Gostaríamos de agradecer à Fundação Canadense para Inovação, a British Columbia Fundo de Desenvolvimento do Conhecimento e das Ciências e Pesquisa de Engenharia National Council (NSERC) do Canadá para apoiar estudos em andamento em regiões de baixo oxigênio das águas oceânicas costeiras e aberto. Além disso, DAW foi apoiado por bolsas de NSERC, Killam ea fundação TULA Centro financiado pela Diversidade Microbiana e Evolution.

Parte 1: Coleta de Água <ol><li> Profundidades são atribuídos a cada elenco. </li><li> Profundidades a amostrar eo volume total a ser recolhido para o elenco atual são determinados. </li><li> É assegurado que o Go-Flo e garrafas Niskin estão abertas, e que as suas válvulas e torneiras estão fechadas. </li><li> O CTD é carregado em parte inferior da linha e menor na água. </li><li> Uma garrafa para a maior profundidade amostrados no primeiro elenco é carregado para a linha após o CTD, é assegurado que a linha está devidamente instalado em suporte e os parafusos são apertados. </li><li> Um mensageiro é anexado a cada garrafa (exceto o mais profundo um) antes de baixar. </li><li> A linha é reduzida a distância entre a profundidade primeiro e no próximo; outra garrafa é então carregado. Note-se que para profundidades onde 20L são coletados para filtração, um total de 2 garrafas de 12L são carregados sobre consecutivamente 0,75 m de distância. </li><li> Uma vez que todas as garrafas estão na linha, a linha é reduzida para a profundidade desejada e 2 minutos são autorizados a passar. </li><li> Um mensageiro é enviado para baixo e alguns minutos são autorizados a passar para garantir que fechar todas as garrafas. </li><li> A linha é reeled e as garrafas são descarregados como eles vêm para cima das prateleiras. </li><li> Para profundidades que exigem amostras de 20L de DNA e RNA, duas garrafas Niskin são coletados. Um frasco da amostra é usada para os parâmetros químicos (veja abaixo), enquanto o outro é drenado para o garrafão. RNA é coletado imediatamente da Niskin segundo. O restante do primeiro também é drenado para o garrafão para dar volume de ~ 20L final. Para amostras de baixa resolução apenas 1L de água é posta de lado para filtração em garrafas estéreis Nalgene. </li></ol> Parte 2: coleta de amostra de gás <ol><li> Se as amostras de oxigênio são necessários, eles são recolhidos em primeiro lugar. </li><li> Um pedaço de tubo fino é anexado ao Niskin / Go-Flo torneira ea outra extremidade do tubo é colocado no lugar de garrafas de vidro de soro. </li><li> O frasco de soro é lavado três vezes com água da amostra, e transbordando de tal forma que nenhuma bolha de ar estão presentes. </li><li> 50μl de cloreto de mercúrio (HgCl 2) são adicionados ao frasco de soro de uma forma que não introduzir bolhas de ar na amostra. </li><li> O frasco é fechado com uma rolha de borracha e frisado com uma cápsula de alumínio, e é então armazenado em gelo em um refrigerador. </li></ol> Parte 3: coleta de amostra de nutrientes <ol><li> Uma seringa cc 60 é lavado com água de amostra, um (0,2 mm) é ligado em linha e um pouco de água é empurrado através da Acrodisc. </li><li> Um frasco de polietileno é lavado com ~ 10 ml de água filtrada 3 vezes (quando apertar garrafa limpeza). </li><li> Amostra de água filtrada é adicionado para as garrafas, cerca de ¾ cheio. </li><li> A amostra é armazenada em gelo no refrigerador. </li></ol> Parte 4: coleta de amostras de amônia <ol><li> Tubos de amostra são lavados com água da amostra. </li><li> Tubos de amostra são preenchidos completamente com água da amostra. </li><li> 5 ml de amostra de água são retirados com uma pipeta de 10ml, o resto da água no tubo é descartado eo 5ml na ponta da pipeta são substituídos dentro do tubo de amostra. </li><li> 7.5ml de reagente de amônio de trabalho é adicionado a cada tubo uma vez amostras de todas as profundidades foram coletadas. </li></ol> Parte 5: Coleta de amostras para contagem de células <ol><li> A pré-rotulados 50 ml tubo Falcon contendo 5,4 ml de formol 37% é cheio até o topo para uma concentração final de 4%. Cuidado é tomado para não derramar. </li></ol> Parte 6: Coleta de amostras para medições de sulfureto de hidrogénio <ol><li> A pré-rotulados 50 ml tubo Falcon contendo acetato de zinco a 20% (Zn (O 2 CCH 3) 2) é cheio até o topo. Cuidado é tomado para não derramar. </li></ol> Parte 7: coleta de pequeno volume para a concentração de biomassa <ol><li> Garrafas de 1 litro são preenchidos até o topo. </li></ol> Parte 8: coleta de grande volume para a concentração de biomassa <ol><li> Garrafões de 20 litros são preenchidos. </li></ol> Parte 9: Resultados Representante O resultado final da água do mar de amostragem resulta em cerca de 12 amostras de oxigênio dissolvido, 32-48 amostras de gás, 16 amostras de nutrientes, 32 amostras de amônio, 16 amostras de sulfeto de hidrogênio, 16 amostras para contagem de células, seis garrafões de 20 litros, 16 garrafas de 1 litro e 4 Sterivex 0.22μm filtros contendo biomassa microbiana preservada com RNA mais 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.

No geral, há um grande esforço de preparação e de planeamento necessários para este tipo de amostragem, de modo orçamento 2 dias antes da viagem para fazer as coisas em ordem e um dia depois para a limpeza de tudo. Em geral, não deixe a água salgada entrar em contacto com a bomba peristáltica ou na caixa de Zarges alumínio usado para transportar o equipamento de campo. Tratar o equipamento com respeito e que terá um bom desempenho por um longo tempo para vir.

<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 vídeo documenta métodos de coleta de amostras de água costeiros marinhos e processá-los para várias aplicações a jusante, incluindo concentração de biomassa, a purificação de ácido nucléico, a abundância de células, nutrientes e gases traço. Para amostras de demonstração de hoje foram coletados a partir do convés do HMS operacional John Strickland em Inlet Saanich. Um quadro A-guindaste, com um guincho multi-propósito e sistema de cabo, é usado em combinação com Niskin ou Go-Flo garrafas de amostragem de água. Condutividade, temperatura e profundidade (CTD) sensores também são utilizados para provar a massa de água subjacente. Para minimizar a saída de gás, amostras de gases-traço são coletados em primeiro lugar. Então, os nutrientes, química da água, e contagem de células são determinados. Finalmente, as águas coletadas para filtração de biomassa. O tempo de set-up e coleta de um elenco único é de aproximadamente 1,5 hora a uma profundidade máxima de 215 metros. Portanto, um total de seis horas é geralmente necessária para completar a série de coleta descrito aqui.

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

Seawater Sampling and Collection

Este vídeo documenta métodos de coleta de amostras de água costeiros marinhos e processá-los para várias aplicações a jusante, incluindo concentração de biomassa, a purificação de ácido nucléico, a abundância de células, nutrientes e gases traço.

A amostragem da água do mar e Cobrança

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 visualizações.  University of British Columbia - UBC. Este vídeo documenta métodos de coleta de amostras de água costeiros marinhos e processá-los para várias aplicações a jusante, incluindo concentração de biomassa, a purificação de ácido nucléico, a abundância de células, nutrientes e gases traço.

Vídeo: 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 produção de ascósporos e Coleta de Experimentos de Microarray.

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

Biologia

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.

Recolha e criopreservação de óvulos e embriões de camundongos Hamster

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.

Volume grande (20L +) Filtração das amostras Seawater Coastal

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.

Volume pequenas (1-3L) Filtragem de Amostras de Água do Mar Coastal

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.

Necropsia de diagnóstico e coleta de tecido e amostra selecionada em ratos e camundongos

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 coleta de leite para Ratos e Reeves Muntjac cervos

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 coleta de sangue não terminal em cobaias

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.

A amostragem de sangue a partir da veia lateral da cauda do rato

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.

Coleta e Identificação de Pólen das Colônias de Abelhas

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.

Coleta remota de dados de tomografia crio-elétron e média de subtomograma

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