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

Os autores agradecem o Swiss National Science Foundation para Grant No. 31003A-132358/1, 31003A_152972 e nº 151948, e Fundação Pierre Mercier pour la Science.

1. Preparação de produtos químicos e equipamentos <ol><li> Adicione 500 ml de um 1% de hexametafosfato de sódio (SHMP) (NaPO3) n solução e esterilizar em autoclave. </li><li> Esterilizar de nitrocelulose (NC) (filtros de tamanho de poro de 0,22 um, 47 mm diâmetro) em autoclave de vidro em placas de Petri fechada. </li><li> Esterilizar filtros NC (dimensão do poro de 0,22 um, 25 mm de diâmetro) em autoclave de vidro em placas de Petri fechada. </li><li> Pesar e anotar o peso vazio da estéreis tubos de 50 ml (com tampa em) (um tubo por amostra). </li><li> Prepare de Tris-EDTA-tampão (tampão TE) 1x: preparar uma solução de Tris 10 mM (tris (hidroximetil) aminometano) e tampão de EDTA 1 mM. Ajustar o pH para 8 e esterilizar em autoclave. </li><li> Preparar solução de lisozima (20 mg / ml) por dissolução de 0,02 g de lisozima em 1 ml de tampão TE. Idealmente, fazer fresco o tempo todo. Armazenar a 4 ° C durante não mais de uma semana. </li><li> Preparar solução fisiológica por dissolução de cloreto de sódio 8 g / L(NaCl) em água destilada. Esterilizar em autoclave. </li></ol> 2. Separação de Biomassa de Sedimentos <ol><li> Adicionar 3 g de amostra de sedimento para pré-pesado estéril tubos de 50 ml usando colheres de metal inflamado a etanol. Execute isso em um, UV esterilizado gabinete de biossegurança limpo para evitar a contaminação. </li><li> Adicionar 15 ml de uma solução estéril (autoclavada) solução SHMP 1% para a amostra usando uma bureta graduada estéril. O SHMP solução também pode ser filtrada para evitar a contaminação. </li><li> Homogeneizar o sedimento e SHMP solução com um dispersor líquido / homogeneizador (por exemplo, homogeneizador Ultra-Turrax). 70% de etanol em autoclave ou esterilizar o rotor dispersão antes da utilização. Executar a homogeneização durante 1 minuto a 17.500 rpm. Deixe o resto da amostra durante 2 minutos e repetir a homogeneização durante 1 min à mesma velocidade do rotor. </li><li> Deixe a amostra repousar durante 10 min. Neste passo, as partículas mais pesadas (minerais) vai assentar. As células e os componentes orgânicos da amostra no entanto Wdoentes permanecem em solução. Em seguida, transferir a solução sobrenadante (contendo biomassa celular) para um tubo limpo 50 ml, enquanto tomando cuidado para não perturbar o pellet sedimento. </li><li> Para o sedimento sedimentos adicionar novamente 15 ml de uma (autoclavada) solução SHMP 1% estéril usando um estéril bureta graduada. Em seguida, repita os passos 2.3 e 2.4. Esta repetição assegura a separação da quantidade máxima de células e partículas orgânicas do componente mineral do sedimento. O sobrenadante desta segunda separação pode ser mesclado com o sobrenadante da primeira separação. Nota: Todos os passos seguintes são feitas no sobrenadante (contendo biomassa celular). O componente mineral (sedimento sedimento) pode ser descartado. </li><li> Centrifugar a amostra a 20 xg durante 1 min. Esta etapa aumenta o g-força suficiente para resolver partículas minerais pequenas enquanto o material biológico celular ainda permanece em solução. Após centrifugação, transferir a solução sobrenadante (contendo biomassa celular) numlimpo tubo de 50 ml. Descartar o sedimento mineral. </li><li> Determinar o volume final da solução contendo biomassa por pesagem da amostra. A determinação do peso evita a necessidade de transferir a amostra para um cilindro graduado e reduz o risco de contaminação. </li></ol> 3. Cobrança de Biomassa no filtro de membrana <ol><li> Prepare unidade de filtração (por 47 mm de diâmetro membranas) e bomba de vácuo. Esterilizar a unidade de filtração quer por autoclavagem ou (se vidro Pyrex) por aspersão com etanol a 70% e em chamas com um bico de Bunsen. Deixe-o esfriar antes de continuar com o protocolo. </li><li> Adicionar membrana NC estéril para a unidade de filtração usando uma pinça esterilizados inflamado a etanol. </li><li> Adicionar metade da amostra de sobrenadante (a partir do passo 2.6) para a unidade de filtração por membrana e recolher as células sobre a membrana usando a bomba de vácuo. </li><li> Quando o líquido tiver totalmente passado através do filtro, parar a bomba de vácuo e cuidadosamente remover a membrana utilizando etanol-chama fórceps esterilizado. Colocar a membrana em uma placa de Petri esterilizada. <ol><li> Corte a membrana ao meio com uma tesoura esterilizada inflamado a etanol. Adicionar cada metade da membrana para um tubo separado de 2 ml. Uma metade da membrana vai ser utilizado para a extracção do ADN e análise de toda a comunidade bacteriana. A outra metade do filtro vai ser armazenado a -80 ° C e serve como uma cópia de segurança. </li></ol></li><li> Coloque nova membrana de NC para a unidade de filtração e recolha de biomassa a partir da segunda metade do volume da amostra (a partir do passo 2.6), utilizando a bomba de vácuo. </li><li> Quando o líquido está totalmente passou através do filtro, parar a bomba de vácuo e retire cuidadosamente a membrana com a pinça esterilizados inflamado a etanol. Coloque toda a membrana, para um tubo separado de 2 ml. Este exemplo vai ser utilizada para o tratamento de separar os esporos a partir de células vegetativas. A amostra pode ser armazenada a -20 ° C até à sua utilização. </li></ol> 4. Lise de células vegetativas <ol><li> Execute otratamento para separar endosporos de células vegetativas da biomassa previamente recolhidas numa membrana de NC (passo 3.6). <ol><li> Se a membrana foi congelado, deixá-lo à TA durante 10 min para descongelar. Em seguida, coloque a membrana em uma placa de Petri estéril e cortá-la (aproximadamente 4 vezes) em pequenos pedaços com uma tesoura esterilizada inflamado a etanol. Em seguida, coloque todas as peças filtro de membrana em um tubo estéril 2 ml. </li></ol></li><li> Adicionar 900 ul de 1x TE (Tris-EDTA) tampão (ver 1.5) para o tubo contendo a amostra de membrana e homogeneiza-se por vortex. Neste passo, a biomassa é removida a partir da membrana para a solução de tampão TE. </li><li> Colocar o tubo em uma incubadora a 65 ° C durante 10 min e 80 rpm. Depois retire o tubo da incubadora e deixe-o esfriar por 15 min. </li><li> Adiciona-se 100 ul de lisozima preparada de fresco (ver 1.5) para atingir uma concentração final de 2 mg / ml. Não adicione a lisozima antes de a amostra ter arrefecido até 37 ° C, pois isso poderia degRade a enzima. </li><li> Incubar a amostra a 37 ° C durante 60 min e 80 rpm, as condições óptimas para a lisozima para lisar as células vegetativas. </li><li> Após a lise estar completa, adicionar 250 ul de 3 N de hidróxido de sódio (NaOH) e 250 uL de solução a 6% de sulfato de dodecilo de sódio (SDS) para a amostra. Ao adicionar este, o volume da amostra atinge 1,5 ml e há uma concentração final de NaOH 0,5 N e a concentração final de 1% de SDS. </li><li> Incubar esta mistura à TA durante 60 min e 80 rpm. Adicionando a base e detergentes vai ajudar na lise celular final. A concentração destes detergentes foi otimizado para não prejudicar os endospores, enquanto lisar células vegetativas. </li><li> Prepara-se uma unidade de filtração estéril, que contém as membranas 25 mm de diâmetro por autoclavagem, ou (se vidro Pyrex) pulverizando-o com etanol a 70% e em chamas com um bico de Bunsen. Deixe-o esfriar. </li><li> Coloque uma membrana NC 0,2 m (25 mm de diâmetro) na unidade de filtração usando uma pinça etanol chama-esterilizados. &lt;/ li&gt; <li> Adicionar a amostra a partir do passo 4.7 sobre a membrana e filtrar o líquido através da bomba de vácuo. Quando o líquido passou por, desligar a bomba de vácuo. Neste passo, o material de célula vegetativa lisadas é removido, uma vez que não é retida na membrana. Apenas endospores permanecerá na membrana. </li><li> Adicionar 2 ml de solução fisiológica estéril para lavar detergentes residuais e filtrar o líquido através da bomba de vácuo. </li><li> Quando o líquido foi totalmente filtrado, desligar a bomba de vácuo. Deixar a membrana da unidade de filtração. </li></ol> 5. O tratamento DNase Nota: realizar o tratamento com DNase directamente na membrana de filtro. É importante que a unidade de filtração não se escapa e a bomba de vácuo é desligada. <ol><li> Adicionar 450 uL de água estéril, 50 ul de tampão de reacção de ADNase (1x) e 0,5 ul de enzima ADNase directamente sobre a membrana de filtro e deixar repousar durante 15 min. Se possível, fazer isso digestão em uma sala que é ligeiramente guerramer que a média RT, uma vez que a enzima funciona melhor a temperaturas de 25 ° C e acima. Nota: Mantendo o bico de Bunsen de lado da unidade de filtração também aumenta a temperatura e tem o benefício adicional de manter o ambiente estéril, por conseguinte, um risco reduzido de contaminação das amostras. </li><li> Quando a digestão de DNase é acabado, em vez da bomba de vácuo para remover o enzima a partir da amostra. </li><li> Lavar enzimática residual por filtragem e adicionando 1 ml de solução fisiológica. </li><li> Se algum líquido tiver passado completamente, desligar a bomba de vácuo e remover a membrana de filtro endosporulante usando uma pinça estéril e colocá-lo em uma placa de Petri estéril. Nota: A amostra de endospores separados está agora pronto para análise a jusante. Armazenar a -20 ° C quando é usado para a extracção de ADN ou, alternativamente, a 4 ° C quando é usado para a germinação e cultivo. </li></ol>

<ol>
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Os autores não têm nada a revelar.

A resistência de endósporos a factores físico-químicos agressivos externos (por exemplo, temperatura ou detergentes) foi utilizado para conceber um método para separar os esporos bacterianos a partir de células vegetativas em amostras ambientais. Este é o primeiro método global para isolar os esporos a partir de amostras ambientais de uma maneira não destrutiva. Os métodos anteriores para quantificar, detectar e analisar os esporos foram em amostras com base na medição de proxies específicos para e...

O objetivo deste trabalho é fornecer um protocolo para a separação de esporos bacterianos, a partir de células bacterianas vegetativas em amostras ambientais. A formação de esporos bacterianos é uma estratégia de sobrevivência, geralmente desencadeado pela inanição, encontrado num número de grupos de bactérias pertencentes ao filo Firmicutes 1. Bactérias formadoras de Endospore são bem estudadas, principalmente porque um número de cepas são agentes patogênicos e, portanto, de importância médica (por exemplo, Bacillus anthracis ou Clostridium difficile). Estirpes ambientais de bactérias formadoras de endospore foram isolados a partir de praticamente qualquer ambiente (solo, água, sedimentos, o ar, gelo, intestino humano, animais intestino, e mais) 1-3. Portanto, Firmicutes são a segunda filo mais abundante em coleções de cultura 4. Por causa de suas proteínas córtex externo e do núcleo de protecção resistentes, endospores podem sobreviver a condições ambientais extremas que vão from dessecação à alta radiação, temperaturas extremas e produtos químicos prejudiciais 5. Este notável resistência torna-se um desafio para extrair DNA de endósporos 6-8. Isso provavelmente explica por que eles têm sido negligenciados nos estudos de sequenciamento ambiental 9,10. Outros métodos, tais como a concentração de endósporos em amostras ambientais por anticorpos fluorescentes 11, a quantificação de ácido dipicolínico (DPA) no solo e nos sedimentos 13 12, por citometria de fluxo 14 ou a pasteurização e cultivo subsequente 15,16 têm sido usados ​​para obter ou quantificar em endosporos amostras ambientais. Em anos recentes, os métodos de extracção de ADN optimizada, bem como iniciadores moleculares específicos para sequências alvo específicas do gene endospore foram desenvolvidos 10,17-20. Isto tem ajudado a revelar mais biodiversidade entre este grupo de bactérias e 21 também levou a aplicações na indústria e medicina para a detecção de endospores, Por exemplo, em leite em pó 19. O protocolo aqui apresentado é baseado na diferença em resistência às condições físico-químicas prejudiciais (tais como o calor e detergentes) de endosporos bacterianos relativos a células vegetativas. Para destruir as células vegetativas em uma amostra, consecutivamente aplicar calor, lisozima e baixas concentrações de detergentes. O tempo e a força destes tratamentos foram optimizadas de forma a não destruir os esporos, mas de lisar todas as células vegetativas. Algumas células em uma piscina de células ambiental são mais resistentes do que outros, por isso, a fim de aumentar a probabilidade de destruir todas as células vegetativas, aplicamos três tratamentos diferentes. A novidade e vantagens deste método é que os endosporos após o tratamento ainda estão intactas e podem ser utilizados para outras análises a jusante. Estes incluem a extração de DNA, PCR quantitativo (qPCR) e amplicon ou seqüenciamento metagenomic (visando especificamente o grupo de endospores e, assim, reduzindo diversity, enquanto o aumento da cobertura). Os endosporos também poderia ser utilizada para o cultivo ou a jusante quantificação por microscopia de fluorescência, citometria de fluxo, ou a detecção de DPA. Uma característica importante deste método é que, ao comparar uma amostra não tratada com uma amostra tratada, pode-se deduzir a quantidade e diversidade de endósporos em uma amostra ambiental, além do componente que corresponde a células vegetativas.

<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.

Os resultados aqui apresentados foram publicados anteriormente 10,21. Por favor, consulte os artigos para a interpretação ambiental e discussão dos dados. O processo global está resumida na Figura 1 e corresponde a três passos principais: em primeiro lugar, a separação da biomassa a partir de sedimentos ou qualquer outra matriz ambiental; Em segundo lugar, a destruição de células vegetativas; e em terceiro lugar, a análise a jusante dos...

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.

O isolamento físico do Endospores de amostras ambientais por alvejado Lise de células vegetativas

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 visualizações.  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.

Vídeo: 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.

Cultivar e Manter<em&gt; Clostridium difficile</em&gt; Em um ambiente anaeróbico

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

Imunologia e Infecção

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, Propagação e Identificação de Espécies Bacterianas com Propriedades Metabolizadoras de Hidrocarbonetos de Habitats Aquáticos

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.

Detecção de Cortex Fragmentos Durante bacteriana germinação de esporos

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.

Visualização do Germinosomes e a membrana interna em esporos de Bacillus subtilis

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

Bioengenharia

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.

Investigando os prejudiciais efeitos de baixa pressão Plasma esterilização na sobrevivência de esporos de Bacillus subtilis usando Live celular 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.

Investigando a germinação de Teliospore usando a análise de 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 Seleção de Fungos Entomopatômicos de Amostras de Solo e Avaliação da Virulência Fúngica contra Pragas de Insetos

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).

Comparação de métodos para isolar fungos entomopatômicos de amostras de solo

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 de Leveduras culturais e moldes de solos para investigar estrutura populacional fúngica

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, caracterização e extração de DNA total para identificação de fungos endofíticos em plantas micoheterotróficas

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