Name: physical isolation of endospores from environmental samples by targeted lysis of vegetative cells
Uploaded: 2016-01-21T13:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

著者は、助成金番号31003A-1分の132358、31003A_152972および第151948スイス国立科学財団を承認し、Fundationピエール・メルシエはラ・サイエンスを注ぎます。

化学物質や機器の作製<ol><li> 1％のヘキサメタリン酸ナトリウム（SHMP）（のNaPO 3）nは溶液500mlを加えて、オートクレーブで滅菌します。 </li><li>密閉ガラスペトリ皿で​​オートクレーブ処理により滅菌ニトロセルロース（NC）フィルター（孔径0.22ミクロン、直径47ミリメートル）。 </li><li>密閉ガラスシャーレでオートクレーブによりNCフィルター（孔径0.22ミクロン、直径25ミリメートル）を滅菌します。 </li><li>滅菌50mlチューブ（キャップ​​付き）（サンプルあたり1チューブ）の空の重量を計量し、注意してください。 </li><li>トリスEDTA緩衝液（TE緩衝液）1Xを準備：10 mMトリス（ トリス （ヒドロキシメチル）アミノメタン）および1mM EDTA緩衝液の溶液を作ります。 pHを8に調整し、オートクレーブで滅菌します。 </li><li> 1ミリリットルのTE緩衝液中のリゾチーム0.02グラムを溶解することによりリゾチーム溶液（20 mg / mlの）を準備します。理想的には、毎回新鮮にします。わずか1週間4℃で保存してください。 </li><li> 8グラム/ Lの塩化ナトリウムを溶解して生理学的溶液を調製します蒸留水に塩化ナトリウム（NaCl）。オートクレーブで滅菌します。 </li></ol>土砂からのバイオマスの2分離<ol><li>予め計量する堆積物試料3gのエタノール燃え上がっ金属スクープを使用して滅菌した50ミリリットルチューブを追加します。汚染を避けるためにきれいな、紫外線滅菌生物学的安全キャビネットの中でこれを実行します。 </li><li>ビュレットを卒業し、滅菌を使用したサンプルの1％（オートクレーブ処理）滅菌SHMP溶液15mlを追加します。 SHMP溶液は、汚染を避けるために濾過することができます。 </li><li>液体分散機/ホモジナイザー（例えば、ウルトラタラックスホモジナイザー）と堆積物とSHMP液を均質化します。 70％エタノール、滅菌または使用前に分散ローターオートクレーブ。 17,500 rpmで1分間均質化を実行します。 2分間のサンプル残りをしようと、同じローター速度で1分間均質化を繰り返します。 </li><li>サンプルは10分間放置してみましょう。このステップでは、最も重い粒子（ミネラル）が沈降します。細胞サンプルの任意の有機成分がW病気の溶液中に残ります。堆積物ペレットを乱さないように注意しながらその後、清潔な50mlのチューブに（細胞バイオマスを含む）上澄み液を移します。 </li><li>堆積物ペレットに滅菌を使用して再度1％（オートクレーブ処理）滅菌SHMP溶液15mlを加えビュレットを卒業。そして、ステップ2.3と2.4を繰り返します。この繰り返しは、堆積物の鉱物成分から細胞や有機粒子の最大量の分離を保証します。この第二の分離の上清を第一の分離からの上清とマージすることができます。 注意：次の手順は、すべての（細胞バイオマスを含む）上清に行われます。ミネラル成分（堆積物のペレット）を廃棄することができます。 </li><li> 1分間20×gでサンプルを遠心。生物細胞材料がまだ溶液中に残っている間このステップでは、小さな鉱物粒子を解決するのに十分な遠心力が増加します。遠心分離した後、へ（細胞バイオマスを含む）上澄み液を移します清潔な50mlのチューブ。ミネラルペレットを捨てます。 </li><li>サンプルを計量することにより、バイオマスを含有する溶液の最終容量を決定します。重量決意をメスシリンダーに試料を移送する必要が回避され、汚染の危険性を減少させます。 </li></ol>フィルター膜上のバイオマスの3コレクション<ol><li>濾過ユニット（直径47mm膜用）と真空ポンプを準備します。 70％エタノールでそれを噴霧し、ブンゼンバーナーでそれを炎によって、オートクレーブまたは（パイレックスガラス場合）のいずれか濾過ユニットを滅菌します。それはプロトコルを続行する前に、冷ましてみましょう。 </li><li>エタノール燃え上がっ滅菌ピンセットを用いて濾過ユニットに無菌NC膜を追加します。 </li><li>膜濾過ユニットに（ステップ2.6）からの上清サンプルの半分を加え、真空ポンプを用いて膜上の細胞を収集します。 </li><li>液体が完全にフィルタを通過したときに、真空ポンプを停止し、慎重にエタンを用いて膜を除去しますOL-炎が鉗子を滅菌しました。滅菌ペトリ皿に膜を置きます。 <ol><li>エタノール燃え上がっ滅菌はさみを使用して半分に膜をカットします。独立した2ミリリットルチューブに膜の各半分を追加します。膜の半分は、全細菌群集のDNA抽出および分析のために使用されます。フィルタの他の半分は、-80℃で保存し、バックアップとして機能します。 </li></ol></li><li>濾過ユニットに新しいNC膜を配置し、真空ポンプを用いて（ステップ2.6から）のサンプル容量の後半からのバイオマスを収集します。 </li><li>液体が完全にフィルターを通過した場合には、真空ポンプを停止し、慎重にエタノール燃え上がっ滅菌ピンセットを用いて膜を除去します。独立した2ミリリットルチューブに膜全体を置きます。このサンプルは、栄養細胞からの内生胞子を分離するために治療のために使用されます。サンプルは使用するまで-20℃で保存することができます。 </li></ol>栄養細胞の溶解4. <ol><li>実行治療は、以前にNC膜（ステップ3.6）に収集されたバイオマスに栄養細胞から内生胞子を分離します。 <ol><li>膜が凍結した場合は、解凍し、室温で10分間そのままにしておきます。その後、滅菌ペトリ皿に膜を配置し、エタノール - 燃え上がっ滅菌はさみを使用して小片に（約4倍）、それをカット。その後、無菌の2mlチューブにすべてのメンブレンフィルター片を配置します。 </li></ol></li><li>サンプル膜を含むチューブに1×TE（トリスEDTA）バッファー（1.5参照）900μlを添加して、ボルテックスでよく混ぜます。このステップでは、バイオマスは、TEバッファー溶液中に膜から除去されます。 </li><li> 10分、80 rpmで65℃のインキュベーター内でチューブを置きます。その後インキュベーターからチューブを取り外し、それを15分間クールダウンしましょう​​。 </li><li> 2 mg / mlの最終濃度に到達するために新たに調製されたリゾチーム（1.5を参照）の100μLを加えます。サンプルは37℃に冷却する前に、これはDEG可能性として、リゾチームを追加しないでください酵素をRADE。 </li><li>リゾチームの最適条件は、栄養細胞を溶解し、60分、80 rpmで37℃でサンプルをインキュベートします。 </li><li>溶解が完了した後、3 N水酸化ナトリウム（NaOH）及び6％のドデシル硫酸ナトリウム（SDS）試料溶液の250μLを250μLを加えます。これを追加することにより、サンプル容量を1.5mlに到達し、0.5 N NaOHを最終濃度1％SDSの最終濃度があります。 </li><li> 60分、80 rpmで室温で、このミックスをインキュベートします。ベースと洗剤を追加すると、最終的な細胞溶解に役立ちます。内生胞子に害を与えないよう栄養細胞を溶解しながら、これらの界面活性剤の濃度は、最適化されています。 </li><li> （パイレックスガラスがあれば）70％エタノールでそれを噴霧し、ブンゼンバーナーでそれを炎オートクレーブ処理により直径25mmの膜を保持している、または滅菌濾過ユニットを準備します。それがクールダウンしましょう​​。 </li><li>エタノール炎滅菌ピンセットを用いて濾過ユニットに0.2μmのNC膜（直径25mm）を配置します。&lt;/ LI&gt; <li>膜上にステップ4.7からのサンプルを追加し、真空ポンプを用いて液体をフィルタリングします。液体が通過した場合には、真空ポンプをオフにします。それは、膜上に保持されないように、この段階で、溶解した栄養細胞物質が除去されます。唯一の内生胞子は、メンブレン上に残ります。 </li><li>残留洗剤を洗い流し、真空ポンプを用いて液体をフィルタリングするために滅菌生理溶液2mlを追加します。 </li><li>液体が完全にフィルタ処理した場合には、真空ポンプをオフにします。濾過ユニットの膜を残します。 </li></ol> 5. DNアーゼ処理注：フィルター膜に直接DNase処理を実行します。これは、濾過ユニットが漏れず、真空ポンプがオフにされることが重要です。 <ol><li>フィルター膜上に直接滅菌水450μlを、DNアーゼ反応緩衝液（1×）、50μlの0.5μlのDNアーゼ酵素を追加し、15分間放置しました。可能な場合は、少し戦争である部屋で、この消化を行います平均室温よりもマー、酵素は、上記の25°Cの温度でうまく動作するので。 注：ブンゼンバーナーを維持することはさておき、濾過ユニットは、温度を上昇させると、無菌環境を維持するという利点を有し、したがって、サンプルの汚染の危険性を減少させました。 </li><li> DNase消化が完了したら、サンプルから酵素を除去するために真空ポンプをオンにします。 </li><li>追加および生理溶液1mlをフィルタリングすることにより、残留酵素を洗い流します。 </li><li>液体が完全に通過した場合には、真空ポンプの電源をオフにし、滅菌ピンセットを用いて内生胞子を含むフィルター膜を除去し、滅菌ペトリ皿にそれを置きます。 注：分離内生胞子のサンプルは現在、下流の分析のための準備ができています。発芽および栽培に使用した場合、-20℃で保存は、DNA抽出のためにまたは代替的に、4℃で使用した場合。 </li></ol>

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外部積極的な物理化学的要因（例えば、温度や洗剤）の内生胞子の抵抗は環境試料中の栄養細胞から細菌内生胞子を分離する方法を考案するために使用されました。これは、非破壊的な方法で環境サンプルから内生胞子を単離するための最初の包括的方法です。サンプル中の内生胞子を、定量検出または分析する従来の方法は、ジピコリン酸または特定のマーカー遺伝子として?...

この研究の目的は、環境試料中の栄養細菌細胞からの細菌の内生胞子を分離するためのプロトコルを提供することにあります。細菌内生胞子の形成は、門ファーミキューテス1に属する細菌群の数で見つかった生き残り戦略、通常は飢餓によって誘発されます。内生胞子形成細菌はよく株の数は、病原体であり、したがって、医療の重要性（例えば、 炭疽菌またはクロストリジウム・ディフィシル ）主な理由は、研究されています。内生胞子形成細菌の環境株は、ほぼすべての環境（土壌、水、底質、大気、氷、人間の腸、動物の腸、およびそれ以上）1-3から単離されています。したがって、ファーミキューテスは、培養コレクション4における第二の最も豊富な門です。 そのため、それらの丈夫な外皮質および保護コアタンパク質の、内生胞子は、FRの範囲の極端な環境条件に耐えることができます高い放射線、極端な温度や有害な化学物質から5 OM乾燥。この驚くべき抵抗は内生胞子6-8からDNAを抽出することが課題になります。彼らは環境配列決定研究9,10に見過ごされてきた理由はここにありそう説明しています。例えば、蛍光抗体11により環境試料中の内生胞子のターゲティングのような他の方法、土壌12と底質13でジピコリン酸の定量（DPA）は、14フローサイトメトリーまたは低温殺菌し、その後の栽培15,16で内生胞子を取得または定量化するために使用されています環境サンプル。近年では、最適化されたDNA抽出法と同様に、特定の分子的プライマー内生胞子特異的遺伝子配列を標的にする10,17-20を開発されています。これは、細菌21のこのグループの中でより多くの生物多様性を明らかにすることができましたし、また内生胞子の検出のための産業や医療への応用につながっています、粉乳19で例えば。 ここで紹介するプロトコルは、栄養細胞からの相対細菌内生胞子の（例えば、熱や洗剤など）の有害な物理化学的条件に対する抵抗の差に基づいています。サンプル中の栄養細胞を破壊するために、我々は連続的に熱、リゾチーム及び界面活性剤の低濃度を適用します。これらの治療の時間と強度は胞子を破壊するが、すべての栄養細胞を溶解しないように最適化されています。環境セル・プール内のいくつかの細胞は他のものより耐性があるので、すべての栄養細胞を破壊する可能性を高めるために、我々は、3つの異なる治療法を適用します。この方法の利点および新規性は、処置後の内生胞子はまだ無傷であり、さらなる下流の分析のために使用することができることです。これらは、DNA抽出、定量PCR（定量PCR）、およびアンプリコンまたはメタゲノムシーケンシング（具体的には内生胞子のグループをターゲットとするため、Dを減らすことが含まれますiversity、）カバレッジ向上させながら。内生胞子はまた、蛍光顕微鏡、フローサイトメトリー、またはDPAの検出によって下流培養または定量化のために使用することができます。この方法の重要な特徴は、処理された試料と未処理試料と比較することにより、一つの細胞を栄養に対応する構成要素に加えて、環境試料中の内生胞子の数と多様性を推定することができることです。

<table><tbody><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 47 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182004 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859 Sigma-Aldrich</td><td>CAS 77-86-1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884 Sigma-Aldrich</td><td>CAS&amp;nbsp; 60-00-4&amp;nbsp;</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>62971 Fluka</td><td>powder, CAS&amp;nbsp; 12650-88-3&amp;nbsp;</td></tr><tr><td>Ultra-Turrax homogenizer T18 basic</td><td>IKA</td><td>3720000</td><td></td></tr><tr><td>Glass filter holders for 47&amp;nbsp;mm membranes, Pyrex glass</td><td>EMD Millipore</td><td>XX10 047 00</td><td></td></tr><tr><td>Chemical duty vacuum pump</td><td>Millipore</td><td>WP6122050</td><td>220 V/50 Hz</td></tr><tr><td>Manifold sampling filtration for 25&amp;nbsp;mm membranes</td><td>Millipore</td><td>1225 Sampling Manifold</td><td>polypropylene</td></tr><tr><td>Dnase</td><td>New England Biolabs</td><td>M0303S</td><td>Rnase free</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881 Sigma-Aldrich</td><td>CAS&amp;nbsp; 1310-73-2&amp;nbsp;</td></tr><tr><td>Sodium dodecyl sulfphate (SDS)</td><td>Sigma-Aldrich</td><td>L3771 Sigma&amp;nbsp;</td><td></td></tr><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 25 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182002 Aldrich</td><td></td></tr></tbody></table>

physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

ここに示された結果は、以前の10,21公開されています。データの環境解釈と議論のためにそれらの記事を参照してください。 全体的な手順を図1にまとめ、三つの主要なステップに対応されます。最初、堆積物からのバイオマスの分離または他の環境マトリックス;第二に、栄養細胞の破壊;そして第三に、分離内生胞子の下流分析。下流の...

Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

栄養細胞の標的溶解によって環境サンプルからの内生胞子の物理的な分離

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

physical-isolation-endospores-from-environmental-samples-targeted

Universidad San Sebastian

Research

JoVE Journal

Environment

14.1K ビュー.  Macquarie University. A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

ビデオ: Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培養およびメンテナンス<em&gt;クロストリジウム·ディフィシル</em&gt;嫌気性環境での

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

免疫・感染学

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

水生生物生息地からの炭化水素代謝特性を有する細菌種の単離・増殖・同定

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

環境学

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

細菌胞子の発芽中に皮質断片を検出

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Germinosomes と枯草菌胞子の内部の膜の可視化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工学

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

生物学

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壌サンプルからの病原性真菌の単離と選択と昆虫害虫に対する真菌性毒性の評価

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

土壌試料からの昆虫病原性菌類の分離方法の比較

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

土壌からの培養酵母・カビの分離による菌群構造調査

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

菌従属栄養植物における内生菌を同定するための単離、特性評価、および全DNA抽出

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...