acid was 12-methyltetradecanoic acid (ai-C15:0). In strain Ben 108T, MK-9(H4), MK-9(H2) and MK-7(H4) were the menaquinones and again the main fatty acid was 12-methyltetradecanoic acid (ai-C15:0). Polar lipids in both strains consisted of phosphatidyl inositol, phosphatidyl glycerol and diphosphatidyl glycerol with two other unidentified glycolipids and phospholipids also present in both. These data, together with the 16S rDNA sequence data, suggest that strain Ben 107T belongs to the genus Friedmanniella which presently includes a single recently described species, Friedmanniella antarctica. Although the taxonomic status of strain Ben 108T is far less certain, on the basis of its 16S rRNA sequence it is also adjudged to be best placed in the genus Friedmanniella. The chemotaxonomic characteristics and DNA-DNA hybridization data support the view that Ben 107T and Ben 108T are novel species of the genus Friedmanniella. Hence, it is proposed that strain Ben 107T (= ACM 5121T) is named as Friedmanniella spumicola sp. nov. and strain Ben 108T (= ACM 5120T) as Friedmanniella capsulata sp. nov.

Bacterial Typing Techniques↗
PubMed2000

Three isolates of novel polyphosphate-accumulating gram-positive cocci, obtained from activated sludge, belong to a new genus, Tetrasphaera gen. nov., and description of two new species, Tetrasphaera japonica sp. nov. and Tetrasphaera australiensis sp. nov.

Two isolates of Gram-positive cocci (Ben 109T and Ben 110) which could accumulate polyphosphate and were microscopically similar in appearance to so-called 'G-bacteria', appearing as tetrads, were isolated from samples of activated sludge biomass by micromanipulation and grown in axenic culture. On the basis of their phenotypic and chemotaxonomic characters and 16S rDNA sequences, these isolates, together with strain T1-X7T isolated and described previously in Japan, belong to a new genus. These isolates are phylogenetically different from Tessaracoccus bendigoensis, Friedmanniella spumicola and Friedmanniella capsulata, Gram-positive cocci isolated previously in this laboratory. They are characterized by type A1 gamma peptidoglycan, with meso-diaminopimelic acid as the diagnostic diamino acid. The main cellular fatty acid of Ben 109T, Ben 110 and T1-X7T is 14-methylpentadecanoic acid (i-C16:0). The major menaquinones of Ben 109T are MK-8(H4), with MK-8(H2) and MK-8 in trace amounts. In Ben 110 MK-8(H4) and MK-6(H4) are the major menaquinones, while T1-X7T has MK-8(H4), MK-7(H4) and MK-6(H4) as its menaquinones. All three contain phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as their polar lipids. These properties, together with 16S rDNA sequence data, suggest that they all belong to a single new genus for which the name Tetrasphaera gen. nov. is proposed. However, the lipid, cellular fatty acid profiles and DNA-DNA similarity data suggest that Ben 109T and Ben 110 are sufficiently different from T1-X7T to represent a different species of the genus Tetrasphaera. Strain T1-X7T represents the type species Tetrasphaera japonica sp. nov. of this new genus, and strains Ben 109T and Ben 110 belong to the other species, Tetrasphaera australiensis sp. nov.

Actinomycetales↗
PubMed2000

'Candidatus Nostocoida limicola', a filamentous bacterium from activated sludge.

Five strains of 'Candidatus Nostocoida limicola' were isolated by micromanipulation from two activated sludge plants. Two (Ben17 and Ben18) were from Sunbury, Victoria, Australia, and three (Ben67, Ver1 and Ver2) were from Verona, Italy. The near complete 16S rDNA sequences were determined for five strains and the phylogenetic location of this important bulking filament in the actinomycete subphylum is reported for the first time. Phylogenetically, the Ben strains formed one group with 99.4% 16S rDNA similarity, and the Ver strains formed another with 99.9% 16S rDNA similarity. The mean similarity between the two groups was 97.4%. By 16S rDNA comparison, the closest relative to all strains was Terrabacter sp. strain DPO1361 (95.0-95.5% identical). On R2A medium, all strains generally grew as short filaments or clumps of cocci, whereas on glucose sulfide (GS) medium, all grew as irregular twisting filaments comprising Gram-positive and Gram-negative cells, which is close to their in situ morphology. Polyphosphate was stored either as granules (R2A) or throughout the trichomes (GS). None of the strains could grow without added nitrogen, reduce nitrate to nitrogen gas or grow anaerobically, whereas all could grow at 15-30 degrees C, produce catalase and reduce nitrate to nitrite. All were inactive in the Hugh & Leifson test. This paper describes 'Candidatus Nostocoida limicola'.

Actinomycetales↗
PubMed2002

Quadricoccus australiensis gen. nov., sp. nov., a beta-proteobacterium from activated sludge biomass.

A gram-negative coccus, designated strain Ben 117T, was obtained in axenic culture by micromanipulation from an Australian activated sludge biomass sample, which had been subjected to chlorination in order to alleviate problems associated with foaming and bulking. This isolate was a strict aerobe and grew in axenic culture, also appearing in biomass samples as cocci or clusters of cocci in tetrads, thus resembling the morphotype 'G-bacteria' seen commonly in activated sludge samples. Strain Ben 117T was non-motile, aerobic, oxidase-negative and catalase-positive and grew between 15 and 30 degrees C, with an optimum of 25-30 degrees C. The pH range for growth was between 6.0 and 8.5, with an optimum of 7.5-8.5. The isolate stained positively for intracellular polyphosphate and poly-beta-hydroxybutyrate and its G+C content was 67 mol%. 16S rDNA sequence analysis suggests that strain Ben 117T is phylogenetically different from members of the genera Amaricoccus, gram-negative 'G-bacteria' isolated previously in this laboratory. Ben 117T is a member of the Rhodocyclus group in the beta-Proteobacteria and equidistantly placed (similarity value of 95%) between Ferribacterium limneticum and Dechloromonas agitata (mean similarity value of 92% with the genus Rhodocyclus). Based on phenotypic and phylogenetic evidence, it is proposed that strain Ben 117T be designated a novel species in a new genus, Quadricoccus australiensis gen. nov., sp. nov.; the type strain is Ben 117T (= NCIMB 13738T = CIP 107055T).

Betaproteobacteria↗
PubMed2002

Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal.

Most filamentous bacteria in biological nutrient removal (BNR) processes have not been identified beyond their morphotype and simple staining reactions. Furthermore, the majority of sludge filaments observed under the microscope do not hybridize to commonly used phylogenetic probes for well characterized bacterial phyla such as the Proteobacteria, Actinobacteria, Firmicutes and BACTEROIDETES: Specific 16S rRNA-targeted oligonucleotide probes were designed for the phylum Chloroflexi (green non-sulfur bacteria) and optimized for use in fluorescence in situ hybridization. Chloroflexi have been implicated in BNR systems by phylogenetic identification of filamentous bacteria isolated by micromanipulation from sludge and culture-independent molecular phylogenetic surveys. The predominant morphotype responding to the probes was filamentous and these filaments were generally abundant in 10 Australian full-scale and two laboratory-scale BNR samples examined. Filamentous bacteria responding to a subdivision 1 Chloroflexi probe were rare in the samples, whereas subdivision 3 Chloroflexi filaments were very common in some sludges. This is in direct contrast to results obtained from molecular phylogenetic surveys of BNR systems where most sludge 16S rDNA clones belong to subdivision 1 and only a few to subdivision 3. It is suggested that filamentous bacteria belonging to the Chloroflexi phylum account for a large fraction of phylogenetically uncharacterized filaments in BNR systems and are likely to be abundant in such systems on a global scale.

Actinobacteria↗
PubMed2005

Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the 'Alphaproteobacteria' from activated sludge.

A novel Gram-negative coccus/coccobacillus, strain Ben 114(T), growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the 'Alphaproteobacteria', with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-beta-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the 'Alphaproteobacteria', namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the 'Alphaproteobacteria', the D. vanus subgroup. The type strain is Ben 114(T) (=NCIMB 13612(T)=CIP 107350(T)).

Bacterial Typing Techniques↗