Funding was made available by the Vermont Water Resources and Lake Studies Center through an agreement with the U.S. Geological Survey. Conclusions and opinions are those of the authors and not the Vermont Water Resources and Lake Studies Center or the USGS.

1. Sample collection
<ol>
	<li>Collect approximately 4 L of soil (or sediment) from desired sites. Collection areas should be relatively small to limit spatial variation in P and soil properties.</li>
	<li>Sieve samples through a coarse (20 mm) screen followed a 2 mm screen. Thoroughly hand-mix samples after sieving.</li>
	<li>Weigh out 100 g of field-moist soil or sediment. Dry in an oven at 105 &#176;C for 24 h and calculate gravimetric water content (soil water mass/dry soil mass).</li>
	<li>Take a 500 mL subsample for chemical analysis. 
	NOTE: Soil pH, organic matter content and labile inorganic P (Pi) concentration are recommended soil tests. Here, labile soil Pi availability was assessed by: 1) Pi extracted by 1.25 mol L-1 ammonium-acetate (pH = 4.8; hereafter referred to as modified Morgan extractable P) measured colorimetrically7,8, 2) Pi extracted by distilled water, and 3) P extracted by 1.25 mol L-1 ammonium-acetate (pH = 4.8) measured by inductively coupled plasma-optical emission spectroscopy (ICP)8.</li>
	<li>Use remaining sieved soil for microcosm studies or store in polyethylene bags at 5 &#176;C for later use. 
	NOTE: Soils dry out when refrigerated for long periods (&#62;30 days) and will require remoistening. Do not freeze soil samples as it affects microbial integrity and P release potential.</li>
</ol>
2. Microcosm construction
<ol>
	<li>Use one-liter (1 L) graduated polypropylene or other non-reactive plastic beakers as individual experimental units (microcosms). Wash beakers in 10% hydrochloric acid and triple rinse with distilled water.</li>
	<li>Measure 2 cm up from the bottom and place a mark next to beaker graduations. Drill a 1.25 cm diameter hole for drainage ports.</li>
	<li>Place a small bead of silicone around the inside edge of hose barb and outer circumference of the bore hole. Carefully insert the drainage port into the hole. 
	NOTE: Allow air-drying for at least 24 h before proceeding to step 2.4.</li>
	<li>Trace the outside circumference of hose barbs onto nylon mesh filter screen and cut out with scissors. Apply a thin bead of silicone around the circumference of each filter on the outside edge and press filters onto hose barb inlets. Allow at least 24 h of drying time before using. 
	NOTE: A 100 &#181;m pore size is recommended for most applications; however, finer-textured soils may require a larger filter pore size to avoid excessively long PW sample collection time.</li>
	<li>Fit a short piece of 0.625 cm diameter latex hose to hose barb ends. Attached a 3.3 cm wide paper binder clip to the hose to prevent flow.</li>
</ol>
3. Conducting a phosphorus release trial
<ol>
	<li>Place 500 mL of sample into duplicate microcosms and gently apply distilled water along beaker walls until FW reaches the 1 L mark. 
	NOTE: Allow microcosms to equilibrate for 24&#8722;48 h before taking initial samples.</li>
	<li>Unclip paper binders to induce PW flow through drainage port. Collect samples by placing clean 30 mL beakers directly beneath PW drainage ports. Allow several mL of PW to drain, discard and use the next 10 mL as a representative sample volume.</li>
	<li>Filter PW samples through 0.45 &#181;m membrane filters and immediately analyze for soluble reactive P (SRP). Record absorbance values and time of measurements. 
	NOTE: SRP is generally assumed to be orthophosphate; however, molybdate-reactive P can also form complexes with colloids and/or nanoparticles that pass through 0.45 &#181;m filters4.</li>
	<li>Take initial FW sample by inserting a 10 mL bulb syringe pipette halfway down the water column and withdraw a sample using a circular motion. Empty into beakers, filter and analyze immediately for SRP.</li>
	<li>Replace sampled water by refilling beakers to the 1 L level with distilled water. 
	NOTE: Evaporative losses will vary. The goal is to consistently maintain a total volume (flooded soil + water column) of 1 L in all microcosms. Replacing evaporative water losses has negligible dilution effects on SRP.</li>
	<li>Repeat steps 3.2 through 3.5 based on the desired number of P release time points for analysis. 
	NOTE: The number of samples taken over time depends on experimenter goals. Sampling one to three times per week is sufficient for many applications assuming incubations are close to 20 &#176;C. Incubating at higher temperatures increases SRP release rates and will require more frequent sampling. The intent here is to show the utility of the microcosm method rather than focusing on data analysis from experiments. Both kinetically-based and empirical models to fit P desorption/sorption data are presented elsewhere9,10. Since the microcosm method relies on a repeated measures design and accommodates replication and differing treatments, generalized linear mixed modeling approaches are also appropriate11.</li>
</ol>

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B., Kronvang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+retention+in+riparian+buffers:+review+of+their+efficiency.">Phosphorus retention in riparian buffers: review of their efficiency.</a> Journal of Environmental Quality. 38 (5), 1942-1955 (2009).</li><li>Bartlett, R. J., Ross, D. S., Tabatabai, M. A., Sparks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+Redox+Processes+in+Soils.">Chemistry of Redox Processes in Soils.</a> Chemical Processes in Soils, SSSA Book Ser. 8. , 461-487 (2005).</li><li>Radcliffe, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+water+extractable+soil+organic+phosphorus+by+phosphatase+hydrolysis.">Characterization of water extractable soil organic phosphorus by phosphatase hydrolysis.</a> Soil Biology &amp; Biochemistry. 34 (1), 27-35 (2002).</li><li>Turner, B. L., Haygarth, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+solubilization+in+rewetted+soils.">Phosphorus solubilization in rewetted soils.</a> Nature. 411, 258 (2001).</li><li>Sparks, D. L., Sparks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+reactions+in+pure+and+mixed+systems.">Kinetics of reactions in pure and mixed systems.</a> Soil Physical Chemistry. , (1986).</li></ol>

The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be interpreted as a potential conflict of interest.

A main technical advantage of the microcosm approach is its ability to simulate in-situ conditions whereby saturated soil or sediment is immediately overlain by FW that may substantially differ in redox and P status. Landscapes with variable source area hydrology such as drainage ditches, flooded cropland, wetlands, and riparian/near-stream zones are all examples of where reduced PW is periodically overlain by more oxidized water with lower Pi concentrations. These redox gradients can strongly affect Pi sorption...

Phosphorus (P) is a critical limiting nutrient for both crop and aquatic biomass productivity. Surface water hydrology is a main driver of P fate and transport, as it controls the physical transport of sediment and P while also affecting remobilization potential during runoff and flooding/ponding events. Various laboratory-based extraction methods are typically used to estimate P release at the field scale under oxidizing conditions. While different mechanisms can contribute to P release, reductive dissolution of iron-phosphates is a well-established reaction mechanism that can lead to large orthophosphate-P fluxes to water1,2,3,4. In a review of mechanisms controlling P biogeochemistry in wetlands, redox status was hypothesized to be the main variable controlling P release to soils and shallow groundwater5. As such, traditional P tests may not be reliable predictors of P release under prolonged saturation.
Given the importance of water residence time and redox status on P fate and transport, laboratory approaches designed to better simulate in situ conditions could lead to improved P transport risk indices for agricultural and wetland ecosystems subject to variable saturation. Since orthophosphate is immediately bioavailable, the rate and extent of desorption during saturation can be used as an index of nonpoint source P pollution risk. Our method was designed to quantify P desorption to porewater (PW) and mobilization to overlying floodwater (FW), a typical condition in areas with variable source area hydrology (e.g., flooded agricultural fields, wetlands, drainage ditches, and riparian/near-stream zones). The method was originally developed to characterize P release potential in seasonally flooded soils from northern New York (USA) and recently applied to quantify P desorption potential of riparian soils from northwestern Vermont&#8217;s Lake Champlain Basin6. Here, we provide a protocol for the laboratory microcosm method and highlight results from a recently published study demonstrating its ability to quantify P desorption potential. We also demonstrate the relationship between P release potential and the reliability of routine soil tests (labile extractable P, pH) to predict release across sites.
Carrying out the method requires access to an analytical laboratory with adequate climate control, ventilation, water, and a proper acid waste disposal system. The method presumes access to routine chemical reagents and laboratory equipment (sinks, hoods, glassware, etc.). Beyond routine laboratory needs, a membrane filtration (&#8804; 0.45 &#181;m) system is required and a UV spectrophotometer to measure P. A pH meter or multiparameter water quality probe are also recommended but not required. Laboratory temperature is an important factor and should be kept constant unless temperature itself is being investigated as an experimental factor (20 &#176;C is recommended). Unhindered access to an adequate analytical laboratory with proper equipment is a prerequisite to perform the method properly and generate meaningful results.

<table><tbody><tr><td>1.25 cm plastic hose barbs</td><td>numerous</td><td>NA</td><td></td></tr><tr><td>Chemical reagents for phosphorus determination</td><td>numerous</td><td>NA</td><td>P analysis capability is assumed; refer to cited references for details on method</td></tr><tr><td>Chordless or electric drill with 1.25 cm bit</td><td>numerous</td><td>NA</td><td></td></tr><tr><td>Graduated plastic beakers (1L)</td><td>numerous</td><td>NA</td><td></td></tr><tr><td>Laboratory with fume hoods, temperature control, and acid waste disposal system</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Nylon mesh filter screen (100um)</td><td>numerous</td><td>NA</td><td></td></tr><tr><td>Silicone</td><td>numerous</td><td>NA</td><td></td></tr><tr><td>UV Spectrophotometer</td><td>numerous</td><td>NA</td><td></td></tr></tbody></table>

measuring phosphorus release in laboratory microcosms for water quality assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Results from a recent study focused on P release potential of riparian areas are highlighted to demonstrate the method&#8217;s ability to characterize site-level P release dynamics6. While some soils showed minimal changes in SRP over time, others had large increases in PW- and FW-SRP concentrations (Figure 1). Two sites with contrasting trends are shown in Figure 1. Soil 7 is a riparian site with low soil pH and characterized by nearly c...

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Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...

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6.5K Views.  United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Institute for Environmentally Integrated Dairy Management. Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Video: Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Biology

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Immunology and Infection

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

Using a pH Meter

Source: Laboratory of Dr. Zhongqi He - United States Department of Agriculture
Acids and bases are substances capable of donating protons (H+) and hydroxide ions (OH-), respectively. They are two extremes that describe chemicals. Mixing acids and bases can cancel out or neutralize their extreme effects. A substance that is neither acidic nor basic is neutral. The values of proton concentration ([H+]) for most solutions are inconveniently small and difficult to compare so that a more practical quantity, pH, has been introduced. pH was originally defined as the decimal logarithm of the reciprocal of the molar concentration of protons <img alt="Equation 1" src="/files/ftp_upload/5500/5500eq1.jpg" />, but was updated to the decimal logarithm of the reciprocal of the hydrogen ion activity <img alt="Equation 2" src="/files/ftp_upload/5500/5500eq2.jpg" />. The former definition is now occasionally expressed as p[H]. The difference between p[H] and pH is quite small. It has been stated that pH = p[H] + 0.04. It is common practice to use the term &#39;pH&#39; for both types of measurements.
The pH scale typically ranges from 0 to 14. For a 1 M solution of a strong acid, pH=0 and for a 1 M solution of a strong base, pH=14. Thus, measured pH values will lie mostly in the range 0 to 14, though values outside that range are entirely possible. Pure water is neutral with pH=7. A pH less than 7 is acidic, and a pH greater than 7 is basic. As the pH scale is logarithmic, pH is a dimensionless quantity. Each whole pH value below 7 is 10x more acidic than the next integer. For example, a pH of 4 is 10x more acidic than a pH of 5 and 100x (10 x 10) more acidic than a pH of 6. The same holds true for pH values above 7, each of which is 10x more basic (or alkaline) than the next lower whole value. For example, a pH of 10 is 10x more basic than a pH of 9.