R2A Agar Plates Research Articles

Phycicoccus cremeus sp. nov., isolated from forest soil, and emended description of the genus Phycicoccus

A bacterial strain, designated V2M29(T), was isolated from forest soil collected in the Changbai Mountains, Heilongjiang Province, China. Cells of strain V2M29(T) were Gram-positive, rod-shaped (0.5-0.8 μm in diameter and 1.5-2.0 μm in length), strictly aerobic and non-motile. Colonies produced on R2A agar plates were creamy, smooth, circular and 0.3-0.8 mm in diameter. Strain V2M29(T) grew at 14-35 °C (optimum, 29 °C), at pH 4.1-10.0 (optimum, pH 7.0-8.0) and in the presence of 0-7.0 % (w/v) NaCl (optimum, 0-2.0 %). Strain V2M29(T) contained MK-8(H(4)) as the major respiratory quinone and iso-C(15 : 0) (20.9 %), iso-C(16 : 0) (18.5 %), C(17 : 1)ω8c (16.7 %) and 10-methyl-C(17 : 0) (9.2 %) as the major cellular fatty acids. The cell-wall peptidoglycan type was based on meso-2,6-diaminopimelic acid and the acyl type of the muramic acid was acetyl. Diphosphatidylglycerol, phosphatidylinositol and six unknown glycolipids were the major polar lipids. The G+C content of the DNA was 72.0 mol% (T(m)). 16S rRNA gene sequence analysis indicated that strain V2M29(T) was phylogenetically related to members of the genus Phycicoccus, with sequence similarities ranging from 96.6-97.8 %. The DNA-DNA relatedness values of strain V2M29(T) to Phycicoccus dokdonensis DS-8(T) and Phycicoccus bigeumensis DSM 19264(T) were 32.5±3.3 % and 29.2±2.3 %, respectively. Based on these results, it is concluded that strain V2M29(T) represents a novel species of the genus Phycicoccus, for which the name Phycicoccus cremeus sp. nov. is proposed, with V2M29(T) (=CGMCC 1.6963(T) =NBRC 104261(T)) as the type strain.

Ricerche sulla biodiversità delle comunità batteriche in tre laghi antartici

The bacterial diversity in three Antarctic lakes located at Crater Cirque (CC), Inexpressible Island (INI) and Luther Peak (LH) in the Victoria Land was investigated by a combination of culture-dependent and culture-independent techniques. The in situ abundance of different bacterial groups was determined by fluorescent in situ hybridization (FISH), while bacterial diversity among the cultivable microflora was investigated by culturing and genetic fingerprinting. Differences in the composition of bacterial assemblages were observed among the lakes investigated, when using the two approaches reported above. Overall, the detection rate by FISH of DAPI-stained cells varied from 48.4% to 68.9% with the general bacterial probe EUB338. Bacteria hybridizing with the group-specific probe CF319a were found to be abundant in the three lakes. A total of 478 strains were isolated from R2A agar plates and grouped by restriction analysis technique; sequencing of representative 16S rDNAs was performed to elucidate the taxonomic positions of isolates. Overall, isolates were placed within five different taxa: ?-Proteobacteria (39%), Bacteroidetes (35%), ?-Proteobacteria (8%), Actinobacteria (7%) and ?-Proteobacteria (5%). Finally, the 5.8% of total isolates shared the highest degree of sequence identity with unclassified bacteria. Members of the ?-Proteobacteria predominated at INI, whereas isolates from CC and LH mainly belonged to the Bacteroidetes. Among the ?-Proteobacteria, the genus Pseudomonas was predominant, whereas Flavobacterium spp. were very common among the Bacteroidetes. Finally, several Actinobacteria were closely related to unknown Antarctic bacteria.

Paracoccus chinensis sp. nov., isolated from sediment of a reservoir

A Gram-negative, short ovoid- to coccus-shaped, aerobic, motile, non-spore-forming bacterium (designated strain KS-11(T)) was isolated from sediment of the eutrophic Guanting reservoir in Beijing, China. Colonies grown on R2A agar plates were circular, convex and colourless to orange. The strain grew in the presence of up to 1 % NaCl (optimum, 0 % NaCl). Growth occurred at 25-40 degrees C (optimum, 28-37 degrees C) and at pH 6.0-9.5 (optimum, pH 7.5-9.0). On the basis of 16S rRNA gene sequence similarity, strain KS-11(T) was shown to belong to the class Alphaproteobacteria, being closely related to Paracoccus marinus (96.9 % 16S rRNA gene sequence similarity), followed by Paracoccus koreensis (96.8 %), Paracoccus solventivorans (96.8 %), Paracoccus alkenifer (96.2 %) and Paracoccus kocurii (95.8 %). The major fatty acids of strain KS-11(T) were summed feature 7 (C(18 : 1)omega7c/omega9t/omega12t) (83.8 %) and C(18 : 0) (6.5 %) and the G+C content of the genomic DNA was 69.0 mol%. Based on comparative analysis of physiological and chemotaxonomic data, it is proposed that strain KS-11(T) represents a novel species of the genus Paracoccus, named Paracoccus chinensis sp. nov. The type strain is KS-11(T) (=CGMCC 1.7655(T)=NBRC 104937(T)).

Detection of bioterror agents in air samples using real-time PCR

To use real-time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction. Bacteria in air were isolated after passive sedimentation onto R2A agar plates and characterized by 16S rRNA sequencing. Real-time PCR was used to identify different bacterial bioterror agents in an artificial air sample consisting of a liquid air sample and a mixture of miscellaneous airborne bacteria showing different colony morphology on R2A agar plates. No time-consuming DNA extraction was performed. Specifically designed fluorescent hybridization probes were used for identification. Fourteen different bacterial genera were classified by 16S rRNA gene sequencing of selected bacterial colonies showing growth on R2A agar plates. Real-time PCR amplification of all the bacterial bioterror agents was successfully obtained in the artificial air sample containing commonly found airborne bacteria and other potential airborne PCR interferences. Bacterial bioterror agents can be detected within 1 h in a liquid air sample containing a variety of commonly found airborne bacteria using real-time PCR. Airborne viable bacteria at Kjeller (Norway) were classified to the genera level using 16S rRNA gene sequencing.

Isolation of Aerobic Heterotrophic and Anaerobic Sulphate Reducing Bacteria from Model Water System by Filtration Method

The purpose of this study was to compare the efficacy of the filtration method with direct method (without filtration) for detection and enumeration of either aerobic heterotrophic bacteria or anaerobic sulphate reducing bacteria (SRB). In addition we compared the results of colony morphology types of heterotrophic bacteria performed by filtration-spread plate experiments with spread plate experiments (i.e. plating on R2A agar plates). We detected that there was no statistically significant difference in the mean of SRB and heterotrophic bacteria counts from unfiltered and filtered samples. The results of the experiments comparing the cultivation and enumeration success of the filtered samples with unfiltered samples clearly demonstrate that filtration method enables the cultivation and enumeration of SRB and heterotrophic bacteria. In addition, the numbers of distinct colony types recovered from filtered samples were usually higher than unfiltered samples.

Acetobacter cibinongensisBacteremia in Human

<i>Acetobacter cibinongensis</i>Bacteremia in Human

Inhibitory effect of PVDF tubes on biofilm formation in dental unit waterlines

It has been reported that dental unit waterlines (DUWLs) are contaminated with bacterial biofilm, and that water discharged from a DUWL contains bacteria that might be opportunistic pathogens. This study aimed to investigate the ability of polyvinylidene fluoride (PVDF) tubing to inhibit bacterial contamination in DUWLs. Newly installed dental units were equipped with either a conventional polyurethane tube (unit A) or a PVDF tube (unit B), and the numbers of bacteria discharged from high- and low-speed handpiece lines were counted using R2A agar plates. Bacterial attachment on surfaces was observed with a scanning electron microscope (SEM) up to 185 days. Bacterial outflow during 1-day clinical service from a DUWL after 1-year usage was also examined. The surface free energy of each tube was determined based on the measurement of contact angles. The number of bacteria discharged from unit B was lower than from unit A at 80 days and thereafter. SEM examination demonstrated that the unit A tube was covered by biofilm constituting rods and filaments after 94 days, while no biofilm was observed in the unit B tube even after 185 days. After 1-year of usage, the unit B released significantly less bacteria than the unit A at every sampling period of 1-day clinic work. Surface free energies, calculated from contact angles measured, of PVDF and polyurethane tubes were 37.7 and 77.8, respectively. The present results indicate that PVDF tubes, which have lower surface free energy than the conventional tubes, were effective in inhibiting biofilm formation and reducing bacterial outflow from DUWLs.

Degradation of antifouling booster biocides in water

The concentrations of booster compounds were surveyed in the port of Osaka, Japan. The concentrations of Sea-Nine 211, Diuron and Irgarol 1051 in water samples from the port of Osaka were in the ranges &lt;0·30–0·55 ng l−1, 13–350 ng l−1, 1·3–77 ng l−1, respectively. Pyrithiones were not detected in water samples. The levels of Diuron and Irgarol 1051 in the port of Osaka were high in the mooring area for small and medium-hull vessels with poor flushing.Susceptibility of bacterial populations in estuarine water to antifouling biocides was studied. Sea-Nine 211, Diuron, Irgarol 1051 and Copper pyrithione dissolved in dimethyl sulfoxide (DMSO) were added to estuarine water and number of colony forming units (CFU) of bacteria in estuarine water was counted using R2A agar plate. The CFU was not decreased at the concentration less than 1·0 mg/l of Diuron, Irgarol 1051 and Copper pyrithione. However, CFU was decreased at 0·1 mg/l of Sea-Nine 211. Degradation of Sea-Nine 211, Diuron, Irgarol 1051 and Copper pyrithione by bacteria in estuarine water was studied using a die-away method. At an initial concentrations of 0·1 mg/l, observed half-lives of Sea-Nine 211 and Copper pyrithione were 10 and 20 days, respectively. In contrast, Diuron and Irgarol 1051 were degraded scarcely during 60 days of culture.Photodegradation of these booster biocides by sunlight and UV light were studied. Under UV, all biocides were below detection limit after one day of irradiation. Under sunlight, Copper pyrithiones were also below detection limits after one day. Drastic decrease of Sea-Nine 211 concentration was observed after one day. Diuron and Irgarol 1051 scarcely decreased during 17 days of sunlight irradiation.

In situ examination of microbial populations in a model drinking water distribution system

A flow cell set-up was used as a model drinking water distribution system to analyze the in situ microbial population. Biofilm growth was followed by transmission light microscopy for 81 days and showed a biofilm consisting of microcolonies separated by a monolayer of cells. Protozoans (ciliates and flagellates) were often seen attached to the microcolonies. The biofilm was hybridized with oligonucleotide probes specific for all bacteria and the α- and β-subclass of Proteobacteria and visualized with a scanning confocal laser microscope. Hybridization showed that the microcolonies primarily consisted of a mixed population of α- and β-Proteobacteria. 65 strains from the inlet water and 20 from the biofilm were isolated on R2A agar plates and sorted into groups with amplified rDNA restriction analysis. The 16S rDNA gene was sequenced for representatives of the abundant groups. A phylogenetic analysis revealed that the majority of the isolated strains from the bulk water and biofilm were affiliated to the family of Comamonadaceae in the β-lineage of Proteobacteria. The majority of the strains from the α-lineage were affiliated to the family of Sphingomonadaceae. We were unable to detect any strains from the Pseudomonas genus and found a low abundance of bacteria affiliated to the γ-subclass of Proteobacteria where Pseudomonas and E. coli are positioned. The analysis revealed a high bacterial diversity in the water phase as well as the biofilm, but no strains were found in both environments.

Evaluation of a hydrogen peroxide disinfectant for dental unit waterlines

The purpose of this study was to investigate the use of a hydrogen peroxide-based dental unit waterline, or DUWL, treatment to reduce the colonization and growth of heterotrophic bacteria. Twenty-three dental units with self-contained water systems were randomly selected. Three of the units and tap water served as controls. Twenty-four water samples were taken at baseline and once a week for five weeks. They were serially diluted, spread-plated in duplicate onto R2A agar plates and incubated at 37 C for seven days. At baseline, the tap water control had a mean count of 0 colony-forming units/milliliter, or CFU/mL, the three control DUWLs had a median count of 8,440 CFU/mL and the 20 treated DUWLs had a median count of 9,760 CFU/mL. By week 1, 19 (95 percent) of the 20 treated DUWLs had counts of less than 200 CFU/mL, and by week 4, the median count for all of the treated DUWLs was 0 CFU/mL. The measurement at week 5 showed that the reduction to below 200 CFU/mL had been maintained. Scanning electron micrographs from processed DUWL tubing samples revealed a similar pattern of results, with biofilm accumulation more evident in the untreated control specimens. Following the parameters of this study, the authors used a hydrogen peroxide-based disinfectant to achieve the ADA goal of no more than 200 CFU of heterotrophic, mesophilic bacteria per milliliter of unfiltered output water. An easy-to-use hydrogen peroxide-based DUWL disinfectant demonstrated effectiveness in improving the quality of water used for intraoral procedures. Protocol compliance meets the ADA year 2000 goal.

Bacterial passage through microfiltration membranes in wastewater applications

Two polyvinylidenefluoride microfiltration membranes (GVHP and GVWP: Millipore, Bedford, MA) with a nominal pore size of 0.22 μm were challenged with mixed microbial cultures present in Milli-Q™ water and in secondary effluent, and with a Gram-negative model bacterium, SW8, to investigate bacterial passage. Total bacterial counts measured microscopically using the DNA fluorochrome DAPI revealed that the small bacteria in Milli-Q™ water passed MF membranes totally. The model bacterium, SW8 and bacteria from secondary effluent were mostly retained with log reduction values (LRV) of 4 and 3.5, respectively. Transmembrane pressure did not influence the levels of bacterial passage significantly. Pore size effects were investigated with track-etched membranes (Poretics™: Osmonics, Minnetonka MN) with nominal pore sizes of 0.2, 0.1 and 0.05 μm. The LRV of 0.2 μm membranes for SW8 and secondary effluent cells was 3 and 1.5, respectively (total counts). Both membranes with pore sizes smaller than 0.2 μm acted similarly, they still transmitted secondary effluent cells with LRV 2 log higher than 0.2 μm membranes, but for SW8 only 50% higher. In contrast to total count results, removal of bacteria was 100% with all membranes when assessed by cultureable counts, i.e. numbers of bacterial colonies recovered on R2A agar plates. Transmitted bacteria failed to grow on standard basal microbiology media most probably because they were injured during passage through the membranes to the extent that recovery in laboratory media did not occur. However, tests with CTC, an indicator of cell viability, indicated that approximately half of the cells of SW8 which passed the membranes had what appeared to be functional electron transfer chains in their membranes. All membranes had a pore size distribution which included pores larger than the nominal value. Field emission scanning electron microscopy (FESEM) provided evidence for entrapment of bacteria within the membrane matrix.

Physiological stress in batch cultures of Pseudomonas putida 54G during toluene degradation.

Physiological stress associated with toluene exposure in batch cultures of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were enumerated on non-selective (R2A agar plates) and a selective minimal medium incubated in the presence of vapor phase toluene (HCMM2). Differential recovery on the two media was used to evaluate bacterial stress, culturability and loss of toluene-degrading capability. A majority of the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol- variants. Numbers of stressed organisms increased with duration of toluene exposure and toluene concentration and coincided with accumulation of metabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indicating a possible relationship between respiring and injured cells. Rate expressions for injury, for formation of Tol- variants and for growth of Tol- variants were determined by calibrating a theoretical model to the results obtained. These rate expressions can be used to calibrate bioreactor models, and provide a basis for better design and control of bioremediation systems.

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.

Comparison of Identification Systems for Classification of Bacteria Isolated from Water and Endolithic Habitats within the Deep Subsurface

One water and three rock samples were taken from a mined tunnel system, U12n, in Rainier Mesa at the Nevada Test Site. Endolithic microorganisms were cultured from ashfall tuff, which was crushed and made into slurries with a formulation of artificial pore water, on R2A agar plates. Microbial counts ranged from 10 to 10 viable cells per g (dry weight) of rock sampled. The cultured water sample yielded 10 viable cells per ml. Many of the isolates were very small (<1 mum) when viewed in the rock matrix and remained small even when cultured. Most were gram-negative rods. Individual isolates were profiled by API-NFT strip number, antibiotic and metal resistance patterns, and colony and cellular morphologies. Three identification systems, API-NFT strips, BIOLOG, and MIDI, were compared. Each system identified only a small percentage of the total isolates, and in only seven cases were the isolates identified the same way by more than one system. The same genus was identified in three of these cases, but different species were indicated. The genus Pseudomonas was the most commonly identified. The isolate profiles and the three identification systems demonstrated that water isolates were considerably different from endolithic isolates.

Serum beta-lipoprotein and other specific protein concentrations in patients with immunocytoma.

Serum beta-lipoprotein and other specific protein concentrations were measured in 56 patients suffering from multiple myelomatosis, "benign" paraproteinaemia or Waldenström's macroglobulinaemia and in 56 control subjects. The mean level of B-lipoprotein in untreated patients with multiple myelomatosis and macroglobulinaemia was significantly lower than that of the controls. Patients who responded to chemotherapy showed a rapid return to normal of the beta-lipoprotein concentration, while the level remained unchanged in most of those who did not.