This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.

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The authors have nothing to disclose.

小型化された形式での藻類の成長。 マイクロタイタープレート中の1ml培養体積に藻類培養物の微細化が増加する実験内での複製を可能にする。それは藻が実験を通して、健康であることを確認することが重要です。藻類の栄養要件が満たされていることを確認するために、様々な藻類メディアを評価するために、マイクロタイタープレートフォーマットを用いて、増殖曲?...

植物プランクトンの生理機能は、伝統的に三角フラスコ中で20ミリリットルからカーボイ1-7 ​​で5 Lの範囲のメソスケールの実験で研究されている。各時点で複製試料を犠牲にすることは手に負えない実験装置を作成し、この実験的なスケールは、実験的な監視のためのサブサンプリングが必要です。 藻類の生理学実験のための実験容積を小型化することによって、同じ日周インキュベータ·スペースを使用しながら独立した実験の数を増加させる能力は、大量のサンプリング擬似複製の制限を軽減または排除する。マイクロタイタープレート形式は、実験変数の条件で藻類を操作するための1ml培養体積を使用して、藻類のバイオアッセイのために開発された。この実験小容積が増加する反復の数を可能に起因複製試料との間の減少した変動性、実験の再現性を増加させる実験、および140日間（ 図2）12のための実験対照（ すなわち、純粋藻類培養液）を維持しながら、真の複製を可能にする。 このマイクロタイタープレート形式で容易のような実験的な質問、各種のために適合される：細菌は、その藻類ホストと、共生中性または病原性の相互作用がありますか？藻類への化合物の刺激の追加や毒性はありますか？これらおよび他の問題は、この新しい形式9-11を用いた迅速なハイスループットの方法で対処することができる。 48ウェルマイクロタイター培養プレートに1mlずつウェル単一の時点でサンプリングされる独立した実験設定にすることができる。各種パラメータを含め、この1ミリリットルボリュームからサ ​​ンプリングしたが、これらに限定されないことができます：水-パルス振幅変調（WATER-PAM）蛍光法（ 材料および機器の表を参照）1を用いたクロロフィル蛍光及び光化学パラメータ3。WATER-PAMの蛍光測定は、藻類13で行われた実験を監視するために使用することができる迅速かつ非侵襲的な技術である。 14,15（WATER-PAMのために4mlの容量- - 2培地で希釈した培養液300μl150）それは小さな培養液量から光合成効率とPSII健康の測定を可能にする。 WATER-PAMの蛍光測定に加えて、この設定を含む他の様々なパラメータを測定するために用いることができるが、これらに限定されない：藻類細胞および藻類細胞形態の変化に付着した細菌を可視化する顕微鏡。単位（CFU）のカウントを形成する細菌のコロニー。と藻類の細胞数と識別する亜集団のためにフローサイトメトリー。

<table border="0" cellpadding="0" cellspacing="0" width="1233">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Name of Material/ Equipment</td>
			<td style="width:328px;">Company</td>
			<td style="width:331px;">Catalog Number</td>
			<td style="width:163px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cu. ft. Diurnal Incubator (6012-1)</td>
			<td>Caron Corporate</td>
			<td>112310-6012-1-11</td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Nunc EasYFlask 25cm2, Vent/Close Cap, 7mL working volume, 200/Cs&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N156340</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell TC Plates &#8211; 48 Well</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>353078</td>
			<td><a href="http://www.bdbiosciences.com/">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P1000 Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F123602</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P10mL Gilson The Pipetting Standard&#8212;Gilson&#39;s Pipetman</td>
			<td>Mandel Scientific Company Inc.</td>
			<td>GF-F161201</td>
			<td><a href="http://www.mandel.ca">www.mandel.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wide Orifice Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-468</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafine Tips nonsterile [100&#8211;1250 &#181;L]</td>
			<td>VWR International</td>
			<td>89079-470</td>
			<td><a href="http://www.ca.vwr.com">www.ca.vwr.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Finntip 10mL [Vol: 1-10mL]</td>
			<td>Thermo Fisher Scientific</td>
			<td>9402151</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WATER-Pulse Amplitude Modulation (Water-ED)</td>
			<td>Heinz Walz GmbH, Effeltrich, Germany</td>
			<td>EDEE0232</td>
			<td><a href="http://www.walz.com">www.walz.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mm diameter quartz glass cuvette (WATER-K)</td>
			<td>Caron Corporate</td>
			<td></td>
			<td><a href="http://www.caronproducts.com">www.caronproducts.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium&#160;Chloride&#160;(Crystalline/Certified ACS), Fisher Chemical</td>
			<td>Thermo Fisher Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td><a href="http://www.fishersci.ca">www.fishersci.ca</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Difco Marine Broth 2216</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Bacto Agar</td>
			<td>BD Biosciences Discovery Labware</td>
			<td>BD Biosciences Discovery Labware</td>
			<td><a href="http://www.bdbiosciences.com">www.bdbiosciences.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L1 Medium Kit, 50L</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td>NCMA [National Center for Marine Algae and Microbiota</td>
			<td><a href="http://www.ncma.bigelow.org">www.ncma.bigelow.org</a></td>
		</tr>
	</tbody>
</table>

a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry

培養物は、実験1-7を通してサブサンプリングすることができるように、微細藻類の実験操作のための従来の方法は、培養物の大量の（5 L 20 ml）を使用している。 1）それは、全体積の変化および表面積を引き起こす：大量のサブサンプリングは、いくつかの理由で問題となることが実験中の培養物の体積比; 2）疑似レプリケーション（ すなわち、同じ治療フラスコ8からサ ​​ンプルを複製する）は、多くの場合、むしろ真の複製（複製処理からのすなわち、サンプリング）も採用されている。 3）実験の期間は、総容量によって制限されます。汚染は、一般的にサブサンプリング中に起こるような、4）純粋培養または通常の細菌叢は、長期間の実験の間に維持することが困難である。 マイクロタイタープレートの使用は、内、最大48の別々の治療法を用いて1ml培養毎に使用するボリュームの複製を可能に12.65 X 8.5×2.2センチプレート、それによって実験的なボリュームを減少させ、任意の治療をサブサンプリングすることなく、大規模な複製を可能にする。 9-11をスクリーニングする細菌藻類共培養物、藻類生理学テスト、および毒素：さらに、この技術は、実験を含む種々の型式に適合するように修正することができる。水 - パルス振幅変調（WATER-PAM）蛍光法、顕微鏡検査、細菌のコロニー形成単位（CFU）：藻類、細菌および/または共培養は、数々の実験手順のためにサンプリングしたが、これに限定されないことができますと、個々のウェルカウントおよびフローサイトメトリー。マイクロタイタープレート形式とWATER-PAMの蛍光測定の組み合わせは、光化学収率および試料間の低い変動性を有する他の光化学的パラメータの複数の迅速な測定を可能にし、高い再現性実験の過程でカーボイまたは三角フラスコをサブサンプリングの多くの落とし穴を回避する。

WATER-PAMの蛍光定量法の測定値。 水 - パルス振幅変調（PAM）蛍光光度法、蛍光（クロロフィル含量のプロキシ）と藻類の文化の光合成収率（PSII健康）を決定するための迅速かつ効率的な方法である。 PAM WinControlソフトウェアは、（以下では暗順応藻類のサンプルのための基本的なパラメータである）のための生データ値のスプレッドシートを生成します。 <p class="jove_cont...

Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

水 - パルス振幅変調（WATER-PAM）蛍光測定を用いて細菌/植物プランクトンの共培養を評価するために小音量バイオアッセイ

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

a-small-volume-bioassay-to-assess-bacterialphytoplankton-co-culture

Research

JoVE Journal

Environment

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

10.4K ビュー.  University of Alberta. The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

ビデオ: A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

水からのウイルス濃度のため、小容量の手順

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

環境学

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

水不飽和システムにおける有機汚染物質の輸送および生物学的利用能を評価するために、全細胞バイオレポーターアプローチ

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

三次元根系のための抽出メトリック：イン土壌のX線コンピュータ断層撮影データからボリュームおよび表面分析

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

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

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

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

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

Oの底生交流<sub&gt; 2</sub&gt;、N<sub&gt; 2</sub小さなコアインキュベーションを使用&gt;と溶存栄養素

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

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

低コスト、小型で、簡単に構築されたフライトのミルを使用してアンブロシア甲虫、カシノナガキクイムシ(村山) の飛行能力を測定

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

無菌状態で小さな分子に対する植物の反応を評価するための柔軟な低コスト化の養液栽培システム

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

再現可能な dsRNA のインジェクションと蚊とハエの産卵生物検定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

組成と異なる環境条件下におけるバイオエアロゾルの分布解析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and increasing the recovery of coalbed methane. A visualized and constant-volume gas-solid coupling system is introduced here to investigate the influence of CO2 sorption on the physical and mechanical properties of coal. Being able to keep a constant volume and monitor the sample using a camera, this system offers the potential to improve instrument accuracy and analyze fracture evolution with a fractal geometry method. This paper provides all steps to perform a uniaxial compression experiment with a briquette sample in different CO2 pressures with the gas-solid coupling test system. A briquette, cold-pressed by raw coal and sodium humate cement, is loaded in high-pressure CO2, and its surface is monitored in real-time using a camera. However, the similarity between the briquette and the raw coal still needs improvement, and a flammable gas such as methane (CH4) cannot be injected for the test. The results show that CO2 sorption leads to peak strength and elastic modulus reduction of the briquette, and the fracture evolution of the briquette in a failure state indicates fractal characteristics. The strength, elastic modulus, and fractal dimension are all correlated with CO2 pressure but not with a linear correlation. The visualized and constant-volume gas-solid coupling test system can serve as a platform for experimental research about rock mechanics considering the multifield coupling effect.

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal&#8217;s physical and mechanical properties induced by CO2 adsorption.

可視化・定量ガス固体結合試験システムを用いたCO2-ベアリング石炭を用いた単軸圧縮実験

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and ...