Funding for this work was provided by the National Science Foundation (grant DEB-1553111) and the Eppley Foundation for Scientific Research. This manuscript is VCU Rice Rivers Center contribution #89.

The authors have nothing to disclose.

This ataxic size spectra protocol can be used to quantify and model size structure within communities of stream fishes and invertebrates. Previous size spectra studies in stream ecosystems have ranged from basic descriptive research39,40 to comparisons along a longitudinal river profile41 and among distinct biogeographic regions42. Seasonal comparisons have been performed43,<sup cl...

Body size scaling relationships, such as the positive association between body mass and metabolic rate, are well-known at the individual organism level and are now being studied at higher levels of organization1,2,3. These allometric relationships are most often power-law functions of the form Y = aMb, where Y is the variable of interest (e.g., metabolism, abundance, or home range size), M is the body mass of a single or average individual, b is a scaling coefficient, and a is a constant. For statistical convenience, Y and M data are often log-transformed prior to analysis then modeled with linear equations of the form log (Y) = log (a) + b log (M), where b and log (a) become the linear model slope and intercept, respectively.
The size spectrum is a type of allometric relationship that predicts density (D, the number of individuals per unit area) or biomass (B, the summed mass of individuals per unit area) as a function of M (See Section 4 for additional information on the use of &#8216;normalized&#8217; D or B estimates.) Like other scaling relationships between M and D or between M and B, the size spectrum plays a central role in basic and applied ecology. At the population-level, biologists often interpret negative D <img alt="Image 1" src="/files/ftp_upload/59945/59945img01.jpg" />&#160;M relationships as evidence of density-dependent survival or as models of ecosystem carrying capacity (i.e., the &#8216;self-thinning rule&#8217;)4,5. At the community-level, B <img alt="Image 1" src="/files/ftp_upload/59945/59945img01.jpg" />&#160;M relationships can be used to study system-level effects of anthropogenic perturbations, such as size-selective fishing6,7. Allometric scaling of D and B with M are also central to recent efforts to unite population, community, and ecosystem ecology2,8,9.
One particularly important characteristic of the size spectrum is the fact that it is entirely ataxic9,10. This point is easy to miss when comparing scatterplots of D <img alt="Image 1" src="/files/ftp_upload/59945/59945img01.jpg" />&#160;M or B <img alt="Image 1" src="/files/ftp_upload/59945/59945img01.jpg" /> M data but the distinction between taxic and ataxic models is a critical one. In taxic models, a single M value is used to represent the average body mass of every individual of a given species or taxa11. In ataxic models, all individuals within a data set are partitioned among a series of body size intervals or M bins, regardless of their taxonomic identity12. The latter, ataxic approach is advantageous in aquatic ecosystems where many taxa exhibit indeterminate growth and experience one or more ontogenetic shifts in feeding behavior; in these instances, a single species-level M average will obscure the fact that a species can fill different functional roles throughout its life history9,13,14.
Here, we present a complete protocol to quantify the size spectrum within wadable streams and rivers. The protocol begins with field sampling methods to collect the necessary fish and benthic macroinvertebrate data. Fish will be collected through a &#8216;three-pass depletion&#8217; sampling process. Abundance will then be estimated from the depletion data with the Zippin method15. In depletion sampling, individual fishes within a closed study reach (i.e., individuals can neither enter nor leave the enclosed reach) are removed from the reach through three successive samples. Thus, the number of remaining fishes will be progressively depleted. From this depletion trend, total abundance within the study reach can be estimated then converted to D (in fish per m2), using the known surface area of the study reach. Benthic macroinvertebrates will be collected with standard fixed-area samplers, then identified and measured in the laboratory.
Next, the combined fish and macroinvertebrate data will be partitioned among size bins. Traditionally, the octave or log2 scale (i.e., doubling intervals) has been used to set size bin boundaries16. Once a list of size bins has been established, partitioning of individual benthic macroinvertebrates among their respective size bins is straightforward because invertebrates are directly enumerated as numbers of individuals per unit area. However, estimating fish abundances within size bins is more abstract because these estimates are inferred from the depletion data. Detailed instructions are therefore provided to estimate fish abundance within size bins, irrespective of taxonomic identity, from depletion sample data.
Finally, linear regression will be used to model the size spectrum. This protocol is fully compatible with the original, general method of Kerr and Dickie16 and identical to the methods used by McGarvey and Kirk, 201817 in a study of fish and invertebrate size spectra in West Virginia streams. By using this protocol, investigators can insure that their results are directly comparable with other studies that build upon Kerr and Dickie16, thereby accelerating a broad and robust understanding of body size scaling relationships in freshwater ecosystems and the mechanisms that drive them.

<table>
	<tbody>
		<tr>
			<td>Chest waders</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Personal protective equipment for use during electrofishing. Do NOT use &#39;breatheable&#39; waders as electrical current will pass through them.</td>
		</tr>
		<tr>
			<td>Rubber lineman&#39;s gloves</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Personal protective equipment for use during electrofishing.</td>
		</tr>
		<tr>
			<td>Dip nets with fiberglass poles</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to capture stunned fishes during electrofishing.</td>
		</tr>
		<tr>
			<td>Backpack electrofishing unit</td>
			<td>Smith-Root; Halltech; Midwest Lake Management; Aqua Shock Solutions</td>
			<td>www.smith-root.com; www.halltechaquatic.com; https://midwestlake.com; https://aquashocksolutions.com/</td>
			<td>Backpack electrofishers are currently manufactured and distributed by four independent companies in North America. Prices and warranty/technical support are the most important factors in choosing a vendor.</td>
		</tr>
		<tr>
			<td>Block nets/seines (&#215;2)</td>
			<td>Duluth Nets</td>
			<td>https://duluthfishnets.com/</td>
			<td>Necessary length will depend on stream width. 3/8 inch mesh is recommended.</td>
		</tr>
		<tr>
			<td>Cam-action utility straps with 1 inch nylon webbing (&#215;4)</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to secure/anchor block nets. Available at auto supply, hardware, and department stores.</td>
		</tr>
		<tr>
			<td>Large tent stakes (&#215;4)</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to secure/anchor block nets. Available at camping and department stores.</td>
		</tr>
		<tr>
			<td>5 gallon plastic buckets (&#215;5)</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to hold and transport fish during electrofishing. Available at hardware and paint supply stores.</td>
		</tr>
		<tr>
			<td>10-20 gallon totes (&#215;3)</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used as livewells, sedation tanks, and recovery bins for captured fishes. Available at hardware and department stores.</td>
		</tr>
		<tr>
			<td>Battery powered &#39;bait bucket&#39; aeration pumps</td>
			<td>Cabelas</td>
			<td>IK-019008</td>
			<td>Used to aerate fish holding bins during field processing.</td>
		</tr>
		<tr>
			<td>Fish anesthesia (Tricaine-S)</td>
			<td>Syndel</td>
			<td>www.syndel.com</td>
			<td>Used to sedate fishes for field processing. Tricaine-S is regulated by the U.S. Food and Drug Administration.</td>
		</tr>
		<tr>
			<td>Folding camp table and chairs</td>
			<td>Cabelas</td>
			<td>IK-518976; IK-552777</td>
			<td>Used to process fish samples.</td>
		</tr>
		<tr>
			<td>Pop-up canopy</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used as necessary for sun and rain protection.</td>
		</tr>
		<tr>
			<td>Fish measuring board</td>
			<td>Wildco</td>
			<td>3-118-E40</td>
			<td>Used to measure fish lengths.</td>
		</tr>
		<tr>
			<td>Battery powered field scale with weighing dish</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to weigh fishes. Must weigh be accurate to 0.1 or 0.01 grams.</td>
		</tr>
		<tr>
			<td>Clear plastic wind/rain baffle</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used to shield scale in rainy or windy conditions. Must be large enough to cover the scale and a weighing dish.</td>
		</tr>
		<tr>
			<td>White plastic or enamel examination trays</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Trays are essential for examining fishes in the field.</td>
		</tr>
		<tr>
			<td>Stainless steel forceps</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Forceps are helpful when examining small fishes and in transfering invertebrates to specimen jars.</td>
		</tr>
		<tr>
			<td>Hand magnifiers</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Magnification is often helpful when identifying fish specimens in the field.</td>
		</tr>
		<tr>
			<td>Fish identification keys</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Laminated keys that are custom prepared for specific locations are most effective.</td>
		</tr>
		<tr>
			<td>Datasheets printed on waterproof paper</td>
			<td>Rite in the Rain</td>
			<td>n/a</td>
			<td>Waterproof paper is essential when working with aquatic specimens.</td>
		</tr>
		<tr>
			<td>Retractable fiberglass field tapes</td>
			<td>Lufkin</td>
			<td>n/a</td>
			<td>Used to measure stream channel dimensions.</td>
		</tr>
		<tr>
			<td>Surber sampler or Hess sampler</td>
			<td>Wildco</td>
			<td>3-12-D56; 3-16-C52</td>
			<td>Either of these fixed-area benthic samplers will work well in shallow streams with gravel or pebble substrate.</td>
		</tr>
		<tr>
			<td>70% ethanol or isopropyl alcohol</td>
			<td>Multiple options</td>
			<td>n/a</td>
			<td>Used as invertebrate preservative.</td>
		</tr>
		<tr>
			<td>Widemouth invertebrate specimen jars (20-32 oz.)</td>
			<td>U.S. Plastic Corp.</td>
			<td>67712</td>
			<td>Any widemouth plastic jars will work but these particular jars are durable and inexpensive.</td>
		</tr>
	</tbody>
</table>

modeling the size spectrum for macroinvertebrates and fishes in stream ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

Exemplar results, including original field data, are presented for Slaunch Fork, West Virginia, a small stream in southern West Virginia. Additional size spectra model results are also presented for two other streams in the same region: Camp Creek and Cabin Creek, West Virginia. These are the three study sites included in McGarvey and Kirk17, but data presented here are from new samples collected in May 2015. A fully worked, manual example of the size spectra modeling process is included for the S...

Watch this Scientific Journal Video about Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems at JoVE.com

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

modeling-size-spectrum-for-macroinvertebrates-fishes-stream

Research

JoVE Journal

Environment

7.3K Views.  Virginia Commonwealth University. This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

Video: Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...