A rapid method for enumerating Salmonella in milk powders

A one-day procedure for enumerating Salmonella in milk powder is described. Milk powder suspension was treated with a mixture of trypsin and Tween 80, then centrifuged to give a sediment of microbial cells that was plated on the xylose lysine desoxycholate agar for the direct enumeration of Salmonella. Known populations (1–200 cfu/25 g of sample) of salmonellas inoculated into milk powder suspensions were recovered with almost 100% efficiency in each of 152 trials of the method, covering 17 different serotypes.     

A method for quantifying conservation value in lentic freshwater habitats using desmids as indicator organisms

A method is presented for quantification of aquatic nature conservation value based on desmid assemblages present. Species richness (indicative of internal structural and functional differentiation of the ecosystem), the occurrence of rare taxa (often indicative of particular environmental conditions) and the presence of species indicative of ecosystem maturity are the parameters chosen to determine conservation value. For the sake of utility, schemes have been developed to transform the values scored for the various parameters to a simple scale ranging from 0 to 10, relative to regional and historical standards. By way of illustration, two examples are presented. The first shows the decline of desmid-related conservation value along a gradient of increasing eutrophication in a series of interconnected shallow lakes, the other illustrates the course of this value through time in a polluted and subsequently restored moorland pool.     

A novel method of enumerating Nocardia amarae in foaming activated sludge

Various experimental investigations were carried out in order to develop a novel method of enumerating Nocardia amarae in foaming activated sludge using n-octadecane as a carbon source (OD medium). Colonies of N. amarae isolate appeared about 3 d earlier on OD medium than on MS medium. N. amarae counts on OD medium with 20 mg/l of nalidixic acid, using both synthetic and practical activated sludges, were always higher than those on MS medium. It is concluded that OD medium with nalidixic acid was more effective for the count of N. amarae in activated sludge than MS medium.     

The role of ciliated protozoa in pelagic freshwater ecosystems

The abundance and biomass of ciliates are both strongly related to lake trophic status as measured by chlorophylla concentrations. Taxonomic replacements occur with increasing eutrophication such that large-bodied forms (predominantly oligotrichs) are progressively replaced by smaller-bodied ciliates (mainly scuticociliates). Highly acidic lakes display a more pronounced dominance of large-bodied forms when contrasted with less acidic lakes of comparable trophy. Community structure of ciliate populations is determined largely by lake trophy with acidic oligotrophic systems being characterized by reduced diversity and species richness compared with hypereutrophic systems. The temporal and spatial distribution of small (< 100m) ciliate populations is ascribed to lake thermal regimes which provide localized concentrations of food resources. Likewise, in extremely productive lakes, very large (> 100m) meroplanktonic ciliates enter the water column during midsummer after the development of thermal stratification and associated profundal deoxygenation. Laboratory studies indicate that large zooplankton (crustaceans) are capable of utilizing ciliates as a food source, but there is little direct evidence from field studies documenting this trophic link. Ciliates can be voracious grazers of both bacterioplankton and phytoplankton, and each species has a distinct range of preferred particle size which is a function of both mouth size and morphology. Myxotrophic ciliates may be important components in some plankton communities, particularly during periods of nutrient limitation or after their displacement from the benthos of eutrophic lakes. Evidence regarding the importance of planktonic ciliated protozoa in nutrient regeneration and as intermediaries in energy flow is discussed.     

Membrane filter method for enumerating Escherichia coli.

A membrane filter procedure for enumerating Escherichia coli was developed and evaluated. The method quantifies E. coli within 24 h without requiring subculture and identification of isolates. It incorporates a primary selective-differential medium for gram-negative, lactose-fermenting bacteria; resuscitation of weakened organisms by incubation for 2 h at 35 degrees C before incubation at 44.5 degrees C for 18 to 22 h; and an in situ urease test to differentiate E. coli from other thermotolerant, lactose-positive organisms. The recovery of E. coli from marine, estuarine, and freshwater samples exceeded 90%. Of the presumptively positive colonies, 91% were verified as E. coli. Less than 1% of all of the verified E. coli colonies failed to react typically.     

Structure of bacterial communities in diverse freshwater habitats

The structures and dynamics of bacterial communities from raw source water, groundwater, and drinking water before and after filtration were studied in four seasons of a year, with culture-independent methods. Genomic DNA from water samples was analyzed by the polymerase chain reaction?- denaturing gradient gel electrophoresis system and by cloning of the 16S rRNA gene. Water samples exhibited complex denaturing gradient gel electrophoresis genetic profiles composed of many bands, corresponding to a great variety of bacterial taxa. The bacterial communities of different seasons from the four sampling sites clustered into two major groups: (i) water before and after filtration, and (ii) source water and groundwater. Phylogenetic analyses of the clones from the autumn sampling revealed 13 phyla, 19 classes, and 155 operational taxonomic units. Of the clones, 66% showed less than 97% similarities to known bacterial species. Representatives of the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were found at all four sampling sites. Species belonging to the phylum Firmicutes were an important component of the microbial community in filtered water. Representatives of Enterobacteriaceae were not detected, indicating the absence of fecal pollution in the drinking water. Differences were found in the bacterial populations that were sampled from the same sites in different seasons. Each water habitat had a unique bacterial profile. Drinking water harbors diverse and dynamic microbial communities, part of which may be active and resilient to chlorine disinfection. This study provides, for the first time, basic data for uncultivable drinking water bacteria in Israel.     

Biomonitoring of freshwater lentic habitats using desmids

Limnology - Freshwater ponds besides being a source of potable water, also serve as a habitat for a range of aquatic organisms. The quality of the pond water is dependent on several interrelated...     

The colonisation of disturbed freshwater habitats byCharaceae

Various species of charophytes are able rapidly to colonise disturbed or cleaned shallow water bodies. This fact may be explained by the long-lasting viability of charophyte oospores, their easy dispersal and, probably, enhanced germination after passage through digestive tracts of waterfowl. Vegetative parts of Charophytes are sometimes resistant to herbicide treatment.     

Vegetation dynamics in temporary and shallow freshwater habitats

The vegetation dynamics in habitats subject to large seasonal fluctuations in water level has been reconsidered in the light of the availabe literature on the ecological requirements of the plants which occur in different ecophases in these habitats. It is suggested that the water-level change is a component of the normal environment for the vegetation of such habitats, and that the changes in the community structure simply reflect the oscillating shifts in the dominance of various constituent species of an otherwise stable community which is adapted to the particular habitat conditions.     

A method for spatial freshwater conservation prioritization

1. Freshwater ecosystems are amongst the most threatened and poorly protected globally. They continue to be degraded through habitat loss, pollution and invading species and conservation measures are urgently needed to halt declining trends in their biodiversity and integrity.
2. During the past decade a suite of decision support tools and computational approaches have been developed for efficient and targeted conservation action in terrestrial or marine ecosystems. These methods may be poorly suited for planning in freshwater systems because connectivity in terrestrial and marine systems is typically modelled in a way unsuitable for rivers, where connectivity has a strong directional component.
3. We modify the conservation prioritization method and software, zonation , to account for connectivity in a manner better suited to freshwater ecosystems. Prioritization was performed using subcatchment/catchment-based planning units and connectivity was modified to have directional upstream and downstream components consistent with the ecology of our target species.
4. We demonstrate this modified method for rivers and streams in the southern North Island of New Zealand. Data included predicted occupancy from boosted regression tree models of species distributions for 18 fish species. The study area covered 2.1 million hectares and included 394 first- to fourth order catchment or subcatchment planning units.
5. Realistic modelling of connectivity had a major influence on the areas proposed for conservation. If connectivity was ignored, recommended conservation areas were very fragmented. By contrast, when connectivity was modelled, high priority conservation targets consisted of entire river basins or headwater subcatchments.
6. The proposed method serves as a starting point for the implementation of reserve selection methods in river ecosystems.     

Disturbance of freshwater habitats by anadromous salmon in Alaska

High densities of habitat modifiers can dramatically alter the structure of ecosystems. Whereas spawning sockeye salmon (Oncorhynchus nerka) dig nests that cover over 2 m² and are at least 20 cm deep, and can spawn at high densities, relatively little attention has been devoted to investigating the impacts of this disturbance. We hypothesized that this temporally and spatially predictable bioturbation has large impacts on the coastal aquatic habitats used by sockeye. We experimentally investigated the impacts of disturbance caused by spawning sockeye in two streams and two lakes in Alaska by excluding salmon from 2.25 m² plots where they traditionally spawn. We sampled exclusions and control plots before, during, and after spawning. During sockeye spawning, fine sediment accumulated in areas where sockeye were excluded from spawning. In addition, sockeye spawning significantly decreased algal biomass by 80% compared to exclusion plots. We found mixed effects of spawning on the invertebrate assemblage. Tricladida and Chironomidae densities increased by 3x in exclusion plots relative to control plots in one creek site. However, for most taxa and sites, invertebrate densities declined substantially as spawning progressed, regardless of experimental treatment. Habitat modification by spawning salmon alters both community organization and ecosystem processes.     

Common pattern of population decline for freshwater cetacean species in deteriorating habitats

1. Freshwater cetacean species, including the baiji (Lipotes vexillifer), Amazon River dolphin (Inia geoffrensis), Ganges/Indus River dolphins (Platanista spp.) and Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis), apex predators in megariver ecosystems, face serious challenges owing to the deterioration of habitat quality. 2. We simulated population change of four freshwater cetacean species under increasing habitat deterioration. Carrying capacity (K) was used to represent the habitat quality, and a logistic model was used to describe the rate of habitat deterioration (dK). 3. An individual‐based Leslie matrix model showed that population declines and extinctions in freshwater cetaceans under increasing habitat deterioration exhibit a consistent pattern irrespective of the initial level of K or population size. When dK is low, population abundance fluctuates stochastically around initial K, but a rapid increase in dK is accompanied by a sharp population decline, with a residual population ultimately declining continuously to extinction. 4. Simulations show that traditional census survey techniques used in cetacean species are unlikely to detect early signs of population decline before a critical level is reached. 5. Empirical data of the likely extinction of baiji strongly agree with our simulation exercise, implying that extinction of other freshwater cetacean species may occur sooner than previously considered. Hence, precautionary approaches for habitat restoration and landscape management should be implemented before freshwater cetacean population declines are detected, and ideally, before habitat quality begins to deteriorate.     

Prevalence of nitrous oxide reducing capacity in denitrifiers from a variety of habitats

Summary A total of 81 strains isolated by T. N. Gamble from soils from eight countries, fresh water lake sediments and nitrified poultry manure were examined for their ability to grow on N₂O as their electron acceptor, as well as for their tendency to produce N₂O from NO ₃ ⁻ in the absence and presence of acetylene. Seventy-seven of the 81 strains were confirmed as denitrifiers. Fifty-nine of the 77 strains grew on N₂O, while 12 strains produced N₂O but could not utilize it. Six strains reduced NO ₃ ⁻ to N₂ but could not grow on N₂O, suggesting that even if N₂O is always an intermediate product of denitrification, it is not always a freely diffusible intermediate. The organisms, however, would consume N₂O that accumulated early in growth and accumulated N₂O in the presence of acetylene. Thus the total number of N₂O users was 65 strains or 83% of the total tested. This implies that the N₂O reducing capacity of denitrifiers occur widely in nature. A high proportion ofPseudomonas fluorescens biotype II reduced N₂O. The accumulation of N₂O from NO ₃ ⁻ in the presence of acetylene provides strong evidence that N₂O is generally an intermediate in denitrification as well as provides additional support for the usefulness of this chemical as a general inhibitor of N₂O reduction.     

A simple micro-growth assay for enumerating bacteria

A simple method for nonspecific determination of bacteria concentrations in a variety of liquid samples was developed. The assay was based on the time required for a sample grown in liquid media to reach a threshold turbidity. Samples were combined with media in a covered 96-well microwell plate and the turbidity was monitored in real time as the bacteria grew in a temperature-controlled plate reader. A significant problem with growth in microwells was condensation on the cover, which prevented accurate turbidity measurement. This problem was overcome by coating the cover with a small amount of surface-active agent. Salmonella and E. coli concentrations could be determined with a relative error of approximately 20% at levels from 10 to 10(6) cells/ml (eight replicates). An assay of 10 samples with standards required 10 min to set up and 20 min for data processing using a computer spreadsheet program. Growth time at 37 degrees C ranged from 4 h for samples at 10(7) cells/ml to 16 h for samples at 10 cells/ml.     

Statistical analysis of the direct count method for enumerating bacteria.

The direct count method for enumerating bacteria in natural environments is widely used. This paper analyzes the sources of variation contributed by the various levels of the method: subsamples, filters, and microscope fields. Based on a nested analysis of variance, we show that most of the variance (less than 80%) is caused by the fields and that the filters contributed nearly all of the remaining variance. The replication at each of the levels determines the total cost and error of a measurement. We compared several sampling schemes, including an optimal strategy which gives the lowest possible variance for a given cost. We recommend that preparing one filter from one subsample is adequate only if the samples are closely spaced in time or distance; otherwise, one filter should be prepared from two or preferably three subsamples. This sampling scheme emphasizes the importance of the highest level of replication. Our analysis shows that the accuracy of the direct count method can be substantially improved (by 20 to 50%) without a large increase in cost when the proper degree of replication at each level is performed.     

How to optimize the drop plate method for enumerating bacteria

The drop plate (DP) method can be used to determine the number of viable suspended bacteria in a known beaker volume. The drop plate method has some advantages over the spread plate (SP) method. Less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar. By distributing the sample in drops, colony counting can be done faster and perhaps more accurately. Even though it has been present in the laboratory for many years, the drop plate method has not been standardized. Some technicians use 10-fold dilutions, others use twofold. Some technicians plate a total volume of 0.1 ml, others plate 0.2 ml. The optimal combination of such factors would be useful to know when performing the drop plate method.This investigation was conducted to determine (i) the standard deviation of the bacterial density estimate, (ii) the cost of performing the drop plate procedure, (iii) the optimal drop plate design, and (iv) the advantages of the drop plate method in comparison to the standard spread plate method. The optimal design is the combination of factor settings that achieves the smallest standard deviation for a fixed cost. Computer simulation techniques and regression analysis were used to express the standard deviation as a function of the beaker volume, dilution factor, and volume plated. The standard deviation expression is also applicable to the spread plate method.