Gli autori ringraziano per il supporto Mattias Gyllborg impianto di calamari e dei commenti a questo manoscritto, Michael Hadfield e il Laboratorio Kewalo Marine per l&#39;assistenza durante la raccolta campo, ei membri del Laboratorio di Ruby e McFall-Ngai per i contributi a questo protocollo. Il lavoro nel laboratorio di Mandel è supportato da NSF IOS-0843633.

1. Preparazione di inoculi batterica <ol><li> Giorno 0 Due giorni prima calamari inoculazione, la piastra dei ceppi batterici rilevanti su LBS 13 agar. </li><li> Incubare batteri a 25-28 ° C durante la notte. </li><li> Giorno 1 Inoculare 3 LBS ml di media cultura in un tubo di vetro con una colonia di ogni V. ceppo fischeri per l&#39;infezione. Preparare duplicare i tubi come backup. </li><li> Giorno 2 (Coordinate passi batteriche 1,4-1,6 con passo calamari 3.7-3.10) 1 h prima dell&#39;inoculo, batteri subcultura 1:80 (37,5 pl) in 3 ml LBS in una provetta di vetro e crescere per 1 h con aerazione. </li><li> Misurare la OD 600 del campione prima dell&#39;inoculo. Misurazioni tipiche sono 0,3-0,6 seconda del ceppo. </li><li> Per un inoculo bersaglio di 3-5 x 10 3 UFC / ml calcolare il volume di inoculo come segue: volume di inoculo (pl) = 1,25 / 60 OD0 (ad esempio, per OD 600 = 0.5, il volume di inoculo calcolato = 1.25/0.5 = 2,5 microlitri). Tale importo è aggiunto direttamente l&#39;acqua di mare contenenti calamari al punto 4.1. Questo calcolo può essere necessario regolare per diversi ceppi di V. fischeri o per l&#39;inoculazione a livelli inferiori o superiori a quelli indicati qui. </li></ol> 2. Preparazione di piastre di agar per il conteggio del inoculi <ol><li> Per ogni trattamento, LBS etichette piastre (2 per il trattamento) a campioni piastra del inoculo nel passaggio 4.1. </li><li> Aggiungere 5 perline placcatura sterili per piastra. </li></ol> 3. Raccolta dei minori Squid <ol><li> Misurare la salinità dell&#39;Oceano istantanea con il rifrattometro e regolare a 35 ‰. </li><li> Filtrare 1 L di Ocean istantaneo utilizzando l&#39;unità di filtrazione e una linea a vuoto allegato o pompa del vuoto, per generare filtro sterilizzato istantanea Ocean (UFAS). Ossigenare l&#39;acqua agitando vigorosamente prima di ogni erogazione. The unità di filtro può essere riutilizzato per 2 giorni. </li><li> Aliquotare 40-50 ml di UFAS in ciascuno di due (2) ciotole campione usa e getta. Etichetta uno come Earlies e una come timelies. </li><li> Preparare un eccesso di pipette di trasferimento in plastica per acquisire calamari giovanile tagliando la pipetta circa 1 cm dalla punta, sopra le creste più bassi (vedi Figura 3). Questo facilita una zona più ampia attraverso la quale il calamaro può passare al momento della raccolta. Eliminare le pipette di trasferimento in cui vi è una superficie ruvida esposta. </li><li> Utilizzo di pipette di trasferimento preparati, raccogliere E. scolopes che nati durante la notte e trasferire alla ciotola Earlies di UFAS. Hatchlings I primi sono stati nel sistema di uova per oltre 1 ora e sono suscettibili di colonizzazione da parte di contaminanti V. fischeri nel sistema uovo. Non utilizzare per gli esperimenti di colonizzazione Earlies sensibili. </li><li> Controllare i serbatoi d&#39;uovo ogni 30-45 min per hatchlings nuovi. Assicurarsi che tutti i neonati vengono eliminati nel corso di ogni check. Rimuovere hatchlings con una pipetta di trasferimento, e il deposito nella ciotola timelies di UFAS. Gli animali raccolti in modo tempestivo sono disponibili per gli esperimenti di colonizzazione. </li><li> Quando la raccolta è terminata (~ 45 min dopo il tramonto), trasferire il calamaro al laboratorio principale. Empiricamente è vantaggioso a colonizzare gli animali in condizioni di luce ininterrotta di laboratorio per 3 vaccinazioni h. </li><li> Per ogni trattamento, preparare una ciotola con 40 ml UFAS. Aggiungi Squid per le ciotole per il saggio (massimo n = 40 per ciotola). </li><li> Preparare una ciotola aggiuntiva come aposymbiotic controllo (negativo). </li><li> Preparare una pipetta di trasferimento dedicato per ciascun trattamento. </li><li> Euthanize calamari extra in 2% di etanolo. </li></ol> 4. Squid Colonization <ol><li> Giorno 2 - Utilizzando una Pipetman P10, dispensare l&#39;aliquota calcolata di batteri (Step 1.5) in ogni ciotola calamari (Step 3.8) per ogni trattamento. Avviare un timer 3 ore subito dopola prima inoculazione. </li><li> Per ogni trattamento, creare un &quot;vortice&quot; nel contenitore con la pipetta di trasferimento dedicato posizionando la pipetta vicino al bordo della tazza e pipettando su e giù ripetutamente per miscelare l&#39;acqua e calamari per circa 10 sec. Accurata miscelazione è fondamentale. </li><li> Tavola 50 microlitri da ogni ciotola su una piastra di agar LBS dal punto 2.2 (per il tecnico replica, due piastre 50 piastre pl per ogni trattamento). Incubare a 25-28 ° C durante la notte. </li><li> Preparare catino (100 ml UFAS / bis) per ogni trattamento. </li><li> Preparare fiale Drosophila (4 ml UFAS / bis) per ogni calamari. </li><li> Dopo esattamente 3 ore, trasferire i calamari ai loro rispettivi catino (completa per tutti i trattamenti). Ciò arresta l&#39;inoculazione. </li><li> Procedere al trasferimento di ogni singolo calamari al suo flacone con il proprio Drosophila UFAS. Utilizzare una pipetta di trasferimento designato per ogni trattamento. </li><li> Spostare vassoi di fiale Drosophila alla struttura calamari per tornare al giorno / night ciclo di luce gli animali sperimentato durante l&#39;embriogenesi. </li><li> Giorno 3 - Prepare fiale Drosophila (4 ml UFAS / bis) per ogni calamari. </li><li> Prima del tramonto a 22-24 ore dopo l&#39;inoculazione, trasferire ogni calamari in un nuovo flacone Drosophila. Utilizzare una pipetta di trasferimento designato per ogni trattamento. </li><li> Giorno 4 - Preparare microcentrifuga etichettata provette da 1,5 ml (1/squid). </li><li> Prima del tramonto a 46-48 ore dopo l&#39;inoculazione, misurare e registrare la luminescenza di ogni calamari nel flaconcino Drosophila (set luminometro 6 s per l&#39;integrazione e l&#39;auto-lettura in chiusura del coperchio). </li><li> Come controllo negativo per luminescenza sfondo, misurare un flaconcino con UFAS che non contiene alcun calamari. </li><li> Trasferire ciascun calamari in un volume di circa 700 pl di una provetta per microcentrifuga 1,5 ml dalla Fase 4,12. Spostarsi in una scatola di cartone freezer. Una volta che il coperchio è posto sulla scatola, non rimuoverlo, come i segnali di luce per l&#39;espulsione i batteri non sono well-compreso. </li><li> Congelare provette da microcentrifuga a -80 ° C per una notte. </li></ol> 5. Determinazione dei livelli di colonizzazione <ol><li> Per ogni calamari, preparare due (2) microprovette, ognuna con 475 microlitri UFAS (o in autoclave 70% Instant Ocean). </li><li> Preparare pestelli in primo luogo utilizzando un Kimwipe per pulire il pestello e rimuovere i detriti lordo e / o tessuto. </li><li> Luogo pestelli tip-down in un bicchiere da 50 ml contenente etanolo al 95%. Etanolo deve essere aggiunto ad una altezza di circa 3 cm. </li><li> Per ogni pestello, togliere dal bicchiere e pulire la punta con un Kimwipe. </li><li> Immergere pestello di nuovo nel bagno di etanolo, rimuovere e inserire (punta verso l&#39;alto) in un rack provetta e lasciare asciugare completamente per circa 15 minuti. </li><li> Scongelare calamari in un portaprovette microcentrifuga (massimo n = 8). </li><li> Se necessario, regolare il volume a 700 microlitri. </li><li> Utilizzo di un pestello dal punto 5.5, a distruggere il tessuto animale finché l&#39;inchiostro sac RUPTures (l&#39;acqua diventa torbida un colore grigio). </li><li> Rimuovere il pestello e garantire tutti tessuto rimane nel tubo. </li><li> Vortex brevemente il tessuto per esattamente 10 secondi utilizzare un timer). </li><li> Lasciare il tessuto riposare per 10 min. Il tessuto si sistemerà ed i batteri e l&#39;inchiostro rimane in soluzione. Per i calcoli che seguono, i batteri o la soluzione di inchiostro è la [A] diluizione (cioè la luce E. scolopes omogenato organo in 700 pl). Diluizioni seriali 1:20 ([B], [C]) sono descritti di seguito. </li><li> Per la [B] diluizione, aggiungere 25 pl [A] alla uno dei tubi da microcentrifuga preparata al punto 5.1. Vortex. </li><li> Per la [C] diluizione, aggiungere 25 pl [B] a uno dei tubi da microcentrifuga preparata al punto 5.1. Vortex. </li><li> Tavola 50 microlitri di ciascuna diluizione su agar LBS, 2 repliche per trattamento. </li><li> Incubare le piastre a 25-28 ° C per una notte. </li></ol> 6. Analisi dei dati <ol><li> Per calcolare il CFU / organo luce (LO), i paesit colonie sulla piastra per ogni trattamento, in cui 10-400 colonie sono presenti, e usare la seguente formula: CFU / LO = (colonie su [A] piastra) x 14; CFU / LO = (colonie su [B] piastra) x 280, oppure CFU / LO = (colonie su [C] piastra) x 5600. </li><li> Tracciare i singoli punti dati e le mediane su una scala logaritmica. </li><li> I dati spesso non sono normalmente distribuiti, con diverse varianze, e gli outliers possono contenere informazioni biologicamente significativo. Pertanto, non parametrici fornire un metodo utile per determinare se i trattamenti differiscono in modo significativo. </li><li> Utilizzare software Prism GraphPad per l&#39;analisi statistica. Per i due trattamenti, utilizzare il Wilcoxon rank test Sum. Per i confronti tra più di due trattamenti, utilizzare il Kruskal-Wallis test con appropriato post-test. </li></ol> 7. Risultati rappresentativi I risultati di un saggio di colonizzazione campione sono mostrati nella Figura 4. Due ceppi di <em&gt; V. fischeri, che presentano diversi livelli relativi di luminescenza sono stati inoculati in ogni sei calamari, insieme a sei calamari che è servito come aposymbiotic (non colonizzata) i controlli. Il E. scolopes simbionte, ES114 14 e brillante è il Sepiola robusta simbionte, SR5 15,16. Livelli di inoculo simili (Fig. 4A) portano al 100% colonizzazione nel raggio di 3 h. A 48 ore, i livelli di luminescenza (Fig. 4B) e conta CFU (Fig. 4C) sono stati determinati per valutare la competenza colonizzazione del ceppo. Determinazione della luminescenza specifico (Fig. 4D; batterio per) consente di determinare la luminosità di ogni ceppo batterico durante la simbiosi. <img alt="Figura 1" src="/files/ftp_upload/3758/3758fig1.jpg" /> Figura 1. Diagramma di flusso della procedura di colonizzazione. Batteri e calamari vengono raccolte separatamente, poi mixato presso l&#39;inoculo specificato. Squid vengono lavate, poi trasferito acqueo a 3 ore, 24 ore, e 4<html> 8 h dopo l&#39;inoculazione. Al 48 h la luminescenza è misurata e gli animali vengono congelati, che serve a superficie sterilizzare gli animali. Light-organo colonizzato i batteri rimangono vitali a -80 ° C attraverso un disgelo (senza ulteriori cicli gelo-disgelo). <img alt="Figura 2" src="/files/ftp_upload/3758/3758fig2.jpg" /> Figura 2. Diagramma di flusso illustrante omogeneizzazione e placcatura diluizione dei batteri. Seriali 20-diluizioni fornire la gamma dinamica adeguata per il conteggio dei batteri colonizzati. <img alt="Figura 3" src="/files/ftp_upload/3758/3758fig3.jpg" /> Figura 3. Trasferire pipette con un cono albero opportunamente stretto un foro stretto (A) che danneggerebbero calamari giovanile. Pretrattamento, tagliando parte più stretta con le forbici o una lametta si ottiene uno strumento adeguato (B) per il trasferimento di calamari giovanile. s/ftp_upload/3758/3758fig4.jpg &quot;/&gt; Figura 4. I dati di esempio per un test di colonizzazione. (A) i livelli di batteri nelle ciotole inoculo. (B) Luminescence di calamari individuale. (C) il conteggio delle colonie di calamari individuale. (D) luminescenza specifico di calamari individuale. Apo, Aposymbiotic (non colonizzata controllo negativo).

<ol>
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Il dosaggio colonizzazione descritto permette l&#39;analisi di un processo naturale simbiotico in un ambiente di laboratorio controllato. Come tale, può essere utilizzato per valutare colonizzazione da ceppi mutanti mediante diversi isolati naturali, e sotto regimi chimici differenti. Variazioni su gli esperimenti descritti sono comunemente usati per valutare diversi aspetti della simbiosi. La cinetica di colonizzazione può essere misurata esaminando luminescenza durante le prime 24 ore, che può essere rilevata autom...

<table border="1"><tbody><tr><td> Nome di reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Tubi di vetro Cultura, diametro 16 mm </td><td> VWR </td><td> 47729-580 </td><td></td></tr><tr><td> Tappi per Tubi cultura del vetro </td><td> Pescatore </td><td> NC9807998 </td><td></td></tr><tr><td> Spettrofotometro visibile per la determinazione di OD 600 </td><td> Biowave </td><td> CO8000 </td><td> Qualsiasi spettrofotometro in grado di misurare OD 600 funzionerà. Questa unità può misurare la OD 600 di liquido direttamente in provette di coltura di vetro. Qualche aggiustamento del calcolo inoculo può essere necessario a seconda dello strumento utilizzato. </td></tr><tr><td> Glomax 20/20 Singolo-Tube Luminometro </td><td> Promega </td><td> E5311 </td><td> Equivalente al BioSystems Turner 20/20n luminometro. Include il supporto provetta. </td></tr><tr><td> Glomax 20/20 standard Luce </td><td> Promega </td><td> E5341 </td><td> Per la calibrazione luminometro. </td></tr><tr><td> Rifrattometro, Handheld </td><td> Foster e Smith Aquatics </td><td> CD-14.035 </td><td> Calibrare prima di ogni utilizzo con acqua deionizzata. Risciacquare dopo ogni uso con acqua deionizzata per evitare l&#39;accumulo di sale. </td></tr><tr><td> Istantanea Ocean (artificiale concentrato di mare) </td><td> Foster &amp; Smith Aquatics </td><td> CD-16.881 </td><td> Preparare a 35 ‰ in acqua deionizzata, utilizzando il rifrattometro, quindi filtrare attraverso un 0,2 micron SFCA filtro. </td></tr><tr><td> Unità di filtraggio </td><td> Nalgene </td><td> 158-0020 </td><td> Tensioattivo-free di acetato di cellulosa (SFCA) membrana, 0,2 micron. Abbiamo osservato risultati variabili con qualche tensioattivo contenenti filtri PES. </td></tr><tr><td> Pipette di trasferimento </td><td> Pescatore </td><td> 13-711 -09:00 </td><td> Utilizzando forbici o lametta, tagliare la punta nettamente al di sopra della cresta prima per aumentare il diametro della punta della pipetta ed evitare che si schiaccino i piccoli calamari. </td></tr><tr><td> Bocce campione monouso (bicchieri di plastica) </td><td> Cometa </td><td> T9s (9 oz.) </td><td> Ciotole per inoculazione, con diametro superiore 3 ¼ &quot;, diametro inferiore 2 ¼&quot;, altezza 3 &quot;. Bocce creare un ambiente omogeneo in quanto non hanno margine di fondo, in cui i calamari possono rimanere intrappolati in un basso di ossigeno di nicchia. La dimensione è ottimizzata per 40 ml di inoculo. Disponibile all&#39;indirizzo webstaurantstore.com, # 619PI9. </td></tr><tr><td> Drosophila Fiale </td><td> VWR </td><td> 89092-720 </td><td> Diametro Vial corrisponde l&#39;apertura sul PMT luminometro. </td></tr><tr><td> Provette da microcentrifuga da 1,5 ml </td><td> ISC Bioexpress </td><td> C-3217-1CS </td><td> I tubi devono adattarsi alla forma dei pestelli. </td></tr><tr><td> Etanolo, 200 Proof </td><td> Pescatore </td><td> BP2818-100 </td><td></td></tr><tr><td> Pestelli </td><td> Kimble Chase / Kontes </td><td> 749521-1500 </td><td></td></tr><tr><td> Beads placcatura, 5 mm di diametro </td><td> Kimble Chase </td><td> 13500 5 </td><td> Preparare 5 per tubo e autoclave. </td></tr></tbody></table>

colonization of euprymna scolopes squid by vibrio fischeri

Batteri specifici sono presenti in associazione con tessuti animali 1-5. Tali host-batteriche associazioni (simbiosi), può essere dannoso (patogeno), non hanno alcuna conseguenza fitness (commensale), o essere di beneficio (mutualità). Mentre molta attenzione è stata data alle interazioni patogeni, si sa poco sui processi che determinano l&#39;acquisizione riproducibile di benefici / commensale batteri dall&#39;ambiente. La luce-organo mutualismo tra la marina batterio Gram-negativo V. fischeri e la Hawaiian Bobtail calamari, E. scolopes, rappresenta una interazione altamente specifica in cui un host (E. scolopes) stabilisce un rapporto simbiotico con una sola specie batteriche (V. fischeri) nel corso della sua vita 6,7. Bioluminescenza prodotta da V. fischeri durante questa interazione fornisce un anti-predatorio beneficio E. scolopes durante le attività notturne 8,9, mentreil tessuto ricco di nutrienti host fornisce V. fischeri con una nicchia protetta 10. Durante ogni generazione host, questo rapporto si riassume, rappresentando così un processo prevedibile che può essere valutata in dettaglio vari stadi di sviluppo simbiotico. In laboratorio, del minore calamari sportello aposymbiotically (colonizzati), e, se raccolti entro i primi 30-60 minuti e trasferito al simbionte priva di acqua, non possono essere colonizzati se non lo sperimentale inoculo 6. Questa interazione fornisce pertanto un sistema utile modello in cui per valutare i singoli passaggi che portano alla acquisizione specifico di un microbo simbiotico dall&#39;ambiente 11,12. Qui si descrive un metodo per valutare il grado di colonizzazione che si verifica quando appena schiusi aposymbiotic E. scolopes sono esposti a (artificiale) contenenti acqua di mare V. fischeri. Questo saggio descrive semplice inoculazione, l&#39;infezione naturale, e il recuperodel simbionte batterico dall&#39;organo luce nascente E. scolopes. cura è presa per fornire un ambiente coerente per gli animali durante lo sviluppo simbiotico, con particolare riguardo alla qualità dell&#39;acqua e segnali luminosi. Metodi per caratterizzare la popolazione simbiotica descritta includono (1) misura della bioluminescenza batteri-derivati, e (2) colonia diretta conteggio di simbionti recuperati.

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Il metodo descrive la procedura con cui il calamaro hawaiano bobtail,<em&gt; Euprymna scolopes</em&gt; E il suo simbionte batterica,<em&gt; Vibrio fischeri</em&gt;, Vengono allevati separatamente e poi introdotta per consentire colonizzazione specifica dell&#39;organo luce calamari dai batteri. Rilevamento colonizzazione da parte di luminescenza batteri derivati ​​e conteggio delle colonie diretta sono descritti.

La colonizzazione delle Euprymna scolopes Squid da Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

colonization-of-euprymna-scolopes-squid-by-vibrio-fischeri

Research

JoVE Journal

Immunology and Infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri

19.8K Views.  Northwestern University. Il metodo descrive la procedura con cui il calamaro hawaiano bobtail, Euprymna scolopes E il suo simbionte batterica, Vibrio fischeri, Vengono allevati separatamente e poi introdotta per consentire colonizzazione specifica dell'organo luce calamari dai batteri. Rilevamento colonizzazione da parte di luminescenza batteri derivati ​​e conteggio delle colonie diretta sono descritti.

Video: Colonization of Euprymna scolopes Squid by Vibrio fischeri

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Valutare alterazioni metaboliche epatiche durante la colonizzazione progressiva di Germe senza mouse da 1 H spettroscopia NMR

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Ricerca

Immunologia e infezioni

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Quantificazione di colonizzazione fungina, sporogenesi, e la produzione di micotossine Uso Bioassays Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - un metodo per modificare geneticamente<em&gt; Vibrio cholerae</em&gt; Sulla base di trasformazione naturale e FLP-ricombinazione

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Indagare gli effetti dei probiotici sul pneumococcico colonizzazione utilizzo di un<em&gt; In Vitro</em&gt; Aderenza Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Sviluppo di un più sensibile e specifica Chromogenic Agar Media per la rilevazione di<em&gt; Parahaemolyticus Vibrio</em&gt; E Altro<em&gt; Vibrio</em&gt; Specie

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Tecniche di laboratorio utilizzate per mantenere e differenziare i biotipi di<em&gt; Vibrio cholerae</em&gt; Isolati clinici ed ambientali

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Induzione della differenziazione cellulare e dell&#39;immagine delle cellule singole di<em&gt; Vibrio parahaemolyticus</em&gt; Nuotatore e cellule Swarmer

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...