Nos gustaría agradecer a la Fundación Canadiense para la Innovación, el British Columbia Fondo de Desarrollo de conocimientos y el Nacional de Ciencias e Ingeniería de Investigación (NSERC) de Canadá para apoyar los estudios en curso en las regiones de bajo nivel de oxígeno de las aguas oceánicas costeras y abiertas. DAW fue apoyado por becas de NSERC, Killam y la Fundación Centro TULA financiado por la diversidad microbiana y la evolución.

<ol><li> Puesta en marcha de aparatos de filtración <ol><li> Antes de filtrar, primero limpie la condensación de las botellas llenas y pesarlos </li><li> Coloque una bandeja de autoclave en un lado de la bomba, y el frasco de residuos en el otro. </li><li> A continuación, coloque una botella de muestra completa en la bandeja de autoclave, sin embargo, no quite la tapa de la botella todavía. </li><li> Pase el tubo a través de la bomba peristáltica para que el extremo luer del tubo está en el frasco. </li><li> Con unas pinzas, retirar el algodón de una pipeta estéril de 25 ml, y adjuntar la pipeta en el otro extremo de la tubería. </li></ol></li><li> Filtración de agua de mar <ol><li> Para empezar a filtrar el agua de mar, abrir la botella e insertar la pipeta. Encienda la bomba y ajustar la velocidad de flujo a 1. Reloj para asegurar que el agua se bombea en la dirección correcta. </li><li> Permitir ~ 100 ml de agua pase a través del tubo entero. </li><li> Apague la bomba y coloque un filtro Sterivex a la conexión luer macho en el extremo libre de la tubería. </li><li> Continuar el filtrado hasta que no quede agua en las botellas. </li></ol></li><li> El almacenamiento de filtros <ol><li> Para almacenar los filtros, en primer lugar expulsar cualquier resto de agua con una jeringa de 30-60. </li><li> Selle la parte inferior del filtro Sterivex con Parafilm. Con una pipeta, añadir 1,8 ml de solución amortiguadora de lisis del filtro, manteniendo ~ 200 ul de espacio en el filtro para la adición posterior de los reactivos. </li><li> Luego, selle la parte superior del filtro Sterivex con un pequeño trozo de Parafilm, y la etiqueta de los filtros con la fecha y la identificación de la muestra. </li><li> Almacenar los filtros de pre-etiquetados tubos Falcon de 50 ml a -80 ° C </li></ol></li><li> Limpiar <ol><li> Inserte la pipeta de 25 ml en un matraz que contenía 0,1% de HCl, y el interruptor de la bomba. </li><li> Después del lavado HCl, enjuague el tubo con agua esterilizada. Por último, medir y registrar la masa de las botellas de 1 litro vacía con el fin de calcular el volumen de agua que pasa por el filtro. </li></ol></li></ol> Resultados representante: Cada muestra de agua de 1 L rinde un Sterivix filtro, que contiene la biomasa mayor de 0,22 micras. Esto se almacena en buffer de lisis para la extracción de ADN y posterior análisis. El resultado final consiste en un filtro de Sterivix por botella de 1 litro almacenados en buffer de lisis y listo para la extracción de ADN. El filtro contiene la biomasa mayor de 0,22 micras. Los filtros pueden ser almacenadas a -80 ° C antes de la extracción Edna.

The authors have nothing to disclose.

Es imprescindible para limpiar los bancos y las bombas con una toalla húmeda después de cada uso para eliminar cualquier rastro persistente de agua salada que de otra manera corroer el equipo. Aunque el procedimiento es simple, el tiempo requerido puede ser extensa en función de la concentración de la biomasa o los residuos de la muestra que se filtra.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>77913-20, 77200-62, 77200-04</td>
<td>&nbsp;</td>
<tr>
<td>Tygon LFL tubing</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>06429-24</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Hose clamps</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>0683204</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>1/4&#x201D; x 3/16&#x201D; pipe adaptors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31702-20</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Polypropylene prefilter housing</td>
<td>&nbsp;</td>
<td>ADVANTEC</td>
<td>501200</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Metal luer fitting (Hose end to Male luer lock)</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31507-27</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>2&#x201C; Magnets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>3 port carboy caps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>GV 0.22 &mu;m STERIVIX filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SVGV01R5</td>
<td>&nbsp;</td>
<tr>
<td>47 mm diameter 2.7 &mu;m GF/D filters</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1823047</td>
<td>&nbsp;</td>
<tr>
<td>Lysis buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>0.1 % HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>
<tr>
<td>Autoclaved water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>		</tbody>
		</table>
		</div>

small volume (1-3l) filtration of coastal seawater samples

El flujo de trabajo comienza con la recolección de las aguas marinas costeras de la comunidad microbiana abajo, análisis de gases en nutrientes y oligoelementos. Para las muestras de la manifestación de hoy s se obtuvieron de la cubierta de la operación HMS John Strickland en Saanich Inlet. Este video documenta pequeño volumen (aproximadamente 1 L) de filtración de la biomasa microbiana de la columna de agua. El protocolo es una extensión del protocolo de muestreo a gran volumen se describió anteriormente, con una diferencia importante: aquí no hay paso de pre-filtración, por lo que todas las clases de tamaño de la biomasa se recogen hasta los 0,22 m de corte del filtro. Las muestras recogidas de esta manera son ideales para el análisis de ácidos nucleicos. Pasos de la instalación, filtración y limpieza de cada toma unos 20-30 minutos. Si se usan dos bombas peristálticas de forma simultánea, hasta 8 muestras pueden ser filtrados al mismo tiempo. Para prevenir la formación de biofilm entre los viajes de muestreo, todo el equipo de filtración debe ser enjuagado con HCl diluido y agua destilada y autoclave inmediatamente después del uso.

Watch this Scientific Journal Video about Small Volume 1-3L Filtration of Coastal Seawater Samples at JoVE.com

Small Volume 1-3L Filtration of Coastal Seawater Samples

Este video documenta pequeño volumen (aproximadamente 1 L) de filtración de la biomasa microbiana de la columna de agua.

Volumen pequeño (1-3L) Filtración de muestras de agua marina costera

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

small-volume-1-3l-filtration-of-coastal-seawater-samples

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Analytical Chemistry

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Unit 1

Small Volume (1-3L) Filtration of Coastal Seawater Samples

Introduction to Separation Methods

SCIENTISTS IN ACTION

11.1K vistas.  University of British Columbia - UBC. Este video documenta pequeño volumen (aproximadamente 1 L) de filtración de la biomasa microbiana de la columna de agua.

Video: Small Volume 1-3L Filtration of Coastal Seawater Samples

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

El agua de mar de muestreo y recolección

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Investigación

Biología

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

De gran volumen (20 litros +) Filtración de muestras de agua marina costera

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Sustrato Generación para endonucleasas de CRISPR / Cas Sistemas

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un método de alto rendimiento para la medición de la tasa de filtración glomerular en ratones conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Aislamiento rápido y purificación de mitocondrias para el trasplante por disociación de tejidos y filtración diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Demostración Laboratorio Fisiología: tasa de filtración glomerular en una rata

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

La medición de volumen de bucles de presión en el ratón

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

Segmentación de volumen y análisis de materiales biológicos con SuRVoS (Super-región segmentación de volumen) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.