Gli autori riconoscono la National Science Foundation svizzero per Grant No. 31003A-132358/1, 31003A_152972 e n 151.948, e Fondazione Pierre Mercier pour la Science.

1. Preparazione di prodotti chimici e attrezzature <ol><li> Fare 500 ml di una esametafosfato di sodio all&#39;1% (SHMP) (NaPO 3) n soluzione e sterilizzare in autoclave. </li><li> Sterilizzare nitrocellulosa (NC) filtri (diametro dei pori 0,22 micron, 47 mm di diametro) in autoclave in vetro chiuso Petri. </li><li> Sterilizzare filtri NC (dimensione dei pori di 0,22 micron, 25 mm di diametro) in autoclave in vetro chiuso piastre di Petri. </li><li> Pesare e prendere nota del peso a vuoto di sterili provette da 50 ml (con tappo) (un tubo per campione). </li><li> Preparare Tris-EDTA-buffer (TE-buffer) 1x: rendere la soluzione di 10 mM Tris (tris (idrossimetil) aminometano) e 1 tampone mM EDTA. Regolare il pH a 8 e sterilizzare in autoclave. </li><li> Preparare la soluzione di lisozima (20 mg / ml) sciogliendo 0,02 g di lisozima in 1 ml di TE-buffer. Idealmente, fare fresco ogni volta. Conservare a 4 ° C per non più di 1 settimana. </li><li> Preparare la soluzione fisiologica sciogliendo 8 g / l di cloruro di sodio(NaCl) in acqua distillata. Sterilizzare in autoclave. </li></ol> 2. Separazione di biomassa da sedimenti <ol><li> Aggiungere 3 g di campione di sedimento di pre-pesare sterili provette da 50 ml con palline di metallo etanolo fiammato. Eseguire questo in una, UV sterilizzata armadio biosicurezza pulito per evitare la contaminazione. </li><li> Aggiungere 15 ml di una sterile (autoclave) Soluzione SHMP 1% al campione utilizzando una sterile buretta. La soluzione può SHMP inoltre il filtraggio per evitare la contaminazione. </li><li> Omogeneizzare il sedimento e la soluzione SHMP con un dispersore liquido / omogeneizzatore (ad esempio, Ultra-Turrax omogeneizzatore). 70% di etanolo o sterilizzare in autoclave il rotore di dispersione prima dell&#39;uso. Eseguire l&#39;omogeneizzazione per 1 min a 17.500 rpm. Lasciare riposare campione per 2 minuti e ripetere l&#39;omogeneizzazione per 1 min alla stessa velocità del rotore. </li><li> Lasciate riposare il campione per 10 min. A questo punto, le particelle più pesanti (minerali) si sistemerà. Le cellule ei componenti organici del campione tuttavia wmalato rimangono in soluzione. Successivamente, trasferire la soluzione surnatante (contenente biomassa cellulare) in una provetta pulita da 50 ml, facendo attenzione a non disturbare il pellet sedimento. </li><li> Per il pellet sedimento aggiungere nuovamente 15 ml di una sterile (autoclave) Soluzione SHMP 1% con una sterile buretta. Poi ripetere i punti 2.3 e 2.4. Questa ripetizione assicura la separazione della quantità massima di cellule e particelle organiche dalla componente minerale del sedimento. Il supernatante di questa seconda separazione può essere fusa con il supernatante dal prima separazione. Nota: Le seguenti operazioni sono tutti fatti sul surnatante (contenente biomassa cellulare). La componente minerale (sedimenti pellet) può essere scartato. </li><li> Centrifugare il campione a 20 xg per 1 min. Questo passaggio aumenta la forza g sufficiente per risolvere piccole particelle minerali mentre il materiale cellula biologica rimane in soluzione. Dopo centrifugazione, trasferire la soluzione surnatante (contenente biomassa cellulare) in unpulito tubo da 50 ml. Eliminare il pellet minerale. </li><li> Determinare il volume finale della soluzione contenente biomassa pesando il campione. La determinazione del peso evita di dover trasferire il campione ad un cilindro graduato e riduce il rischio di contaminazione. </li></ol> 3. Raccolta di biomassa su membrana filtrante <ol><li> Preparare unità di filtrazione (per 47 mm di membrane di diametro) e pompa del vuoto. Sterilizzare l&#39;unità di filtrazione sia in autoclave o (se Pyrex vetro) spruzzando con il 70% di etanolo e fiamme con un becco Bunsen. Lasciate raffreddare prima di continuare con il protocollo. </li><li> Aggiungere sterili membrana NC all&#39;unità di filtrazione con pinze sterilizzate etanolo fiammato. </li><li> Aggiungere metà del campione surnatante (dal punto 2.6) sul gruppo di filtrazione a membrana e raccogliere le cellule sulla membrana utilizzando la pompa a vuoto. </li><li> Quando il liquido è completamente passata attraverso il filtro, arrestare la pompa per vuoto e rimuovere con cautela la membrana utilizzando ethanol-fiamma pinza sterilizzata. Posizionare la membrana in una piastra di Petri sterile. <ol><li> Tagliare la membrana a metà con le forbici sterilizzate etanolo fiammato. Aggiungere ogni metà della membrana su un tubo separato 2 ml. Una metà della membrana sarà utilizzata per l&#39;estrazione del DNA e l&#39;analisi dell&#39;intera comunità batterica. L&#39;altra metà del filtro sarà conservato a -80 ° C e serve come backup. </li></ol></li><li> Posizionare nuova membrana NC sul gruppo di filtrazione e raccogliere biomassa dalla seconda metà del volume del campione (dal punto 2.6) con la pompa a vuoto. </li><li> Quando il liquido è completamente passata attraverso il filtro, arrestare la pompa per vuoto e rimuovere con cautela la membrana con pinze sterili etanolo fiammato. Posizionare tutta la membrana in un tubo separato 2 ml. Questo campione verrà utilizzato per il trattamento di separare endospore da cellule vegetative. Il campione può essere conservato a -20 ° C fino al momento dell&#39;uso. </li></ol> 4. Lisi di cellule vegetative <ol><li> Eseguire latrattamento per separare endospore dalle cellule vegetative sulla biomassa precedentemente raccolto su una membrana NC (passo 3.6). <ol><li> Se la membrana è stato congelato, lasciarlo a temperatura ambiente per 10 minuti per scongelare. Poi posto la membrana in una piastra di Petri sterile e tagliarlo (circa 4 volte) in pezzi più piccoli con le forbici sterilizzate etanolo fiammato. Poi posto tutti i pezzi del filtro a membrana in una provetta sterile da 2 ml. </li></ol></li><li> Aggiungere 900 ml di 1x TE (Tris-EDTA) Buffer (vedi 1.5) per il tubo contenente la membrana campione e mescolare accuratamente vortice. A questo punto, la biomassa viene rimosso dalla membrana nella soluzione TE-buffer. </li><li> Porre il tubo in un incubatore a 65 ° C per 10 min e 80 rpm. Successivamente rimuovere il tubo dal termostato e lasciarlo raffreddare per 15 minuti. </li><li> Aggiungere 100 ml di lisozima preparata di fresco (vedi 1.5) per ottenere una concentrazione finale di 2 mg / ml. Non aggiungere il lisozima prima che il campione è raffreddato a 37 ° C, in quanto questo potrebbe degRade l&#39;enzima. </li><li> Incubare il campione a 37 ° C per 60 min e 80 rpm, le condizioni ottimali per il lisozima di lisare cellule vegetative. </li><li> Dopo lisi è completa, aggiungere 250 ml di idrossido di 3 N di sodio (NaOH) e 250 ml di soluzione al 6% sodio dodecilsolfato (SDS) al campione. Aggiungendo questo, il volume del campione raggiunge 1,5 ml e vi è una concentrazione finale di 0,5 N NaOH e concentrazione finale di 1% SDS. </li><li> Incubare la miscela a temperatura ambiente per 60 min e 80 rpm. Aggiunta la base e detergenti aiuterà nella lisi cellulare finale. La concentrazione di questi detergenti è stato ottimizzato per non danneggiare le endospore, mentre lisi cellule vegetative. </li><li> Preparare un&#39;unità di filtrazione sterile che contiene 25 mm di diametro membrane in autoclave, o (se Pyrex vetro) spruzzandolo con 70% etanolo e fiamme con un becco Bunsen. Lasciate raffreddare. </li><li> Posizionare un NC membrana 0,2 micron (diametro 25 mm) in unità di filtrazione con pinze etanolo fiamma sterilizzati. &lt;/ li&gt; <li> Aggiungere il campione dal punto 4.7 sulla membrana e filtrare il liquido con la pompa a vuoto. Quando il liquido è passato attraverso, spegnere pompa a vuoto. A questo punto, il materiale cellula vegetativa lisato viene rimosso, in quanto non è trattenuto sulla membrana. Solo endospore rimarranno sulla membrana. </li><li> Aggiungere 2 ml di soluzione fisiologica sterile per lavare detergenti residue e filtrare il liquido con la pompa a vuoto. </li><li> Quando il liquido è completamente filtrato, spegnere la pompa del vuoto. Lasciare la membrana sul gruppo di filtrazione. </li></ol> 5. Il trattamento DNasi Nota: eseguire il trattamento DNasi direttamente sulla membrana del filtro. È importante che l&#39;unità di filtrazione non perde e la pompa a vuoto è spento. <ol><li> Aggiungere 450 ml di acqua sterile, 50 ml di tampone di reazione DNasi (1x) e 0,5 microlitri DNasi enzima direttamente sulla membrana del filtro e lasciar riposare per 15 minuti. Se possibile, fare questo digestione in una stanza che è un po &#39;di guerramer della media RT, poiché l&#39;enzima funziona meglio a temperature di 25 ° C e oltre. Nota: Mantenere il becco Bunsen parte l&#39;unità di filtrazione aumenta anche la temperatura e ha il vantaggio di mantenere le frazioni sterile, quindi ridotto rischio di contaminazione dei campioni. </li><li> Quando la digestione DNasi è terminato, accenda la pompa del vuoto per rimuovere l&#39;enzima dal campione. </li><li> Lavare enzimatica residua aggiungendo e filtrando 1 ml di soluzione fisiologica. </li><li> Se del liquido è completamente superato, spegnere pompa del vuoto e rimuovere la membrana filtro contenente endospore con pinza sterile e posizionarlo in una piastra di Petri sterile. Nota: Il campione di endospore separati è ora pronto per l&#39;analisi a valle. Conservare a -20 ° C se utilizzato per l&#39;estrazione del DNA oppure a 4 ° C se utilizzato per la germinazione e la coltivazione. </li></ol>

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La resistenza di endospore a fattori fisico-chimici aggressivi esterni (ad esempio, temperatura o detergenti) è stato usato per elaborare un metodo per separare endospore batteriche da cellule vegetative in campioni ambientali. Questo è il primo metodo completo per isolare endospore da campioni ambientali in modo non distruttivo. I metodi precedenti per quantificare, rilevare o analizzare endospore in campioni erano basati sulla misurazione delle deleghe specifiche per endospore come l&#39;acido dipicolinico ...

L&#39;obiettivo di questo lavoro è quello di fornire un protocollo per la separazione di endospore batteriche da cellule batteriche vegetative in campioni ambientali. La formazione di endospore batteriche è una strategia di sopravvivenza, di solito innescato da inedia, trovato in un certo numero di gruppi di batteri appartenenti al phylum Firmicutes 1. Batteri che formano endospore sono ben studiati, soprattutto a causa di un numero di ceppi sono patogeni e quindi di importanza medica (per esempio, il Bacillus anthracis o Clostridium difficile). Ceppi ambientali di batteri che formano endospora sono stati isolati da praticamente ogni ambiente (suolo, acqua, sedimenti, aria, ghiaccio, intestino umano, gli animali intestino, e altro) 1-3. Pertanto, Firmicutes sono la seconda phylum più abbondante nel collezioni di colture 4. A causa delle loro proteine ​​resistenti corteccia esterna e nucleo protettivo, endospore possono sopravvivere a condizioni ambientali estreme che vanno from essiccazione a radiazioni elevate, temperature estreme e sostanze chimiche nocive 5. Questo notevole resistenza lo rende una sfida per estrarre il DNA da endospore 6-8. Questo probabilmente spiega perché sono stati trascurati negli studi di sequenziamento ambientale 9,10. Altri metodi, come ad esempio il targeting di endospore in campioni ambientali da anticorpi fluorescenti 11, la quantificazione di acido dipicolinico (DPA) nel suolo e nei sedimenti 13 12, citofluorimetria 14 o la pastorizzazione e la successiva coltivazione 15,16 sono stati utilizzati per recuperare o quantificare endospores in campioni ambientali. Negli ultimi anni, metodi di estrazione del DNA ottimizzato così come primers molecolari specifici per indirizzare sequenze geniche specifiche endospora sono stati sviluppati 10,17-20. Ciò ha contribuito a rivelare di più la biodiversità tra questo gruppo di batteri 21 e ha portato anche ad applicazioni nell&#39;industria e in medicina per il rilevamento di endospore, Per esempio in latte in polvere 19. Il protocollo qui presentata si basa sulla differenza di resistenza alle condizioni fisico-chimiche nocive (quali calore e detergenti) di endospore batteriche relativi alle cellule vegetative. Per distruggere le cellule vegetative in un campione, noi applichiamo consecutivamente calore, lisozima e basse concentrazioni di detergenti. Il tempo e la forza di questi trattamenti sono stati ottimizzati in modo da non distruggere le spore, ma per lisare tutte le cellule vegetative. Alcune cellule in un pool di cellule ambientale sono più resistenti di altre, per cui al fine di aumentare la probabilità di distruggere tutte le cellule vegetative, si applicano tre diversi trattamenti. Il vantaggio e la novità di questo metodo è che i endospore dopo il trattamento sono ancora intatte e possono essere utilizzati per ulteriori analisi a valle. Questi includono l&#39;estrazione del DNA, PCR quantitativa (qPCR) e amplicone o sequenziamento metagenomica (di mira in particolare il gruppo di endospore e riducendo diversity, aumentando la copertura). Le endospore potrebbero essere utilizzati anche per la coltivazione a valle o la quantificazione mediante microscopia a fluorescenza, citometria a flusso, o il rilevamento di DPA. Una caratteristica importante di questo metodo è che confrontando un campione non trattato con un campione trattato, si può dedurre la quantità e la diversità di endospore in un campione ambientale, oltre alla componente corrispondente al vegetative cellule.

<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.

I risultati presentati qui sono stati pubblicati in precedenza 10,21. Si prega di fare riferimento a tali articoli per l&#39;interpretazione ambientale e discussione dei dati. La procedura generale è riassunto in Figura 1 e corrisponde a tre fasi principali: in primo luogo, la separazione della biomassa da sedimenti o qualsiasi altra matrice ambientale; secondo, la distruzione di cellule vegetative; e terzo, l&#39;analisi a valle delle endospore ...

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.

Isolamento fisico di Endospore da campioni ambientali da mirata Lisi di cellule vegetative

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 Views.  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.

Video: 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.

Coltura e manutenzione<em&gt; Clostridium difficile</em&gt; In un ambiente anaerobico

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

Ricerca

Immunologia e infezioni

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.

Isolamento, propagazione e identificazione di specie batteriche con proprietà metabolizzanti di idrocarburi da habitat acquatici

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

Ambiente

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.

Rilevamento Cortex Frammenti Durante la germinazione delle spore batteriche

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.

Visualizzazione di Germinosomes e la membrana interna in Bacillus subtilis spore

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

Bioingegneria

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.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

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

Biologia

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.

Indagando Teliospore germinazione utilizzando analisi Microrespiration e Microdissection

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.

Isolamento e selezione di funghi entomopatogeni da campioni di suolo e valutazione della virulenza fungina contro gli insetti nocivi

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).

Confronto dei metodi per isolare i funghi entomopatogeni da campioni di suolo

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.

Isolamento di lieviti e muffe coltivabili dai terreni per studiare la struttura della popolazione fungina

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.

Isolamento, caratterizzazione ed estrazione totale del DNA per l'identificazione di funghi endofiti in piante micoeterotrofe

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