アンソニー Weinhold と私たちの昆虫飼育の手順を改善するための提案と同様、テクニカル サポートの年にわたって私たちと働いている多数の学生に感謝したいと思います。

1. 採卵 (図 1)
<ol><li>Egging カップを用意し、暖かい (~ 40 ° C) 水で満たされた ~ 500 mL ビーカーに布の片方の端を置きます。カップの両側をオーバー ラップし、輪ゴムで固定します。カップの上に戻って布の緩い端を折る。</li>
	<li>Egging カップ ケージ ~ 2 h. 妊娠女性の安定した古い 8-10 日ハエの飛ぶ場所が布のプレハブハウスします。</li>
	<li>スクイズ洗浄ボトルを小さな鍋に egging 布オフ ケージとリンスの卵から egging カップを削除します。</li>
</ol>2. 幼虫飼育のパンを準備します。
<ol><li>
メディア (1 飼育パンの量) の準備します。
	<ol>
<li>小麦ふすま (500 g)、木の削りくず (200 g) と魚の食事 (115 g) 10 L プラスチックの皿パンで組み合わせるし、よく混ぜます。</li>
		<li>(~ 25 ° C、1600 mL) の水に炭酸水素アンモニウム (50 g) を加え、溶けるまでを混ぜます。</li>
		<li>水ソリューションを乾燥した原料および unwetted 材料がないことを確認するためにミックスに追加します。パンの中のレベルが、圧縮しません。</li>
	</ol>
</li>
	<li>
安定した卵のフライを追加します。
	<ol>
<li>パンの長さ中の浅い溝を作る。</li>
		<li>卵ピペット (図 2) を溝に安定して飛ぶ卵 (卵 ~ 8,000) 1 mL を入金します。 注: 必要な時点で、100 を挿入するプラスチック卒業ピペットを切削で作られ卵ピペット メッシュ スクリーンと戻って一緒に接着のピペット。</li>
		<li>卵の乾燥を防ぐために培地の薄層をカバーしてください。</li>
		<li>カバー、ピローケース、パン、ラバー バンドとラベルを閉じます。幼虫の部屋で鍋を配置 (23 ± 2 ° C、30-50% RH 12:12 [L:D] h 日長)。 注: 幼虫の発育は、産卵の時から 10-14 日かかります。</li>
	</ol>
</li>
</ol>3. 蛹処理、幼虫は媒体の端へ移動 13 または 14 日産卵と Pupariate の後の 7-9 日
<ol><li>主にパンを培地の表面に形成し、きれいなフライパンに地殻の下の端のまわりにある puparia をスクープします。</li>
	<li>水の完全な 3/4 に ½ puparia で鍋を埋めます。Puparia 中小の塊を破る。培地の小麦ふすまとタンパク質成分シンク puparia ながら > 1 日古いフロート19。</li>
	<li>浮遊物、puparia、ザルやふるい (#5、7 #、#12 #20) 残りの媒体を削除する一連の洗浄といくつかの中のコンポーネントを削除します。#12 ふるい収集、puparia し #20 ドレインを入ることから固形廃棄物を保持します。シンク噴霧器とふるい間 puparia を洗います。</li>
	<li>#12 ふるいにきれいなフライパンに、puparia をすすぐ。水の ½ の完全にパンをご記入ください。ザルにフローティング puparia を注ぐでき、余分な水分を排出します。</li>
	<li>転送はファンと乾燥スクリーンに puparia を排水し、ドライ (図 3) まで残します。</li>
</ol>4. 蛹棚、Puparia を収集するための代替メソッドは、蛹の棚18
注: 棚は、10 L 料理鍋 (31.9 cm x 10.2 cm x 10.2 cm) の端からプラスチックのカットの部分から行われます。
<ol><li>2.1 で説明されるように媒体を準備します。テーパー中 ~7.5 cm ~2.5 cm 深さの 1 つの端からのパンの他に深い。</li>
	<li>
飼育のパンと幼虫が棚 (図 4) 横をクロールするを防ぐためにテープの浅い端の中に棚を配置します。
	<ol>
<li>スポンジ (~14.5 × 9.0 x 4.5 cm3、再生セルロース) を飽和水に浸した布 (30.5 x 42 cm2綿) や媒体からの約 1 つの半分インチの棚に場所で包む。</li>
	</ol>
</li>
	<li>
2.2 で説明されているように、卵を追加し、前に進みます。
	<ol>
<li>スポンジもしっとりしていることを毎日鍋を確認します。</li>
	</ol>
</li>
	<li>棚、スポンジ、きれいな皿に布からリンス puparia はパンおよび 14 日産卵後ザルに注ぐ。</li>
</ol>5. 品質管理
<ol><li>パン (総重量) で生産された puparia のすべての重量を量る。</li>
	<li>100 puparia を分離、重量を量る 9 cm シャーレに配置されます。</li>
	<li>カウントとセックス大人 〜 5-8 日後に出現率と性比を決定します。必要な場合、後日カウントするため冷凍庫に大人が登場し、シャーレを格納します。ハエが彼らの目の形と眼窩板20または低倍率下で性器の幅によって性欲を刺激します。</li>
	<li>レコードの総重量、重量 100 puparia、現れた大人の男性と女性の数。</li>
</ol>6. 血液の準備
<ol><li>70 g 水 500 mL にクエン酸ナトリウム第三水和物を含む 19 L バケツにローカルの屠畜場から新鮮な牛の血液を収集します。攪拌は、5-10 分は積極的に血液や研究室に戻った。</li>
	<li>削除するザルを通してひずみ血液凝固し、コレクションと冷凍庫 (-20 ° C) に保管の日付とラベルを 2 L 容器に分割します。</li>
	<li>冷凍庫、冷蔵庫内の場所から削除コンテナー 2 日前に必要です。〜 2 週間一度解凍し、1 年間冷凍保存血を使用できます。</li>
</ol>7. 成人のメンテナンス
<ol><li>ライン ケージ下部洗浄を容易にする肉屋の紙にケージを準備します。</li>
	<li>各ケージに 50 g の乾燥安定したハエ蛹 (~ 3,500) を配置します。大人 eclose 蛹処理後数日に数時間。</li>
	<li>場所新鮮な血液を供給するため各ケージの上に女性のナプキンを浸した。成虫の羽化の 24 h 内で給餌を開始し、産卵 (出現後 9-10 日) の前日まで毎日繰り返します。</li>
	<li>(セクション 1)、産卵後 4-8 時間冷凍庫にケージを置くし、紙を取り外し、frass の側面お湯で床からを洗浄および洗剤と塩素系漂白剤で表面を洗浄してきれい。お湯でケージを洗うし、乾燥することができます。</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+collecting+clean+stable+fly+(Diptera:Muscidae)+pupae+of+known+age.">A new method for collecting clean stable fly (Diptera:Muscidae) pupae of known age.</a> Southwest. Entomol. 34 (4), 469-476 (2009).</li><li>Champlain, R. A., Fisk, F. W., Dowdy, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+improvements+in+rearing+stable+flies.">Some improvements in rearing stable flies.</a> J. Econ. Entomol. 47 (5), 940-941 (1954).</li><li>Zumpt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The stomoxyine biting flies of the world. , (1973).</li><li>Wienhold, B. J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+properties+of+stable+fly+(Diptera:+Muscidae)+developmental+sites+associated+with+round+bale+hay+feeding+sites+in+eastern+Nebraska.">Substrate properties of stable fly (Diptera: Muscidae) developmental sites associated with round bale hay feeding sites in eastern Nebraska.</a> Environ. Entomol. 41 (2), 213-221 (2012).</li><li>Friesen, K., Berkebile, D. R., Wienhold, B. J., Durso, L., Zhu, J., Taylor, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+parameters+associated+with+stable+fly+(Diptera:+Muscidae)+development+at+hay+feeding+sites.">Environmental parameters associated with stable fly (Diptera: Muscidae) development at hay feeding sites.</a> Environ. Entomol. 45 (3), 570-576 (2016).</li><li>Albuquerque, T. A., Zurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+the+bacterial+community+of+animal+feces+and+their+correlation+with+stable+fly+oviposition,+larval+development,+and+adult+fitness.">Temporal changes in the bacterial community of animal feces and their correlation with stable fly oviposition, larval development, and adult fitness.</a> Front. Microbiol. 5 (590), 1-9 (2014).</li><li>Bailey, D. L., Whitfield, T. L., LaBrecque, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+biology+and+techniques+for+mass+producing+the+stable+fly,+Stomoxys+calcitrans+(L.)+(Diptera:+Muscidae).">Laboratory biology and techniques for mass producing the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae).</a> J. Med. Entomol. 12 (2), 189-193 (1975).</li><li>Smith, J. 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著者が明らかに何もありません。

安定したハエは、さまざまな自然の中の基板の開発を見つけることができますや、研究室では、メディアの多くの種類で飼育することができます。充填剤として木の削りくずやバーミキュ ライトを使いました。バーミキュ ライトがうまく、しかし、飼育困難な媒体から分離 puparia 製高価 (~$0.60/pan)。おそらく puparia を培地から分離する追加の厳しさのためには、羽化もバーミキュ ライト、木...

安定したハエ、第(l.)、吸血性のハエが痛みを伴う刺され家畜の放牧行動を混乱させる、痛みを引き起こすとに苦しんでいる動物を仲間し世界人間のレクリエーション活動を中断します。発酵栄養物、しばしば動物の排泄物で汚染された未熟な安定したハエを開発します。農業慣行の変化と作物作物残渣、オーストラリア1の野菜、ブラジル2サトウキビとパイナップル コスタリカ3で安定したハエの深刻な流行を生み出した。動物あたりのちょうど 14 の安定したハエは経済のしきい値の4になることが、最近の発生5の中に動物あたりの以上 2,000 のハエの観測が行われました。このような寄生レベルはゼロに近いホスト生産性が低下し、死亡率6があります。農学関連の集団発生、結果として安定したハエは更新された興味を受け取っているし、研究所の植民地の需要が大幅に増加します。
すべての holometamorphic の昆虫に関しては安定したハエは未熟か幼虫の段階で成長に必要な栄養素のすべてを取得します。したがって、飼育システムの重要なコンポーネントは、幼虫のダイエットや基板です。フィールド7で基板の広い範囲の開発、安定したハエの幼虫を観察されているし、彼らは基板8,9の微生物のコミュニティに依存しています。天然幼虫基板は、分解または多くの場合窒素廃棄物で汚染された植物原料を発酵の主に構成されます。
研究所の飼育、安定したハエ幼虫基板、通常加えられた窒素源となる植物素材構成されます。多数の材料を安定して飛ぶ幼虫ダイエットのため使用されています。最初の幼虫ダイエット自然基板を真似た、エンバクわらと馬や牛の肥料10,11を発酵します。炭水化物源にはが含まれて小麦にはふすまには12 には,13,14, アルファルファの食事12,13,14と特殊化学品が開発した商用の処方製造業者協会 (csma 方式、33% 小麦ふすま、27% アルファルファの食事, 40% ビール酵母粒)13,14,15,16。酵母懸濁液12魚粉、炭酸水素アンモニウム17窒素源が含まれます。不活性材料の充てんは、エンバク殻12、バガス13、バーミキュ ライト16、木材チップ13,18小球形にされたピーナツの殻14などダイエットで含まれています。
飼育室の主な目的は、実験の結果がフィールドの人口のそれらを反映するために、生理学的、できるだけ「野生型」に似ている製品を生産するです。これは繁殖で、選択を最小化する必要があります遺伝的に維持するために多様性と栄養のリソース フィールドのそれらに匹敵します。副次目的は労働および費用を最小限に抑えるです。費用を最小限に抑えることの主要なコンポーネントは、ローカルで使用できる食事成分の。サシバエの飼育紹介システムは、これらの目標を満たすために開発されました。

<table>
	<tbody>
		<tr>
			<td>Diamalt</td>
			<td>Premier Malt Products, Inc., Saddle Brook, NJ</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>CSMA Fly media</td>
			<td>Purina Animal Nutrition, Arden Hills, MN</td>
			<td>5S6Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Maxi Pad</td>
			<td>The Tranzonic Co., Cleveland, OH, USA</td>
			<td>5001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Manna</td>
			<td>MannaPro, Chesterfield, MO, USA</td>
			<td>Manna Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Spectrum Chemical Manufacturing Corp, Gardena, CA</td>
			<td>A1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat bran, Coarse</td>
			<td>Siemer Milling Company, Teutopolis, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>Tractor Supply Company, Brentwood, TN</td>
			<td>502770699</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishmeal</td>
			<td>Consumer Supply Distributing, North Sioux City, SD</td>
			<td>F1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult cages</td>
			<td>All Aluminum Window Company, Lincoln, Ne</td>
			<td>Custom</td>
			<td>45 &#215; 45 &#215; 45 cm, 18 &#215; 16 mesh aluminum screen, stockinette access</td>
		</tr>
		<tr>
			<td>9 &#215; 28 cm black cotton cloth</td>
			<td>Robert Kaufman Fabrics, Los Angeles, CA</td>
			<td>K040-114</td>
			<td>Egging cloth</td>
		</tr>
		<tr>
			<td>10 liter plastic dish pans</td>
			<td>Rubbermaid, Saratoga Springs, NY</td>
			<td>FG2951ARWHT</td>
			<td>Larval pans</td>
		</tr>
		<tr>
			<td>Stockinette, Cotton, 12&#34; X 25 yd roll</td>
			<td>Tex-Care Medical Company, Burlington, NC</td>
			<td>91311-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

laboratory rearing of stable flies and other muscoid diptera

安定したハエ、第は、家畜、人間、伴侶動物および野生動物の世界の深刻な害虫です。最後の 20 + 年の間に農業慣行の変更は、いくつかの国で安定したハエの深刻な発生になった。これらの流行は、家畜の生産、この害虫の研究、管理作業を向上するための公開の要求の結果として人間のレクリエーションを中断しました。研究室の研究のための安定したハエを飼育のためのシンプルで安価なプロシージャが表示されます。プロシージャは、ローカルで使用できる食事成分、機器および消耗品を使用します。他有翅のハエの顔フライ (イエバエ autumnalis)、ノサシバエ (ノサシバエ製剤)、イエバエ (イエバエ) などを飼育の手順を適用できます。手順は、平均 12.5 mg と 〜 35% 卵成虫生存に安定して飛ぶ puparia を生成します。約 3000 のハエは、各パンの生産されています。

幼虫 pupariate 10-14 日間大人は 14-16 日産卵後出てくる。生成時間、卵には 〜 24 日です。図 5に 3 つの異なる増量エージェントと 2 人のコロニー 2013年 5 月 1 月 2017 年までの飼育データが掲載されています。コットンウッドを与えた最高の収量、3867 ±1442 (<img alt="Equation" src="/files/ftp_upload/57341/57341eq1.jpg"> </img> ± 標準偏差) 大人 2,872 ±1294 を生産研究所...

Watch this Scientific Journal Video about Laboratory Rearing of Stable Flies and Other Muscoid Diptera at JoVE.com

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

安定したハエ (第) を飼育する手順が表示されます。手順は、食事成分、機器および消耗品のローカルで利用可能な材料を使用します。

研究所は、安定したハエや他の有翅の双翅目の飼育

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

laboratory-rearing-of-stable-flies-and-other-muscoid-diptera

Research

JoVE Journal

Biology

8.5K ビュー.  ARS-USDA. 安定したハエ (第) を飼育する手順が表示されます。手順は、食事成分、機器および消耗品のローカルで利用可能な材料を使用します。

ビデオ: Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Microbial Communities in Nature and Laboratory - Interview

自然と実験室での微生物群集 - インタビュー

生物学

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

モスキート子育てのためのプロトコル（A. gambiae）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

収穫と準備ショウジョウバエ</em電気生理学的記録とその他の手続のための>胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

プロービングDNA安定同位体（DNA - SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

子育てに最適化されたプロトコル Fopius arisanus、Tephritid果実の寄生ハエ

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

無菌実験技術：めっき法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

フルーツハエを扱うシンプルな自家製ツール -ショウジョウバエメラノガスター

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>マウスの内リンパ嚢の解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

マウスの内リンパ嚢の解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

大腸菌およびその他の細菌からの細胞外小胞のスケーラブルな単離および精製(英語)

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...