Tryptophanase-Positive Bacteria in the Marine Environment

The prevalence of indole-positive organisms in the gut and environment of marine animals was studied. Indole formation by a group of the isolates was found to occur only in the presence of tryptophan. The isolates examined were all assigned to the genus Vibrio.     

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.     

Enumeration of Autotrophic Ammonium-oxidizing Bacteria in Marine Waters by a Direct Method

A membrane filter method was developed for enumerating, in mixed populations, autotrophic nitrifying bacteria which transform ammonium to nitrite. It gave counts in good agreement with estimates obtained by using a most-probable-number procedure. Compared to the most-probable-number procedure, the membrane method is convenient and precise. A means of extending the sensitivity of the membrane method is presented.     

Enumeration of Particle-Bound and Unattached Respiring Bacteria in the Salt Marsh Environment

Proportions of respiring bacteria determined with a 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride dye-epifluorescent technique were significantly elevated in the 300-μm surface layer of a salt marsh estuary. Almost all the detectably respiring bacteria in the particle-laden surface layer and a significant proportion in subsurface waters were attached to particles.     

Silica Gel Medium for Enumeration of Petroleumlytic Microorganisms in the Marine Environment

A silica gel medium was developed for the enumeration of petroleumlytic microorganisms in the marine environment. The medium was satisfactorily used for the investigation of the vertical distribution of bacteria in seawater from the surface to 1,000 m depth of western north Pacific central water as well as the neritic region of Japan.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Crypsis in the Pelagic Environment

Pelagic aquatic environments differ from terrestrial environmentsin being three-dimensional and relatively homogeneous, ratherthan two-dimensional and heterogeneous. The present paper examinesthe causes and consequences of these differences in the contextof their influence on the interactions of animals with environmentallight. Particular emphasis is placed on light as a determinantof effective modes of crypsis in the two different habitats.The terrestrial world has selected for the expression of crypticityin the form of superficial color patterns. The heterogeneityof this habitat has resulted in evolutionary divergence of thesesuperficial color patterns, often in very closely-related animals.In contrast, in the homogeneous pelagic aquatic habitats, evolutionaryconvergence on three main forms of crypsis is evident: (1) transparency;(2) reflection of most, if not all visible wavelengths; and,(3) ventral bioluminescence as counterillumination; thus, tobe cryptic most animals in these habitats use one or a combinationof these modalities to variously transmit, reflect or mimicenvironmental light. In the present paper, special attentionis given to transparency as the most prevalent, yet least understood,of these mechanisms that are used in predator-prey interactions.     

Sampling Design and Enumeration Statistics for Bacteria Extracted from Marine Sediments

The spatial and temporal distributions of marine bacteria were studied at both a muddy and a sandy subtidal site in North Inlet, S.C. The sampling design was hierarchical, since subsampling (by a dilution series) of the sediments was necessary to count bacterial cells using acridine orange epifluoresence microscopy. The cell count data fit a log-normal distribution. The abundance of bacteria was 10¹¹ g⁻¹ (dry weight) of mud and 10⁹ g⁻¹ (dry weight) of sand. Variance component analyses demonstrated that variation due to the subsampling procedures was always statistically significant. Thus the common practice of counting 20 fields from one filter preparation is inadequate for estimating the true bacterial population variance in marine sediments. It is recommended that replication of the subsampling level be performed. Standardization of data (by dry weight of sediment) decreased sampling variance at the mud site but not at the sand site, implying that bacteria are more homogeneously distributed in sand than in mud.     

Physiological Properties of Nitrogen-scavenging Bacteria from the Marine Environment

Nine selected strains of marine bacteria from the marine environment, although taxonomically heterogeneous in character, exhibited a capacity for prolonged growth on 'nitrogen-free' media; only one strain, a Klebsiella sp., fixed dinitrogen under any of a considerable range of test conditions. The non-nitrogen-fixing bacteria were able to scavenge low levels of nitrogenous materials, principally ammonia, from the atmosphere. Contrary to previous suggestions, this growth did not have an abnormally low cellular protein and nitrogen content. Some strains with apparently low intracellular protein contents, as determined on a cellular dry weight basis, showed accumulations of carbonaceous storage products which distorted the cytochemical analyses. Representative strains grown in chemostat culture showed a high affinity (i.e. low Ks values—0.35–0.52 μmol/1) for the NH₄+ ion, compared with Escherichia coli (1.75 μmol/1) and preliminary studies of their glutamine synthetases suggested that the affinities ( K_m values—0.25–0.29 μmol/1) of this enzyme for hydroxylamine were similar to the few values reported for other marine bacteria.     

Succession of Pelagic Marine Bacteria during Enrichment: a Close Look at Cultivation-Induced Shifts

Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S−) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S−, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant γ-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable γ-proteobacteria could thus be described as a “feast and famine” existence involving different activation levels of substrate concentration.     

Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.     

Enumeration of Airborne Bacteria in Hospital

An investigation of the “normal” bacterial content of the air of a large general hospital is described. In many different places within five different areas 70 to 200 settle-plate or slit-sampler bacterial counts were carried out. Average counts were most often of the same order as or lower than other published results and were proportional to human activity. The use of the logarithms of the counts showed no advantage, and conventional statistics should be applied with caution in evaluating such studies. Slitsampler and settle-plate counts of all bacteria showed no correlation, whereas those of Staph. aureus were correlated. There is a lack of parallelism between hospital infection and air bacteria counted by current methods, which are, therefore, not suitable for routine use.     

Enumeration of Bacteria in Sea Water

The choice of methods to be used for the enumeration of bacteria in sea water must depend on the objectives of the investigators. Comparisons of data based on different methods may be invalid. An operational definition is given for a colony-generating unit (CGU) which, under some conditions, may be a single viable bacterium. Such units can conveniently be enumerated by a modification of the “minimum probable number” method currently employed in aquatic bacteriology. Counts of “viable bacteria” (CGU) in surface sea water samples from the region of the North Pacific Gyre (28° N, 155°W) were estimated to range from 10,000 to 200,000 per litre.     

Isolation and Characterization of Methanesulfonic Acid-Degrading Bacteria from the Marine Environment

Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur.     

Urea Uptake and Carbon Fixation by Marine Pelagic Bacteria and Archaea during the Arctic Summer and Winter Seasons

How Arctic climate change might translate into alterations of biogeochemical cycles of carbon (C) and nitrogen (N) with respect to inorganic and organic N utilization is not well understood. This study combined ¹⁵N uptake rate measurements for ammonium, nitrate, and urea with ¹⁵N- and ¹³C-based DNA stable-isotope probing (SIP). The objective was to identify active bacterial and archeal plankton and their role in N and C uptake during the Arctic summer and winter seasons. We hypothesized that bacteria and archaea would successfully compete for nitrate and urea during the Arctic winter but not during the summer, when phytoplankton dominate the uptake of these nitrogen sources. Samples were collected at a coastal station near Barrow, AK, during August and January. During both seasons, ammonium uptake rates were greater than those for nitrate or urea, and nitrate uptake rates remained lower than those for ammonium or urea. SIP experiments indicated a strong seasonal shift of bacterial and archaeal N utilization from ammonium during the summer to urea during the winter but did not support a similar seasonal pattern of nitrate utilization. Analysis of 16S rRNA gene sequences obtained from each SIP fraction implicated marine group I Crenarchaeota (MGIC) as well as Betaproteobacteria, Firmicutes, SAR11, and SAR324 in N uptake from urea during the winter. Similarly, ¹³C SIP data suggested dark carbon fixation for MGIC, as well as for several proteobacterial lineages and the Firmicutes. These data are consistent with urea-fueled nitrification by polar archaea and bacteria, which may be advantageous under dark conditions.     

Inactivation of Viable Ascaris Eggs by Reagents during Enumeration

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H₂SO₄) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-μm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO₄, MgSO₄, and NaCl) or during incubation (0.1 N H₂SO₄ and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

A Note on the Total Viable Counts and Selective Enumeration of Anaerobic Bacteria in the Caecal Content, Soft and Hard Faeces of Rabbit

Total viable counts, proteolytic, ureolytic, cellulolytic and NH⁺₄ utilizing anaerobic bacteria in caecal contents, soft and hard faeces of rabbits were studied. No significant differences between the bacterial counts from the caecum and soft faeces were observed ( P < 0·05) but there were significant differences between the bacterial counts of the soft and hard faeces ( P < 0·01).