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. Además, DAW fue apoyado por becas de NSERC, Killam y la Fundación Centro TULA financiado por la diversidad microbiana y la evolución.

Parte 1: Recolección de Agua <ol><li> Profundidades son asignados a cada elenco. </li><li> Profundidades para el muestreo y el volumen total que deben recopilarse para el reparto actual se determinan. </li><li> Se garantiza que el Go-Flo y botellas Niskin están abiertas, y que sus válvulas y grifos están cerrados. </li><li> El CTD se carga en el fondo de la línea y bajar en el agua. </li><li> Una botella de lo más profundo muestra en primer molde se carga en la línea después de la CTD, se garantiza que la línea esté correctamente ajustado en el soporte y los tornillos estén firmemente ajustadas. </li><li> Un mensajero se une a cada botella (con excepción de la más profunda) antes de bajar. </li><li> La línea se reduce la distancia entre la primera profundidad y el siguiente, otra botella se carga. Cabe señalar que para las profundidades donde se recogen 20 litros para la filtración, un total de 2 botellas de 12L se cargan consecutiva de 0,75 m de distancia. </li><li> Una vez que todas las botellas están en la línea, la línea se baja hasta la profundidad deseada y 2 minutos se les permite pasar. </li><li> Un mensajero es enviado ya pocos minutos se permite el paso para asegurarse de que todas las botellas de cerca. </li><li> La línea se tambaleaba y las botellas se descargan a medida que surgen en los bastidores. </li><li> Para profundidades que requieren muestras de 20 litros para el ADN y el ARN, dos botellas Niskin se recogen. Una botella se utiliza para tomar muestras de los parámetros químicos (ver más abajo), mientras que el otro se vierte en la bombona. ARN se obtiene inmediatamente de la segunda Niskin. El resto de los primeros es también vierte en la bombona para dar ~ volumen final de 20 litros. Para muestras de baja resolución sólo 1 litro de agua se destina a la filtración en botellas estériles de Nalgene. </li></ol> Parte 2: recolección de gas de muestra <ol><li> Si las muestras de oxígeno se necesitan, son recogidos por primera vez. </li><li> Un pedazo de tubo delgado se une a la Niskin / Go-Flo grifo y el otro extremo del tubo se coloca en lugar de las botellas de vidrio de suero. </li><li> La botella de suero, se enjuaga tres veces con agua de la muestra, y lleno a rebosar de tal manera que no queden burbujas de aire. </li><li> 50μl cloruro de mercurio (HgCl 2) se añade a la botella de suero en una forma que no introducir burbujas de aire en la muestra. </li><li> Se cierra la botella con un tapón de goma y ondulado con una tapa de aluminio, y se almacena en hielo en una hielera. </li></ol> Parte 3: recogida de muestras de nutrientes <ol><li> Una jeringa de 60 cc se enjuaga con agua de la muestra, uno (0,2 m) está conectado en línea y un poco de agua es empujada a través de la Acrodisc. </li><li> Una botella de polietileno, se enjuaga con agua ~ 10 ml de filtrado 3 veces (cuando se agite la botella de enjuague). </li><li> El agua filtrada de la muestra se añade a las botellas, alrededor de ¾ de su capacidad. </li><li> La muestra se almacena en hielo en el refrigerador. </li></ol> Parte 4: colección de amoníaco de la muestra <ol><li> Los tubos de muestra se enjuagan con agua de la muestra. </li><li> Tubos de muestra se llena completamente con agua de la muestra. </li><li> 5 ml de muestra de agua se saca con una pipeta de 10 ml, y el resto del agua en el tubo se descarta y el 5 ml en la punta de la pipeta se sustituyen en el tubo de la muestra. </li><li> 7,5 ml de reactivo de amonio de trabajo se añade a cada tubo una vez muestras de todas las profundidades han sido recogidos. </li></ol> Parte 5: La toma de muestras para el recuento de células <ol><li> Un pre-etiquetados 50 ml tubo Falcon que contiene 5,4 ml de formaldehído al 37% se ha llenado hasta la parte superior para una concentración final del 4%. Hay que tener cuidado de no derramar. </li></ol> Parte 6: toma de muestras de sulfuro de hidrógeno, las mediciones <ol><li> Un pre-etiquetados 50 ml tubo Falcon que contiene 20% de acetato de zinc (Zn (O 2 CCH 3) 2) se llena hasta el tope. Hay que tener cuidado de no derramar. </li></ol> Parte 7: colección de pequeño volumen para la concentración de biomasa <ol><li> Botellas de 1 litro se llena hasta el tope. </li></ol> Parte 8: colección de gran volumen para la concentración de biomasa <ol><li> Garrafones de 20 litros se llena. </li></ol> Parte 9: Resultados de Representante El resultado final de muestreo de agua de mar resulta en aproximadamente 12 muestras de oxígeno disuelto, 32-48 muestras de gas, 16 muestras de nutrientes, de 32 muestras de amonio, 16 muestras de sulfuro de hidrógeno, 16 muestras para el recuento celular, 6 garrafones de 20 litros, 16 botellas de 1 litro y 4 Sterivex 0.22μm filtros que contienen biomasa microbiana conservado con el ARN más tarde.

<ol>
	<li>Cline, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+hydrogen+sulfide+in+natural+waters.">Spectrophotometric determination of hydrogen sulfide in natural waters.</a> Limnology &#38; Oceanography. 14, 454-458 (1969).</li><li>Holmes, R. M., Aminot, A., Kerouel, R., Hooker, B. A., Peterson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+precise+method+for+measuring+ammonium+in+marine+and+freshwater+ecosystems.">A simple and precise method for measuring ammonium in marine and freshwater ecosystems.</a> Canadian Journal of fisheries and Aquatic Science. 56, 1801-1808 (1999).</li></ol>

The authors have nothing to disclose.

En general, hay una gran cantidad de preparación y planificación requerida para este tipo de muestreo, por lo económico de 2 días antes del viaje para hacer las cosas en orden y un día después de limpiar todo. En general, no deje que el agua salada entra en contacto con la bomba peristáltica o en la caja de aluminio Zarges utilizado para transportar el equipo de campo. El tratamiento de los equipos con respeto y que tenga un buen rendimiento durante mucho tiempo por venir.

<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>20L Carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-960-15</td>
<td>&nbsp;</td>
<tr>
<td>3/8" Tygon</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-36</td>
<td>&nbsp;</td>
<tr>
<td>Sterivex filters</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SVG010RS</td>
<td>&nbsp;</td>
<tr>
<td>Prefilter Housing Unit</td>
<td>&nbsp;</td>
<td>AMD</td>
<td>501200</td>
<td>&nbsp;</td>
<tr>
<td>Luer Fitting</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>31507-27</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>50ml Plastic tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass Serum Bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Glass Sample Collection</td>
</tr>
<tr>
<td>Septa and aluminum caps for glass serum bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hand Crimper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20-200ul Pipette and Tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass Tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ammonium Sample Collection</td>
</tr>
<tr>
<td>30ml Plastic Bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Nutrient Collection</td>
</tr>
<tr>
<td>Acrodisc Filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5 ml Tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Nitrite Sample Collection</td>
</tr>
<tr>
<td>37% Formaldehyde</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cell count samples</td>
</tr>
<tr>
<td>20% Zinc Acetate</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For sulfide samples</td>
</tr>
<tr>
<td>Mercuric Chloride</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For gas samples</td>
</tr>
<tr>
<td>milliQ water</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Working Reagent</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Borate buffer, sodium sulfite, orthophaladialdehyde</td>
</tr>
<tr>
<td>Sulfanilamide</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5g into 450ml water and 50ml HCl</td>
</tr>
<tr>
<td>N-(1-naphthy)-ethylenediamine dihydrochloride solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.5g into 500ml water</td>
</tr>
</tbody>
</table>

seawater sampling and collection

Este video documenta los métodos de recogida de muestras de agua marina costera y su procesamiento para diversas aplicaciones de aguas abajo como la concentración de biomasa, la purificación de ácidos nucleicos, la abundancia de células, los nutrientes y análisis de trazas de gas. Para las muestras de la manifestación de hoy se obtuvieron de la cubierta de la operación HMS John Strickland en Saanich Inlet. Un marco de A-torre, con un cabrestante de usos múltiples y un sistema de cable, se utiliza en combinación con Niskin o Go-Flo botellas de muestreo de agua. Conductividad, temperatura y profundidad (CTD), los sensores se utilizan también para probar la masa de agua subyacente. Para minimizar la emisión de gases, las muestras de gases traza son recogidos por primera vez. Entonces, los nutrientes, la química del agua, y el recuento de células se determinan. Finalmente, las aguas son recogidas para la filtración de la biomasa. El tiempo de preparación y de recolección de un reparto único es de ~ 1,5 horas a una profundidad máxima de 215 metros. Por lo tanto, un total de 6 horas es necesario en general para completar la serie de la colección se describe aquí.

Watch this Scientific Journal Video about Seawater Sampling and Collection at JoVE.com

Seawater Sampling and Collection

Este video documenta los métodos de recogida de muestras de agua marina costera y su procesamiento para diversas aplicaciones de aguas abajo como la concentración de biomasa, la purificación de ácidos nucleicos, la abundancia de células, los nutrientes y análisis de trazas de gas.

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 ...

Research

JoVE Journal

Biology

19.8K vistas.  University of British Columbia - UBC. Este video documenta los métodos de recogida de muestras de agua marina costera y su procesamiento para diversas aplicaciones de aguas abajo como la concentración de biomasa, la purificación de ácidos nucleicos, la abundancia de células, los nutrientes y análisis de trazas de gas.

Video: Seawater Sampling and Collection

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae ascósporas de producción y de recolección de experimentos de microarrays.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Investigación

Biología

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Recolección y criopreservación de ovocitos de hámster y de embriones de ratón

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

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, ...

Small Volume (1-3L) 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 today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

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 ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

La necropsia de diagnóstico y selecta colección de tejidos y muestras en ratas y ratones

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Métodos de recolección de leche Ratones y Reeves Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Técnicas de muestreo de sangre no terminal en los conejillos de Indias

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Toma de muestras de sangre de la vena lateral de la cola de la rata

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Recolección e identificación de polen de colonias de abejas melíferas

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Recopilación remota de datos por tomografía crioelectrónica y promedio de subtomografía

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...