Sampling for Regional Monitoring of Nematode Communities in Agricultural Soils

Regional assessment of nematode communities to monitor the condition or ecological health of agricultural soils requires sampling programs with measures of known reliability and the ability to detect differences over time. Numbers of fields sampled in a region, samples taken per field, and subsamples assayed per sample must be balanced with cost to provide the best sampling scheme. We used components of variance from statewide surveys in North Carolina (1992) and Nebraska (1993) to estimate number of (i) fields to be sampled; (ii) 20-core, composite soil samples to be obtained for each field; and (iii) subsamples to be assayed for each composite sample to detect a specified amount of change in index values within a geographic region. Variances for these three components were used to estimate the degree of reliability for five ecologically based indices (four measures of maturity and one of diversity) of nematode communities. Total variance for maturity and diversity indices, based upon communities of free-living nematodes, was greater in North Carolina than in Nebraska; the opposite was true for indices based strictly upon maturity of communities of plant-parasitic nematodes or of all nematodes in soil. Variability within samples was greater in North Carolina than in Nebraska, especially for maturity indices based only upon free-living nematodes. We identified two possible sampling strategies for a regional survey: Option 1, with two independent samples per field and a single subsample assayed per sample, which would provide a reliability ratio value ≥0.6 for most indices; and Option 2, with three independent samples per field and two subsamples assayed per sample, which would provide a reliability ratio value ≥0.7 for several indices. When cost was considered, Option 1 was the better strategy. Number of fields to be sampled within a region or state varied with the index chosen; with specific indices, however, a 10% change in mean index value could be detected with a sample of 50 to 100 fields.     

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.     

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10(6) cells filter(-1). In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10(5), the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10⁶ cells filter⁻¹. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10⁵, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Testing and evaluation of off-gas filters for bioreactors by a new bacterial aerosol challenge test method (TBAC).

A TNO bacterial aerosol challenge (TBAC) filter test rig was developed for direct assessment of the effectiveness of bioreactor off-gas filters as an alternative to routinely applied indirect wet integrity testing (IT). This TBAC test rig is based on bacterial aerosol challenging with Pseudomonas diminuta and dual monitoring by laser particle counting (LPC) and Andersen microbial sampling (AMS) of viable cells. The TBAC filter test rig is able to reproduce the various conditions encountered in fermentation processes. In experiments with several filters from one class, it was demonstrated that some filters were actually penetrated by up to 3,000 viable cells per test, despite their approval by commercially available IT test equipment. Repetitive filter use, prolonged use, and autoclaving of filters resulted in an increase in pressure drop over the filter but improved the performance of leaking/deviant filters due to building up of a filter cake (this phenomenon was identified by electron microscopy). The integrity tests used were found inadequate for accurate assessment of filter quality. Certification of filter lots by random tests of commercially available off-gas filters using sensitive direct methods such as those presented here might be advisable, as not all filters purchased were of appropriate quality.     

Modelling habitat associations with fingernail clam (Family: Sphaeriidae) counts at multiple spatial scales using hierarchical count models

1. Macroinvertebrate count data often exhibit nested or hierarchical structure. Examples include multiple measurements along each of a set of streams, and multiple synoptic measurements from each of a set of ponds. With data exhibiting hierarchical structure, outcomes at both sampling (e.g. within stream) and aggregated (e.g. stream) scales are often of interest. Unfortunately, methods for modelling hierarchical count data have received little attention in the ecological literature. 2. We demonstrate the use of hierarchical count models using fingernail clam (Family: Sphaeriidae) count data and habitat predictors derived from sampling and aggregated spatial scales. The sampling scale corresponded to that of a standard Ponar grab (0.052 m²) and the aggregated scale to impounded and backwater regions within 38–197 km reaches of the Upper Mississippi River. Impounded and backwater regions were resampled annually for 10 years. Consequently, measurements on clams were nested within years. Counts were treated as negative binomial random variates, and means from each resampling event as random departures from the impounded and backwater region grand means. 3. Clam models were improved by the addition of covariates that varied at both the sampling and regional scales. Substrate composition varied at the sampling scale and was associated with model improvements, and reductions (for a given mean) in variance at the sampling scale. Inorganic suspended solids (ISS) levels, measured in the summer preceding sampling, also yielded model improvements and were associated with reductions in variances at the regional rather than sampling scales. ISS levels were negatively associated with mean clam counts. 4. Hierarchical models allow hierarchically structured data to be modelled without ignoring information specific to levels of the hierarchy. In addition, information at each hierarchical level may be modelled as functions of covariates that themselves vary by and within levels. As a result, hierarchical models provide researchers and resource managers with a method for modelling hierarchical data that explicitly recognises both the sampling design and the information contained in the corresponding data.     

A membrane filtration method for estimating the number of viable bacteria in solutions containing antibiotic

The method involves filtration of dilutions of the mixture through a membrane filter followed by replication on ox blood agar and enumeration of organisms. This method was compared with the conventional drop count method, and a significant correlation was found (r = 0.985). When solutions containing antibiotic were used, the membrane filtration method gave a more reliable estimate of the viable count at the time of sampling.     

Glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.     

ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.     

Spectral analysis of electroencephalographic recordings of long duration by means of band-pass filters

A method is proposed for spectral analysis of EEG recordings of long duration, using a set of band-pass filters. The sensitivity of the filters varies continuously over their full range, and the input signal is sampled at every sensitivity level. The full spectrum is presented as an array of numbers where the columns are the frequencies and the rows the sensitivity levels, printed as a percentage of the total possible responses of the filter (percent of activity). The logarithm of the numbers expressing the activity of a filter are used for the calculation of the regression line. Thus, the activity in each frequency is characterized by two parameters, the slope and the ordinate at origin, the slope being the more interesting one.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

The Essential Complexity of Auditory Receptive Fields

Encoding properties of sensory neurons are commonly modeled using linear finite impulse response (FIR) filters. For the auditory system, the FIR filter is instantiated in the spectro-temporal receptive field (STRF), often in the framework of the generalized linear model. Despite widespread use of the FIR STRF, numerous formulations for linear filters are possible that require many fewer parameters, potentially permitting more efficient and accurate model estimates. To explore these alternative STRF architectures, we recorded single-unit neural activity from auditory cortex of awake ferrets during presentation of natural sound stimuli. We compared performance of > 1000 linear STRF architectures, evaluating their ability to predict neural responses to a novel natural stimulus. Many were able to outperform the FIR filter. Two basic constraints on the architecture lead to the improved performance: (1) factorization of the STRF matrix into a small number of spectral and temporal filters and (2) low-dimensional parameterization of the factorized filters. The best parameterized model was able to outperform the full FIR filter in both primary and secondary auditory cortex, despite requiring fewer than 30 parameters, about 10% of the number required by the FIR filter. After accounting for noise from finite data sampling, these STRFs were able to explain an average of 40% of A1 response variance. The simpler models permitted more straightforward interpretation of sensory tuning properties. They also showed greater benefit from incorporating nonlinear terms, such as short term plasticity, that provide theoretical advances over the linear model. Architectures that minimize parameter count while maintaining maximum predictive power provide insight into the essential degrees of freedom governing auditory cortical function. They also maximize statistical power available for characterizing additional nonlinear properties that limit current auditory models.     

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Comparison of endotoxin exposure assessment by bioaerosol impinger and filter-sampling methods

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.     

Microplate fecal coliform method to monitor stream water pollution.

A study has been carried out on the Moselle River by means of a microtechnique based on the most-probable-number method for fecal coliform enumeration. This microtechnique, in which each serial dilution of a sample is inoculated into all 96 wells of a microplate, was compared with the standard membrane filter method. It showed a marked overestimation of about 14% due, probably, to the lack of absolute specificity of the method. The high precision of the microtechnique (13%, in terms of the coefficient of variation for log most probable number) and its relative independence from the influence of bacterial density allowed the use of analysis of variance to investigate the effects of spatial and temporal bacterial heterogeneity on the estimation of coliforms. Variability among replicate samples, subsamples, handling, and analytical errors were considered as the major sources of variation in bacterial titration. Variances associated with individual components of the sampling procedure were isolated, and optimal replications of each step were determined. Temporal variation was shown to be more influential than the other three components (most probable number, subsample, sample to sample), which were approximately equal in effect. However, the incidence of sample-to-sample variability (16%, in terms of the coefficient of variation for log most probable number) caused by spatial heterogeneity of bacterial populations in the Moselle River is shown and emphasized. Consequently, we recommend that replicate samples be taken on each occasion when conducting a sampling program for a stream pollution survey.     

Microplate fecal coliform method to monitor stream water pollution

A study has been carried out on the Moselle River by means of a microtechnique based on the most-probable-number method for fecal coliform enumeration. This microtechnique, in which each serial dilution of a sample is inoculated into all 96 wells of a microplate, was compared with the standard membrane filter method. It showed a marked overestimation of about 14% due, probably, to the lack of absolute specificity of the method. The high precision of the microtechnique (13%, in terms of the coefficient of variation for log most probable number) and its relative independence from the influence of bacterial density allowed the use of analysis of variance to investigate the effects of spatial and temporal bacterial heterogeneity on the estimation of coliforms. Variability among replicate samples, subsamples, handling, and analytical errors were considered as the major sources of variation in bacterial titration. Variances associated with individual components of the sampling procedure were isolated, and optimal replications of each step were determined. Temporal variation was shown to be more influential than the other three components (most probable number, subsample, sample to sample), which were approximately equal in effect. However, the incidence of sample-to-sample variability (16%, in terms of the coefficient of variation for log most probable number) caused by spatial heterogeneity of bacterial populations in the Moselle River is shown and emphasized. Consequently, we recommend that replicate samples be taken on each occasion when conducting a sampling program for a stream pollution survey.     

Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.     

Spatial and Temporal Variation of Phenanthrene-Degrading Bacteria in Intertidal Sediments

Phenanthrene-degrading bacteria were isolated from a 1-m² intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera Vibrio and Pseudomonas. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in Pseudomonas putida NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m² site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.     

Evaluation and simplification of the assimilable organic carbon nutrient bioassay for bacterial growth in drinking water.

A modified assimilable organic carbon (AOC) bioassay is proposed. We evaluated all aspects of the AOC bioassay technique, including inoculum, incubation water, bioassay vessel, and enumeration technique. Other concerns included eliminating the need to prepare organic carbon-free glassware and minimizing the risks of bacterial and organic carbon contamination. Borosilicate vials (40 ml) with Teflon-lined silicone septa are acceptable incubation vessels. Precleaned vials are commercially available, and the inoculum can be injected directly through the septa. Both bioassay organisms, Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX, are available from the American Type Culture Collection and grow well on R2A agar, making this a convenient plating medium. Turbid raw waters need to be filtered prior to an AOC analysis. Glass fiber filters used with either a peristaltic pump or a syringe-type filter holder are recommended for this purpose. A sampling design that emphasizes replication of the highest experimental level, individual batch cultures, is the most efficacious way to reduce the total variance associated with the AOC bioassay. Quality control for the AOC bioassay includes an AOC blank and checks for organic carbon limitation and inhibition of the bioassay organisms.