Wir möchten den kanadischen Stiftung für Innovation, der British Columbia Knowledge Development Fund und der National Sciences and Engineering Research Council (NSERC) of Canada für die Unterstützung laufender Studien auf niedrigem Sauerstoffgehalt Regionen der Küsten-und offenen Meer zu danken. Darüber hinaus wurde DAW durch Stipendien von NSERC, Killam und die TULA Stiftung finanziert Zentrum für Mikrobielle Diversität und Evolution unterstützt.

Teil 1: Wasser-Sammlung <ol><li> Die Tiefen werden auf jeden cast zugeordnet. </li><li> Tiefen bis abgetastet und das Gesamtvolumen für die aktuellen Besetzung gesammelt werden ermittelt. </li><li> Es ist sichergestellt, dass die Go-Flo und Niskin Flaschen geöffnet sind, und dass ihre Ventile und Zapfen sind geschlossen. </li><li> Die CTD ist auf Grund der Zeile geladen und unteren ins Wasser. </li><li> Eine Flasche für die tiefste Tiefe in gegossenen Proben zunächst auf der Linie nach dem CTD geladen, es ist sichergestellt, dass die Linie richtig in die Halterung montiert und die Schrauben angezogen sind. </li><li> Ein Bote ist zu jeder Flasche angebracht (mit Ausnahme der tiefste) vor dem Absenken. </li><li> Die Linie ist der Abstand zwischen der ersten Tiefe und das nächste gesenkt, eine weitere Flasche wird dann geladen. Anzumerken ist, dass für die Tiefen, in denen 20L für die Filtration gesammelt, insgesamt 2 12L Flaschen nacheinander über 0,75 m geladen auseinander liegen. </li><li> Wenn alle Flaschen auf der Linie ist die Linie in die gewünschte Tiefe abgesenkt und 2 Minuten, werden durchgelassen. </li><li> Ein Bote ist ab und an ein paar Minuten werden durchgelassen, dass alle Flaschen in der Nähe zu gewährleisten. </li><li> Die Linie wird in taumelte und die Flaschen werden entladen, da sie bis auf die Regale kommen. </li><li> Für Tiefen, 20L Proben für DNA-und RNA benötigen, sind zwei Niskin Flaschen gesammelt. Eine Flasche wird verwendet, um die chemischen Parameter (siehe unten) Probe, während der andere in den Ballon abgelassen wird. RNA wird sofort aus der zweiten Niskin gesammelt. Der Rest der ersten ist auch in den Kanister auf ~ 20L Endvolumen geben abgelassen. Für Low-Resolution-Proben nur 1L Wasser beiseite für die Filtration in sterile Nalgene Flaschen eingestellt. </li></ol> Teil 2: Gas Probennahme <ol><li> Wenn Sauerstoff Proben benötigt werden, werden sie gesammelt erste. </li><li> Ein Stück dünnen Schlauch wird auf die Niskin befestigt / Go-Flo Zapfen und das andere Ende des Schlauches ist Platz ins Glas Serumflaschen platziert. </li><li> Das Serum Flasche wird 3 mal mit der Probe Wasser gespült und gefüllt bis zum Überlaufen, so dass keine Luftblasen vorhanden sind. </li><li> 50 ul Quecksilberchlorid (HgCl 2) sind die Serum-Flasche in einer Weise, die nicht vorstellen tut Luftblasen in der Probe zugegeben. </li><li> Die Flasche wird mit einem Gummistopfen und gekräuselten mit einer Aluminium-Kappe verschlossen, und wird dann auf Eis in einem kühleren gespeichert. </li></ol> Teil 3: Nährstoff-Probennahme <ol><li> A 60 cc Spritze mit Probe Wasser gespült, ist ein (0,2 pm) in-line befestigt und etwas Wasser wird durch die Acrodisc geschoben. </li><li> Ein Polyethylen-Flasche wird mit ca. 10 ml gefiltertes Wasser 3-mal gespült (schütteln Flasche beim Spülen). </li><li> Filtrierten Probe Wasser, um die Flaschen hinzu, etwa ¾ voll. </li><li> Die Probe wird auf Eis in den kühleren gespeichert. </li></ol> Teil 4: Ammoniak Probennahme <ol><li> Proben werden mit der Probe gespült. </li><li> Probenröhrchen sind komplett mit der Probe gefüllt. </li><li> 5 ml Probe Wasser mit Hilfe einer 10ml Pipette aufgenommen, der Rest des Wassers in der Röhre wird verworfen und die 5ml in der Pipettenspitze in die Probenröhrchen ersetzt. </li><li> 7.5ml von Ammonium arbeiten Reagenz in jedes Röhrchen gegeben, wenn Proben aus allen Tiefen gesammelt wurden. </li></ol> Teil 5: Probenentnahme für die Zell-Zählung <ol><li> Ein pre-markierten 50 ml Falcon Röhrchen mit 5,4 ml 37% Formaldehyd ist an der Oberseite für eine Endkonzentration von 4% gefüllt. Es wird darauf geachtet nicht zu verschütten. </li></ol> Teil 6: Probenahme für Schwefelwasserstoff Messungen <ol><li> Ein pre-markierten 50 ml Falcon-Röhrchen mit 20% Zinkacetat (Zn (O 2 CCH 3) 2) ist an der Oberseite gefüllt. Es wird darauf geachtet nicht zu verschütten. </li></ol> Teil 7: Kleines Volumen Kollektion für Biomasse-Konzentration <ol><li> 1-Liter-Flaschen sind auf dem Rand gefüllt ist. </li></ol> Teil 8: Großvolumige Kollektion für Biomasse-Konzentration <ol><li> 20-Liter-Kanister gefüllt sind. </li></ol> Teil 9: Repräsentative Ergebnisse Das endgültige Ergebnis des Meerwassers Probenahme Ergebnisse in etwa 12 gelösten Sauerstoff Proben, 32-48 Gasproben, 16 Nährstoff-Proben, 32 Ammonium-Proben, 16 Schwefelwasserstoff Proben, 16 Proben für die Zellzahlen, 6 20-Liter-Kanister, 16 1-Liter-Flaschen und 4 Sterivex 0,22 &amp;mgr; m-Filter enthalten, mikrobielle Biomasse mit RNA erhalten später.

<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.

Insgesamt gibt es ein hohes Maß an Vorbereitung und Planung für diese Art der Probenahme erforderlich, so Budget 2 Tage vor der Reise, die Dinge in Ordnung und 1 Tag später bekommen für die Reinigung alles auf. In der Regel lassen Sie sich nicht von Salzwasser in Kontakt mit der Schlauchpumpe oder in den Aluminium-Zarges-Box verwendet, um den Feldgeräten Transport kommen. Behandeln Sie die Geräte mit Respekt und es tadellos für eine lange Zeit zu kommen.

<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

Das Video dokumentiert Methoden zur Erhebung Coastal Marine Wasserproben und deren Verarbeitung für verschiedene Downstream-Anwendungen einschließlich Biomasse-Konzentration, Aufreinigung von Nukleinsäuren, Zell-Hülle und Fülle, Nährstoff-und Spurengasen analysiert. Für die heutige Demonstration Proben wurden vom Deck der HMS John Strickland, die in Saanich Inlet gesammelt. Ein Ein-Rahmen-Bohrturm, mit einem Mehrzweck-Winde und Kabel-System wird in Kombination mit Niskin verwendet oder Go-Flo Wasser Probenflaschen. Leitfähigkeit, Temperatur und Tiefe (CTD) Sensoren werden auch verwendet, um die zugrunde liegenden Wassermassen Probe. Zur Minimierung der Ausgasung, sind Spurengas gesammelten Proben zuerst. Dann werden Nährstoffe, Wasser Chemie und Zellzahlen bestimmt. Schließlich werden die Gewässer für Biomasse Filtration gesammelt. Das Set-up und Abholzeit für einem Guss ist ~ 1,5 h bei einer maximalen Tiefe von 215 Metern. Daher ist insgesamt 6 Stunden in der Regel erforderlich, um die Sammlung Serie hier beschriebenen abzuschließen.

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

Seawater Sampling and Collection

Das Video dokumentiert Methoden zur Erhebung Coastal Marine Wasserproben und deren Verarbeitung für verschiedene Downstream-Anwendungen einschließlich Biomasse-Konzentration, Aufreinigung von Nukleinsäuren, Zell-Hülle und Fülle, Nährstoff-und Spurengasen analysiert.

Meerwasser Probenahme und Sammlung

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

Nanyang Technological University

Research

JoVE Journal

Biology

19.9K Aufrufe.  University of British Columbia - UBC. Das Video dokumentiert Methoden zur Erhebung Coastal Marine Wasserproben und deren Verarbeitung für verschiedene Downstream-Anwendungen einschließlich Biomasse-Konzentration, Aufreinigung von Nukleinsäuren, Zell-Hülle und Fülle, Nährstoff-und Spurengasen analysiert.

Serie: 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 Ascosporen Produktion und Sammlung für Microarray-Experimente.

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

Forschung

Biologie

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.

Sammlung und Kryokonservierung von Oozyten Hamster und Maus Embryonen

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.

Large Volume (20L +) Filtration von Coastal Meerwasser Proben

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.

Small Volume (1-3L) Filtration von Coastal Meerwasser Proben

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.

Diagnostic Necropsy und ausgewählte Gewebe und Probenentnahme bei Ratten und Mäusen

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 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.

Milchauffangmethoden für Mäuse und 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 ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Sammlung und Analyse von<em&gt; Arabidopsis</em&gt; Phloemexsudaten Mit der EDTA-Methode erleichtert

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

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.

Nicht-Terminal-Blut-Stichprobenverfahren bei Meerschweinchen

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

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Wiederholte Blutentnahme für Bluttests in Adult Zebrafisch

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Gamete Collection und In-Vitro-Fertilisation von Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...