Name: paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis
Uploaded: 2018-03-23T14:00:00
Description: Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

この作品は、NSF のキャリア賞を受賞 1553023 と NIH/NIAID 賞 R01AI103369 によって支持されました。

1. 緑膿菌コロニー バイオ フィルムの成長
<ol><li>中二層プレートの準備<ol>
<li>トリプトン 10 g/L、寒天 10 g/L を準備脱イオン水 (材料の表を参照) ソリューション。</li>
		<li>水浴中で 20 分 50-60 ° C に涼しいオートクレーブ。</li>
		<li>トリプトン寒天培地溶液 45 mL を注ぎ 100 mm × 100 mm 角皿 (材料の表を参照してください) 50 mL コニカル チューブを使用します。寒天を固め...

<ol>
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パラフィン包埋、薄切片組織サンプルは微細形態構造のイメージングを有効にし、一般に真核生物の組織、使用、微生物サンプル8 にいくつかの成功と適用されている古典的な組織学的手法 ,9。Cryoembedding 内因性および蛍光信号の強力な保持できますが、パラフィン包埋は一般に望ましい形態16のより良い保全を提供します。この?...

ほとんど微生物バイオ フィルム フォームする能力があります、セルのコミュニティは、セルフ プロデュースの行列で一緒に開催。バイオ フィルムは、栄養素と基板の提供の様々 な体制で物理的なセットアップの多くの種類で栽培できます。再現可能な多細胞構造を生成する傾向がある特定のアッセイのバイオ フィルム形成とコミュニティまたは巨視的レベルで系統発生的に多様な種のための一般的なアーキテクチャが観察されます。微生物は雰囲気下で固体媒体の植民地で栽培される、肉眼形態はマトリックス生産能力についての情報を伝えるし、他の特徴1,2,3とよく相関します。微生物コロニーの内部アーキテクチャは、バイオ フィルムに固有の化学および生理学に関する手がかりを提供することもが特徴付けることは困難されています。細菌のコロニーに cryoembedding、セクショニングの技術の最近のアプリケーションは、画像と前例のない解像度4,5,6で特定の機能の可視化に有効にしています。しかし、動物組織を用いた研究は、パラフィン包埋 cryoembedding 7と比較されたとき形態の優れた保存を提供し、組織8,9の細菌を視覚化するために使用されていることを示しています。したがって、固定、パラフィン包埋、微生物コロニー バイオ フィルムの薄切片法にプロトコルを開発しました。ここでは、緑膿菌PA14 コロニー バイオ フィルム中薄いセクション10,11, の作製について述べるが、の細菌によって形成されるバイオ フィルムにこの手法を行ったも正常にSynxantha 緑膿菌、枯草菌、とコレラ12。
パラフィン包埋、薄切片のバイオ フィルムの簡単な論理は次のとおりです。まず、バイオ フィルムは処理の間に形態を維持する寒天の層に包まれています。第二に、包まれてバイオ架橋高分子に定着剤に沈んでいるし、形態を維持します。アルコールで脱水しより非極性溶媒でクリアし、し、液体のパラフィン ワックスを浸透させた。潜入、一度サンプルは区分のためのワックスのブロックに埋め込まれています。セクションは、カット、スライド、マウントより本来の状態に戻すために復元します。この時点から、染色したり顕微鏡分析のためのメディアをマウントで覆われています。
このプロトコルには、組織学的解析に適した微生物バイオ フィルムの薄い部分が生成されます。コロニー バイオ フィルムの部分は、このメソッドを使用して薄い切片を光学顕微鏡検査によってイメージが表示されます。バイオ フィルムの個々 の機能のために特定のメディア含む蛍光汚れに成長してまたは退避手順の (ステップ 9.5 9.6) をマウントする前にすぐに染色もできます。最後に、その場でこれらのコミュニティ内での細胞分布や遺伝子発現の報告を許可する構成または規制的に蛍光蛋白質を生成する微生物を設計することができます。コロニー バイオ フィルム深度、セル分布、マトリックス分布、成長パターン、時空間の遺伝子発現を決定するこれらのメソッドを使いました。

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paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis

真核生物において広く確立された技術は、パラフィン包埋による区分します。ここでの埋め込み・灌流のパラフィン ワックスを使用してそのまま微生物コロニー バイオ フィルムの断面、固定法を実施します。コロニー バイオ フィルムで使用するためこのメソッドを適応し、我々 はその成長基板の各サンプルを維持すると、寒天オーバーレイヤーのラミネート技術を開発し、固定液にリジンを追加しました。これらの最適化は、微細形態機能のサンプルの保存/保護を向上させます。この方法で作製した試料は、薄膜の断面と光、蛍光、および透過電子顕微鏡によるイメージングに適しています。緑膿菌 synxantha 菌、枯草菌、コレラ菌のコロニー バイオ フィルムにこの手法を用いています。ディテールはこのメソッドによって生成されるサンプルでの高レベル レポーターひずみと組み合わせて工学または特定の染料の使用は生理学と微生物の開発にエキサイティングな洞察力を提供することができます。

このメソッドは、明瞭な形態学的特徴と遺伝子発現のゾーンを DIC、蛍光顕微鏡、電子顕微鏡によって視覚化される前記バイオ フィルム薄いセクションを生成します。油浸対物レンズ × 40 を使用して DIC イメージングは、いくつかの形態学的特徴 (図 2 e) を表示するのに十分なことができますが、我々 はその蛍光を発見した恒常エクスプレス蛍?...

Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

固定、パラフィン包埋、および微生物コロニー バイオ フィルムの薄い断面技法について述べる。作製した試料では、顕微鏡によるバイオ フィルムの基礎部分やレポーターの発現パターンを視覚化できます。

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

The overall goal of this procedure is to generate paraffin embedded biofilm sections that can be used to assess the distribution of gene expression as reported by fluorescent protein production within intact native morphologies. This method can help answer key questions in the study of microbial communities such how the expression of specific genes in the production of exopolymeric substances distributed in the context of native biofilm architecture. The main advantage of this technique is that is allows for analysis of gene expression within the preserved morphology of biofilm cross-sections while also being amenable to downstream treatments such as staining of distinct features and high-resolution imaging such confocal or electron microscopy.After preparing agar tryptone solution according to the text protocol, use a 50-milliliter conical tube to pour 45 milliliters of the solution into a 100-millimeter by 100 millimeter square dish. Allow the agar to solidify for approximately 20 to 30 minutes. Pour a second 15-millimeter layer on top of the first layer, then let it solidify overnight, and if necessary, use a lint-free tissue to remove condensation from the lids.Next, to spot colony biofilms, after growing an isolated single P.Aeruginosa colony from a frozen stock according to the text protocol, pipette 2.5 to 10 microliters of cell suspension onto a medium bi-layer plate. Colonies may be incubated in the dark at 25 degrees Celsius in 80 to 100%relative humidity for up to four days. Gently pour 15 milliliters of the agar solution over the growth medium and colony and allow the agar to form a gel at room temperature for five minutes.Then, use a sharp razor blade to cut a square, three-layered chuck with the colony laminated between the top two layers. If preparing multiple samples, ensure that each chuck is cut to a comparable size. Gently remove any excess agar from the colony containing chuck.Then, wet the flat head of a spatula in PBS or water and gently insert it between the top and bottom layers of agar. Immediately transfer the laminated colony into an embedding cassette labeled with a chemical-resistant marking pen. Place the embedding cassette into a glass slide mailer containing previously prepared fixative.Process the samples. Use 1X PBS to wash them twice for one hour each. For best results, automate reagent homogenization using the low setting on the spin function of an automatic tissue processor.To embed the samples, fill a wax mold with molten paraffin wax heated to 55 degrees Celsius, then use a heated flat spatula to quickly transfer the infiltrated chuck from the embedding cassette into the wax mold ensuring that the chuck rests parallel to the base of the mold. Allow the wax to solidify overnight at four degrees Celsius. Samples molded in solid wax may be stored indefinitely at this temperature.Once the wax has solidified, excise the sample from the mold and use a razor blade to trim excess wax from around the sample. Leave some wax extending from one end of the sample that can be used to clamp it into the microtome. It is important to trim the wax-embedded sample such that its shape facilitates the collection of smooth linear ribbons.Small imperfections in trimming can produce artifacts in the ribbon and compromise the integrity of the section. Next, heat a water bath to 42 degrees Celsius. Then, clamp the sample into the microtome with the surface of the colony oriented perpendicularly to the edge of the blade.Trim the sample in 50-micrometer intervals until the desired plane of the colony has been reached from which sections will be collected using a microtome set to a sectioning velocity of 75 to 80 RPM and a clearance angle of six to 10 degrees. Cut a ribbon of the desired number of 10-micrometer-thick sections. Then, use a fine-tipped paint brush to detach the ribbon from the blade.Now, with forceps or a drop of water at the tip of a Pasteur pipette, gently transfer the ribbon to the water bath. Immediately insert a slide into the water bath and position it below the ribbon at a 45-degree angle. Touch the narrow edge of the ribbon to the slide just below the frosted label.The ribbon should adhere. Pull the slide out of the water bath and adjust the angle so it's perpendicular to the surface of the water allowing the ribbon to lay flat against the slide along its length. Avoid trapping excess water beneath the ribbon.Gently stand the slide onto an absorbent lint-free tissue wicking excess water from the section. Then, lay the slide on a paper towel and allow it to dry in the dark at room temperature overnight. Heat a leveled hot plate to 45 degrees Celsius and place the slide on the plate for 30 to 60 minutes.The wax will become semi-molten and flatten against the slide. Gently lift the slide from the hot plate and lay it flat onto a smooth level room temperature surface for approximately one minute until the wax solidifies. Ensure that the molten wax does not pull to either side of the slide.With the samples inside a glass slide mailer, de-wax the slides in four washes of clearing agent for five minutes each. Use a Buchner aspirator to remove solution between washes. Use 100%ethanol to wash the slides three times for one minute each.Then, after rehydrating the slides according to the text protocol, immediately mount the sections in a tris-buffered mounting medium and apply a coverslip. The overlay agar around the section does not react with the fixative, so it does not adhere to the chard slide. This agar can dislodge during rehydration and damage the section, so care should be taken not to agitate the solution during rehydration.Allow the mounting medium to polymerize at room temperature overnight. Once polymerized, use clear nail polish to seal the coverslip to the slide. Finally, store the sealed slides indefinitely in the dark at four degrees Celsius.While DIC imaging using a 40 times oil immersion objective can be sufficient to show some morphological features of biofilm thin sections, fluorescence microscopy of strains engineered to constitutively express fluorescent proteins provides enhanced visualization of cellular distribution within the samples. As shown in these panels, images of individual sections can stitched together to generate a cross-section of the entire colony and provides context for the localization of structural features within the overall morphology at the macroscopic level. Using a strain engineered to express fluorescent protein under the control of a specific promoter enables the visualization of the distribution of gene expression.In addition, colonies can be grown on medium containing dyes, or dyes can be added post-sectioning to stains'specific polysaccharides. Finally, samples can also be prepared and imaged at higher resolution using transmission electron microscopy as presented here. It's important when attempting this procedure to be mindful of the samples'vagility and of the potential to introduce fluorescent and structural artifacts either through fixation, sectioning or rehydration which may influence your interpretation of the data.This procedure allows for a quantitative image analysis such as correlating the expression profiles of fluorescent reporters with morphological features and the Z-axis of a biofilm. This protocol can be applied to diverse species of biofilm-forming microbes with medical and industrial significance such as Pseudomonas aeruginosa, Vibrio cholerae and Bacillus subtilis. After watching this video, you should have a good understanding of how to grow and prepare colony biofilms for sectioning via paraffin embedding.

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

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ヒト造血細胞のためのコロニー形成細胞（CFC）アッセイ

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の成長結核バイオフィルム

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&#39;シングルウイルスゲノミクス&#39;を使用してシングルビリオンの単離とゲノム解析

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

歯科バイオフィルムにおける細胞外のpHのレシオメトリックイメージング

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共焦点顕微鏡および電子顕微鏡を用いて糸状菌コロニー上の細菌のバイオフィルムの定性的マルチスケール解析のための新しい方法

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

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細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

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The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

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Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

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