Ricerca sostenuta da New Hampshire-INBRE attraverso un Premio di Sviluppo Istituzionale (IDeA), P20GM103506, presso l&#39;Istituto Nazionale delle Scienze Mediche Generali del NIH.

Nota: le considerazioni di tempo per ogni dosaggio devono essere effettuate poiché le singole preparazioni per i supporti richiedono tempi diversi. Ad esempio, i supporti a lastre di agarro solido devono essere abbassati e asciugati (1-2 giorni). Ulteriori considerazioni di tempo ( cioè la colonia singola e la crescita della coltura durante la notte) sono specificate in ciascun protocollo e si trovano nella tabella 2 . 1. Preparazione dei supporti <ol><li> 1x salina a tampone fosfato (PBS) <ol><li> Pesare 4,0 g NaCl, 0,1 g KCl, 0,72 g Na 2 HPO 4 e 0,12 g KH 2 PO 4 . Combinare i costituenti in un pallone Erlenmeyer da 1 L, regolare il volume a 500 mL con acqua deionizzata, regolare il pH a 7,2 utilizzando un misuratore di pH e aggiungere HCl in goccia alla soluzione e sterilizzare con autoclave o filtro usando un filtro da 0,22 μm. 1x PBS può essere conservato a temperatura ambiente a tempo indeterminato. ATTENZIONE: HCl è un acido concentrato e deve essere manipolato secondoAlle norme istituzionali, locali, statali e federali. Per le linee guida appropriate per la manipolazione fare riferimento alla scheda di dati di sicurezza fornita dal produttore. </li></ol></li><li> Luria-Bertani (LB) brodo <ol><li> Pesare 5,0 g di tryptone, 2,5 g di estratto di lievito e 2,5 g di NaCl. Combinare i componenti in una bottiglia da 1 L, regolare il volume a 500 mL con acqua deionizzata, autoclave e conservare a temperatura ambiente fino a 6 mesi. Per le condizioni indotte da virulenza classica biotipo, regolare il pH a 6,5 ​​con HCl prima dell&#39;autoclavamento. </li></ol></li><li> AKI Medium contenente 0,03% (w / v) NaHCO 3 <ol><li> Per il componente 1 (medium AKI): pesare 7,5 g di peptone, 2,0 g di estratto di lievito e 2,5 g di NaCl. Combinare i componenti in una bottiglia da 1 L, regolare il volume a 450 mL con acqua deionizzata e autoclave. Il mezzo AKI può essere conservato a temperatura ambiente fino a 6 mesi. </li><li> Per il componente 2 (NaHCO 3 ): pesare 1,5 g di NaHCO 3 e regolare il volumeA 50 mL con acqua deionizzata in una vescicola sterile. Filtro sterilizzare NaHCO 3 utilizzando un filtro da 0,22 μm. NaHCO3 deve essere preparato immediatamente prima dell&#39;uso. </li><li> Asepticamente combinare i due componenti creando un rapporto 1:10 filtrando il componente 2 nella componente 1 prima dell&#39;uso. </li></ol></li><li> Luria-Bertani (LB) piatti di agar <ol><li> Pesare 5,0 g di tryptone, 2,5 g di estratto di lievito, 2,5 g di NaCl e 7,5 g di agar. Combinare i componenti in un pallone Erlenmeyer da 1 L, regolare il volume a 500 mL con acqua deionizzata, autoclave e preparare in piatti standard Petri di 100 mm x 15 mm sterili. I supporti placcati possono essere immagazzinati in plastica per un massimo di 6 mesi, con coperchio fino a 4 ° C. </li></ol></li><li> LB Agar Piatti Supplementati con Polymyxin B <ol><li> Per il componente 1 (agar LB): pesare 5,0 g di tryptone, 2,5 g di estratto di lievito, 2,5 g di NaCl e 7,5 g di agar. Combinare i costituenti in un pallone Erlenmeyer da 1 L, regolare il volume a 500 mL wiL&#39;acqua deionizzata e l&#39;autoclave. </li><li> Per il componente 2 (polimicina B): sciogliere la polimicina B in acqua deionizzata in un tubo sterile di microcentrifuga da 2 ml a una concentrazione di 50.000 UI / μl e sterilizzare con filtro utilizzando un filtro da 0,22 μm. </li><li> Una volta che il componente 1 è fresco al tocco, ma non ancora solidificato, aggiungere asetticamente 500 μL di componente 2 al componente 1 creando una concentrazione finale di 50 UI / μL e mescolare accuratamente. Preparare piatti standard Petri di 100 mm x 15 mm versando agar mischiati nei piatti di Petri. I supporti placcati possono essere immagazzinati in plastica per un massimo di 3 mesi, con coperchio fino a 4 ° C. </li></ol></li><li> Piatti minimi di agar di citrato <ol><li> Per il componente 1 (agar con blu bromotimolo): pesare 7,5 g di agar e 0,02 g di bromotimolo blu. Combinare i costituenti in un pallone Erlenmeyer da 1 L, regolare il volume a 450 mL con acqua deionizzata e autoclave la miscela. </li><li> Per il componente 2 (10x VBMM): pesare 10,1 g di MgSO 4 7H 2 O, 10,0 g di acido citrico acido H 2 O, 50,0 g di K 2 HPO 4 anidro e 17,5 g di NaNH 4 HPO 4 4 H 2 O. Combinare i costituenti in un pallone Erlenmeyer da 1 L, regolare il volume a 500 ml Con acqua deionizzata e sterilizzazione autoclave o filtro usando un filtro da 0,22 μm. 10x VBMM può essere conservato a temperatura ambiente fino a 6 mesi. </li><li> Aggiungere 50 ml di componente 2 al componente 1 e preparare in piatti standard Petri da 100 mm x 15 mm versando agar mischiati nei piatti di Petri. I supporti placcati possono essere immagazzinati in plastica per un massimo di 6 mesi, con coperchio fino a 4 ° C. </li></ol></li><li> Piatti di Agar Milk <ol><li> Per il componente 1 (latte): pesare 8,0 g di latte secco non grasso istantaneo in un pallone Erlenmeyer da 1 L, regolare il volume a 200 ml con acqua deionizzata e autoclave. </li><li> Per il componente 2 (agar con infusione cerebrale-cuore): pesare 3,68 g di infusione cerebrale-cuore e 6,0 g di agar. Combina il conIn un pallone Erlenmeyer da 500 mL, regolare il volume a 200 mL con acqua deionizzata e autoclave. </li><li> Combinate i due componenti versando il componente 2 nella componente 1, dopo aver autoclavato e mescolato accuratamente. Preparare in piatti di Petri sterili da 150 mm x 15 mm. Le piastre di latte possono essere conservate fino a una settimana avvolte in plastica, con coperchio verso il basso a 4 ° C. Preparare in piatti di Petri sterili da 150 mm x 15 mm versando agar mischiati nei piatti di Petri. </li></ol></li><li> Piastre di agaricità <ol><li> Per il componente 1 (LB agar): pesare 5,0 g di tryptone, 2,5 g di estratto di lievito, 2,5 g di NaCl e 2,0 g di agar. Combinare i costituenti in un pallone Erlenmeyer da 1 L, regolare il volume a 500 mL con acqua deionizzata e autoclave. </li><li> Per il componente 2 (1% (w / v) cloruro di trifeniltetrazolium; TTC): pesare 0,25 g di TTC. Regolare il volume a 25 ml con acqua deionizzata in una vescicola sterile e sterilizzare con filtro utilizzando un filtro da 0,22 μm. Conservare fino a 6 mesi a temperatura ambiente avvoltoIn foglio per minimizzare l&#39;esposizione alla luce. </li><li> Aggiungere 2,5 ml di 1% (w / v) TTC sterile ai supporti autoclavati. </li><li> Versare il mezzo più spesso possibile (≥50 mL / piastra) in grandi piatti di Petri sterili 150 mm x 15 mm. I supporti placcati possono essere immagazzinati in plastica fino a una settimana, con coperchio fino a 4 ° C. Preparare in piatti di Petri sterili da 150 mm x 15 mm versando agar mischiati nei piatti di Petri. </li></ol></li><li> Voges-Proskauer (VP) <ol><li> Per il componente 1 (Methyl Red-Voges-Proskauer broth; MR-VP): pesare 1,7 g di supporto MR-VP in un pallone Erlenmeyer da 500 ml, regolare il volume a 100 ml con acqua deionizzata e autoclave. Il brodo sterile MR-VP può essere conservato a temperatura ambiente fino a 6 mesi. </li><li> Per il componente 2 (5% (w / v) alfa (α) naphtolo): pesare 1,0 g di α-naftolo in una vescicola sterile e regolare il volume a 20 ml con 95% etanolo. L&#39;α-naftolo può essere conservato a temperatura ambiente fino a 1 settimana avvolto in fogli per ridurre al minimo la luceosure. </li><li> Per il componente 3 (40% (w / v) idrossido di potassio; KOH): pesare 20,0 g di KOH in una vescicola sterile, regolare il volume a 50 ml con acqua sterile deionizzata. KOH può essere conservato fino a 6 mesi a temperatura ambiente in un recipiente di plastica. </li></ol></li></ol> 2. Manutenzione e crescita di V. Ceppi di colera <ol><li> Preparazione e utilizzo di colture congelate <ol><li> Pellet 1.8 ml di coltura di notte per 2 min per centrifugazione (≥8.600 xg) in un tubo sterile di microcentrifuga da 2 ml, rimuovere il surnatante e riposizionare il pellet cellulare in 900 μL di brodo fresco di LB pipettando. </li><li> Aggiungere 900 μL di glicerolo sterile al 60% (v / v) alla coltura e mescolare mediante vortexing. </li><li> Trasferire la miscela in un tubo criogenico sterile da 2 ml e conservare a tempo indeterminato a -80 ° C. </li><li> Per utilizzare, rimuovere una piccola quantità di stock congelata usando un ciclo inoculatore sterile e una striscia per singole colonie su LB agAr. Immediatamente rimettere a magazzino congelato a -80 ° C dopo l&#39;uso per evitare che le culture si scongiurino completamente. Incubare le piastre con il coperchio lungo il basso per 12 a 16 ore a 37 ° C. </li></ol></li><li> Crescita di culture di overnight in liquido medio <ol><li> Striature per colonie singole (secondo la sezione 2.1.4) da scorte surgelate su piastre LB agar. Incubare le piastre con il coperchio laterale per 12 a 16 ore a 37 ° C. </li><li> Inoculare 4 ml di liquido LB liquido in un tubo di coltura sterile da 10 ml con una singola colonia toccando la superficie della singola colonia con un bastone applicatore sterile e trasferendo la colonia nel brodo liquido. </li><li> Incubare con aerazione in un incubatore a scuotitore a 225 giri / min per 12 a 16 h a 37 ° C. </li></ol></li><li> Curva di crescita <ol><li> Preparare la coltura durante la notte in brodo LB liquido come precedentemente indicato nel protocollo 2.2. </li><li> Fai una diluizione 1: 100 di coltura durante la notte trasferendo 250 μL di coltura overnight a 25 ml di brodo fresco di LB in un pallone Erlenmeyer da 250 ml sterile. </li><li> Misurare la densità ottica delle colture liquide a 600 nm (OD 600 ) ogni ora a partire dall&#39;inoculazione (T 0 ). </li><li> Incubare una diluizione 1: 100 di coltura durante la notte con aerazione in un incubatore a scuotitore a 225 giri / min fino a 30 h a 37 ° C o fino a raggiungere la massima densità della coltura. </li><li> Grafico OD 600 vs. tempo e utilizzare regressione lineare per determinare il tempo di raddoppio approssimativo per ogni ceppo. </li></ol></li><li> El Tor Biotipo Virulenza Induzione delle Condizioni <ol><li> Striata per singole colonie da scorta congelata su piastre LB agar. Incubare le piastre verso il basso con coperchio, per 12 a 16 ore a 37 ° C. </li><li> Inoculare 10 mL di mezzo AKI contenente 0,03% (w / v) NaHCO 3 con una sola colonia. </li><li> Incubare la coltura senza aerazione a 37 ° C per 3,5 ore. </li><li> Rimuovere 7 mL di coltura e incubare le restanti 3 mL di cultuCon aerazione in un incubatore a scuotitore a 225 giri / min per altri 4 h. </li></ol></li><li> Condizioni di inducimento della virulenza di biotipo classico <ol><li> Striata per singole colonie da scorta congelata su piastre LB agar. Incubare le piastre con il coperchio laterale per 12 a 16 ore a 37 ° C. </li><li> Inoculare 4 mL di liquido LB liquido (pH 6,5) con una singola colonia. </li><li> Incubare con aerazione in un incubatore a scuotitore a 225 giri / min per 12 a 16 h a 30 ° C. </li></ol></li><li> Pellet cella di lavaggio in 1x PBS <ol><li> Pellet 1.8 ml di coltura durante la notte per 2 min per centrifugazione (≥8.600 xg) in un tubo sterile di microcentrifuga da 2 ml e rimuovere il surnatante usando una pipetta. Riposizionare il pellet di cellula in 1,8 ml di 1x PBS pipettando. </li><li> Ripetere la procedura di lavaggio tre volte seguita da una resuspensione finale in 1,8 mL di 1x PBS. </li></ol></li></ol> 3. Caratterizzazione V. Biotipi del colera <ol><li> PSchermate genetiche basate su CR utilizzando ctxB e tcpA <ol><li> Progettare un insieme di primer, che annealizzano circa 50-70 bp a monte ea valle degli start e stop di traslazione di ctxB e tcpA. </li><li> Preparare la coltura durante la notte in brodo LB liquido come precedentemente indicato nel protocollo 2.2. </li><li> Isolare il DNA cromosomico utilizzando un kit commercialmente disponibile per i batteri Gram-negativi. Il DNA cromosomico purificato può essere immagazzinato indefinitamente a -20 ° C. L&#39;isolamento cromosomico del DNA usando i kit disponibili in commercio richiede generalmente circa 4 h. </li><li> Utilizzare un spettrofotometro a micro-volume per assicurare che il campione del DNA cromosomico sia di alta qualità (A 260/280 &gt; 1,8). </li><li> Per ciascun isolamento cromosomico del DNA, preparare le reazioni di polimerasi (PCR) su ghiaccio nel tubo sterile di 200 μl di PCR e amplificare le regioni di ctxB e tcpA usando componenti standard PCR: Taq polimerasi e buFfer (o equivalenti), soluzione dNTP e primer avanti / indietro. Utilizzare parametri standard PCR (~ 3 h). Ad esempio, utilizzare il seguente protocollo: <ol><li> Denatura iniziale a 95 ° C per 120 s. </li><li> Denatura a 95 ° C per 60 s. </li><li> Applicare primer a 60 ° C per 45 s. </li><li> Estendere a 72 ° C per 90 s. </li><li> Ripetere i passaggi da 3.1.5.2 a 3.1.5.4 per 34 cicli. </li><li> Estensione finale 72 ° C per 600 s. </li><li> Tenuta infinita a 4 ° C. </li></ol></li><li> Dye 5 μL di rispettivi prodotti PCR usando un colorante di caricamento a gel di DNA standard 6x e caricare circa 10 μL di scala 1 kb e 10 μL di prodotto PCR su un gel agarosico al 1% in 1x Tris-Borato-EDTA (TBE) buffer. </li><li> Eseguire l&#39;elettroforesi a gel a 130 V finché il frontale del colorante non raggiunge la fine, ma non fuori, del gel (circa 90 minuti). Si consiglia di monitorare costantemente come tensione e tempi di funzionamento possono variare a seconda dell&#39;apparecchiatura. </li><li> Alla verifica delUna amplificazione PCR di successo alla dimensione prevista, purificare il restante 45 μl di prodotto PCR usando un kit di pulizia e concentratore del DNA disponibili in commercio. </li><li> Sequenza di prodotti PCR puliti utilizzando gli stessi primer utilizzati per l&#39;amplificazione PCR e prepararsi secondo le linee guida locali di sequenziamento. </li><li> Confronta il formato FASTA di sequenze di gene da ogni isolato alla sequenza pubblicata disponibile sul sito NCBI (http://www.ncbi.nlm.nih.gov/gene/), cercando i seguenti numeri di accesso nella casella della query di ricerca. </li><li> CtxB : regione (classica) tra VC0395_A1059 a VC0395_A1060 (El Tor) VC_1456 </li><li> TcpA : (classico) VC0395_A0353 (El Tor) VC_0828 </li></ol></li><li> Analisi fenotipiche per la classificazione di biotipo mediante spotting <ol><li> Preparare la coltura durante la notte in brodo LB liquido come precedentemente indicato nel protocollo 2.2. </li><li> Lavare le pellicole di cellule come specificato nel protocollo 2.6. </li><li> Usare una pipetta aSpot 1 μl di coltura lavata in corrispondente mezzo. Per le specifiche di selezione e incubazione medio, fare riferimento alla Tabella 2. </li></ol></li><li> Analisi della carenza <ol><li> Preparare la coltura durante la notte in brodo LB liquido come precedentemente indicato nel protocollo 2.2. </li><li> Lavare le pellicole di cellule come specificato nel protocollo 2.6. </li><li> Inoculare le piastre di agarità agarico inserendo l&#39;inoculazione dello straccio nella coltura liquida lavata e &quot;pungere&quot; verticalmente nei supporti. In mezzo a ogni inoculazione, sterilizzare il coltello con un bruciatore di Bunsen e, quando pigiate l&#39;agar, assicurati che il pungo inoculato non si piega, in quanto ciò può alterare i risultati. </li><li> Incubare le piastre con il coperchio verso l&#39;alto per 14 a 24 ore a 37 ° C. Dopo 14 ore, controllare le piastre di movimento per evitare la crescita eccessiva di culture. </li></ol></li><li> Analisi Voges-Proskauer (VP) <ol><li> Preparare la coltura durante la notte in brodo LB liquido come precedentemente indicato nel protocollo 2.2. </li><li> Inoculare 4Ml di Voges-Proskauer rosso o rosso di metile, o brodo MR-VP, pipettando 10 μL di coltura overnight in precedenza preparata in 4 mL di brodo MR-VP in un tubo di coltura sterile e incubato con aerazione in un incubatore a scuotitore a 225 giri / min per 12 a 16 H a 37 ° C. </li><li> Aggiungere 150 μl di α-naftolo al 5% (w / v) e 50 μl 40% (w / v) KOH in 1 ml aliquote di coltura overnight di MR-VP in tubi di coltura sterile rispettivamente. </li><li> Brevemente vortice e lasciate stare a temperatura ambiente fino a 4 h fino a quando non cambia colore. </li></ol></li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+spread+of+cholera:+genetic+diversity+and+relationships+within+the+seventh+pandemic+clone+of+Vibrio+cholerae+determined+by+amplified+fragment+length+polymorphism.">Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism.</a> J. Clin. Microbiol. 40 (1), 172-181 (2002).</li><li>Nair, G. B., Faruque, S. M., Bhuiyan, N. A., Kamruzzaman, M., Siddique, A. K., Sack, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+variants+of+Vibrio+cholerae+O1+biotype+El+Tor+with+attributes+of+the+classical+biotype+from+hospitalized+patients+with+acute+diarrhea+in+Bangladesh.">New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh.</a> J. Clin. Microbiol. 40 (9), 3296-3299 (2002).</li><li>Nair, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vibrio+cholerae+O1+strains+similar+to+pre-seventh+pandemic+El+Tor+strains+during+an+outbreak+of+gastrointestinal+disease+in+an+island+resort+in+Fiji.">Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji.</a> J. Med. Microbiol. 55 (11), 1559-1562 (2006).</li><li>Nair, G. B., Mukhopadhyay, A. K., Safa, A., Takeda, Y., Faruque, S. M., Nair, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+hybrid+variants+of+Vibrio+cholerae+O1.">Emerging hybrid variants of Vibrio cholerae O1.</a> Vibrio cholerae—Genomics and Molecular Biology. , 179-190 (2008).</li><li>Safa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+characteristics+of+Matlab+variants+of+Vibrio+cholerae+O1+that+are+hybrids+between+classical+and+El+Tor+biotypes.">Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes.</a> J. Med. Microbiol. 55 (11), 1563-1569 (2006).</li><li>Nusrin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+CTX+phages+among+toxigenic+Vibrio+cholerae+O1+and+O139+strains+isolated+between+1994+and+2002+in+an+area+where+cholera+is+endemic.">Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic.</a> J. Clin. Microbiol. 42 (12), 5854-5856 (2004).</li><li>Carignan, B. M., Brumfield, K. D., Son, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphisms+in+regulator-encoding+genes+have+an+additive+effect+on+virulence+gene+expression+in+a+Vibrio+cholerae+clinical+isolate.">Single nucleotide polymorphisms in regulator-encoding genes have an additive effect on virulence gene expression in a Vibrio cholerae clinical isolate.</a> mSphere. 1 (5), e00253 (2016).</li><li>Iwanaga, M., Yamamoto, K., Higa, N., Ichinose, Y., Nakasone, N., Tanabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+stimulating+cholera+toxin+production+by+Vibrio+cholerae+O1+El+Tor.">Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor.</a> Microbiol. Immunol. 30 (11), 1075-1083 (1986).</li><li>DiRita, V. J., Claude, P., Georg, J., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+cascade+controls+virulence+in+Vibrio+cholerae.">Regulatory cascade controls virulence in Vibrio cholerae.</a> Proc. Natl. Acad. Sci. USA. 88 (12), 5403-5407 (1991).</li><li>Kovacikova, G., Lin, W., Skorupski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duel+regulation+of+genes+involved+in+acetoin+biosynthesis+and+motility/biofilm+formation+by+the+virulence+activator+AphA+and+the+acetate-responsive+Lys-R+type+regulator+AlsR+in+Vibrio+cholerae.">Duel regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive Lys-R type regulator AlsR in Vibrio cholerae.</a> Mol. Microbiol. 57 (2), 420-433 (2005).</li><li>Vogel, H. J., Bonner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylornithinase+of+Escherechia+coli:+partial+purification+and+some+properties.">Acetylornithinase of Escherechia coli: partial purification and some properties.</a> J. Biol. Chem. 218 (1), 97-106 (1956).</li><li>Son, M. S., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+and+biochemical+assays+to+characterize+Vibrio+cholerae+O1+biotypes:+classical+and+El+Tor.">Genetic screens and biochemical assays to characterize Vibrio cholerae O1 biotypes: classical and El Tor.</a> Curr. Protoc. Microbiol. , 6A.2.1-6A.2.17 (2011).</li><li>Kovacikova, G., Skorupski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+virulence+gene+expression+in+Vibrio+cholerae+by+quorum+sensing:+HapR+functions+at+the+aphA+promoter.">Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter.</a> Mol. Microbiol. 46 (4), 1135-1147 (2002).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+functional+quorum-sensing+systems+in+recently+emerged+Vibrio+cholerae+toxigenic+strains.">The prevalence of functional quorum-sensing systems in recently emerged Vibrio cholerae toxigenic strains.</a> Environ. Microbiol. Rep. 3 (2), 218-222 (2011).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques.">Aseptic laboratory techniques.</a> J. Vis. Exp. (63), e3064 (2012).</li><li>Martinez, R. M., Megli, C. J., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+laboratory+maintenance+of+Vibrio+cholerae.">Growth and laboratory maintenance of Vibrio cholerae.</a> Curr. Protoc. , 6A.1.1-6A.1.6 (2010).</li></ol>

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Dei oltre 200 identificati V. Colerae, solo O1 e O139 hanno un potenziale epidemico. Il serogroup di O1 può essere suddiviso in due biotipi: classici e El Tor. Tuttavia, sono emersi i ceppi ibridi, denominati varianti El Tor 13 , 17 , che possiedono il background Biotipo El Tor e le caratteristiche classiche del porto 8 , 9 , 10 , ...

Il colera è una malattia dell&#39;intestino tenue distale causata dal consumo di alimenti contaminati o di acqua contenente il batterio Gram-negativo acquatico Vibrio cholerae . I sintomi del colera includono vomito e diarrea acquosa incontrollabile, portando ad una grave disidratazione che, se non trattata correttamente, provocherà la morte. V. Il colerae può essere diviso in oltre 200 serogruppi basati sulla struttura del lipopolysaccharide O-antigene della superficie cellulare. Tuttavia, solo 2 serogruppi, O1 e O139, hanno mostrato potenziale epidemico o pandemico 1 , 2 . Inoltre, il serogroup O139 è stato principalmente isolato per l&#39;Asia sud-orientale 3 , 4 , mentre il sierogruppo O1 è distribuito in tutto il mondo. Inoltre, il serogroupo O1 può essere diviso in 2 biotipi principali: classici e El Tor. Il biotipo classico era responsabile delle prime 6 pandemie del coleraTra il 1817 e il 1923. La peggiorata settima pandemia è il risultato del biotipo El Tor, che ha spostato globalmente il biotipo classico nell&#39;ambiente 5 , 6 , 7 . Sono stati recentemente presentati ceppi che contengono caratteristiche distintive di biotipi classici e El Tor 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 e da allora sono stati denominati varianti El Tor 13 , 17 . Alcune varianti di El Tor hanno dimostrato elevate capacità di virulenza con una progressione più rapida e grave della malattia rispetto a quanto precedentemente osservato, sottolineandoLa necessità di un approccio più completo all&#39;identificazione degli agenti e alla prevenzione e trattamento delle malattie 8 , 9 , 18 . Mentre l&#39;identificazione del biotipo non impone immediatamente il trattamento, ulteriori progressi nello sviluppo del vaccino e nei futuri agenti terapeutici possono beneficiare della distinzione tra biotipo. La prima serie di protocolli elencati qui consentirà agli investigatori di mantenere correttamente V. Ceppi di colera in un ambiente di laboratorio. La coerenza e l&#39;analisi successiva richiedono la preparazione e la crescita degli stock di isolati, che non dipendono dal biotipo. Tuttavia, per indurre in modo ottimale l&#39;espressione genica di virulenza, sono necessarie tecniche indipendenti di coltura biotipo 19 . Inoltre, la preparazione per diversi dosaggi genetici e biochimici è descritta in questo manoscritto. Tossina del colera (CT) e la tossina co-re(TCP) sono due principali fattori di virulenza controllati dal regolatore master ToxT in entrambi i biotipi del V. Il colerae O1 sierogruppo 20 . CT è una tossina bipartita composta da cinque CtxB Subunità che circondano una singola subunità CtxA ed è responsabile della rapida perdita di elettroliti associata al colera. Il TCP è un pilone di tipo IV codificato dal tcp operon ( tcpABQCRDSTEF ) ed è coinvolto nell&#39;attaccamento e nella colonizzazione dell&#39;intestino tenue distale. TcpA è il primo gene dell&#39;operone tcp che codifica le singole subunità piline essenziali per la costruzione del pilus 8 . La sequenza genica per ctxA è completamente conservata tra biotipi classici e El Tor, mentre ctxB e tcpA si differenziano tra i due biotipi ma sono conservati all&#39;interno di ciascun biotipo 8 . CtxB è completamente conservato tra i biotipi ad eccezione di due posi base(115 e 203). Nel biotipo El Tor, la timina risiede nelle posizioni base 115 e 203, mentre il biotipo classico contiene citosina a queste basi. TcpA è completamente conservato all&#39;interno di ogni biotipo, ma differisce da molteplici basi tra biotipi. Queste distinzioni genetiche servono come marcatori di identificazione biotipo primari, e dopo la sequenza del prodotto di amplificazione della reazione di polimerasi (PCR), inclusi questi siti, le sequenze di isolamento possono essere confrontate con il classico O395 o WT El Tor N16961 di tipo wild (WT) per determinare lo sfondo del biotipo Di CT e TCP rispettivamente, in un dato isolamento di V. cholerae . Numerosi protocolli sono stati sviluppati per caratterizzare le distinzioni fenotipiche tra i biotipi classici e El Tor 21 , 22 , 23 . Polimicina B è un antibiotico peptidico che compromette l&#39;integrità della membrana cellulare esterna in bacTeria e la resistenza alla polimicina B possono essere visualizzati attraverso il saggio di resistenza della polimigina B 21 . Il citrato è un substrato primario del ciclo di Kreb, e la capacità di metabolizzare il citrato come fonte di carbonio unica può essere determinata usando il saggio del metabolismo citrato 22 . HapR codifica un regolatore globale e il regolatore di sensori quorum master in V. cholerae, HapR, che si lega a varie regioni del promotore e regola l&#39;espressione del gene e dell&#39;operone 24 . Alcuni ceppi patogeni di V. cholerae hanno una mutazione in movimento naturale nel gene hapR che ha causato la perdita di questa regolazione dipendente dalla densità dell&#39;espressione genetica di virulenza 24 , 25 . La misurazione dell&#39;attività di proteasi regolata da HapR utilizzando il supporto agarico del latte permette al ricercatore di identificare se un particolare isolato contiene un HapR 23 funzionale. IlI test di hemolysis per una capacità del ceppo di secernere enzimi emolitici che lissano i globuli rossi; Il grado di emolisi può essere visualizzato sulle piastre di agar sangue 23 . La motilità è spesso associata alla virulenza di V. cholerae e può essere analizzata usando le piastre agarico agar 23 . Il test Voges-Proskauer esamina la capacità di uno strain di fermentare il glucosio come una sola fonte di carbonio e produrre l&#39;acetoina sottoprodotto 21 . Con l&#39;emergere di varianti di El Tor, è difficile prevedere i risultati di un determinato dosaggio fenotipico senza estesa screening genotipica e prima di dedurre lo sfondo biotipo di V. Isolati di colerae, si raccomanda di eseguire questo insieme di dosaggi 23 e confrontare i risultati con i ceppi di riferimento come nella tabella 2 . In questo contesto, abbiamo avanzato una serie di protocolli, utilizzando collettivamente l&#39;aforemeI test genotipici e fenotipici per un approccio più completo alla caratterizzazione di V. Biotipi di colera. Inoltre, abbiamo descritto le distinzioni genotipiche e fenotipiche del V noto. Varianti del colerae El Tor (MQ1795 e BAA-2163), rispetto ai ceppi di riferimento di biotipo comunemente usati (WT classico O395, WT El Tor C6706 e WT El Tor N16961, Tabella 1 ). L&#39;emergere di varianti El Tor ha presentato sfide all&#39;affidabilità dei protocolli di individuazione biotipo di singolo dosaggio precedentemente impiegati; Tuttavia, questo sistema di identificazione multipla consentirà una caratterizzazione più affidabile dei criteri clinici e ambientali. Isolati di colera .

<table border="0" cellpadding="0" cellspacing="0" width="1403">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1 kb DNA Ladder</td>
			<td style="width:183px;">New England Biolabs</td>
			<td style="width:171px;">N3232S</td>
			<td style="width:683px;">https://www.neb.com/products/n3232-1-kb-dna-ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60% Glycerol</td>
			<td>Calbiochem</td>
			<td>356352</td>
			<td>http://www.emdmillipore.com/US/en/product/Glycerol%2C-Molecular-Biology-Grade---CAS-56-81-5---Calbiochem,EMD_BIO-356352</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Becto, Dickinson and Co.</td>
			<td>214030</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=214030&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D214030%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar with brain-heart infusion</td>
			<td>Becto, Dickinson and Co.</td>
			<td>237500</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=237500&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D237500%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Peqlab</td>
			<td>732-2789</td>
			<td>https://de.vwr.com/store/catalog/product.jsp?catalog_number=732-2789&#38;_DARGS=/store/cms/de.vwr.com/de_DE/header_2016111711383215.jsp_AF&#38;_dynSessConf=1766917479792147141&#38;targetURL=/store/catalog/product.jsp%3Fcatalog_number%3D732-2789&#38;lastLanguage=en&#38;/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=&#38;_D%3AcurrentLanguage=+&#38;currentLanguage=en&#38;_D%3AlastLanguage=+&#38;_D%3A/vwr/userprofiling/EditPersonalInfoFormHandler.updateLocale=+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anhydrous K2HPO4</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-phosphate-dibasic-anhydrous-crystalline-powder-certified-acs-fisher-chemical-5/p288500?searchHijack=true&#38;searchTerm=P288500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Agar Plates</td>
			<td>Remel</td>
			<td>R01200</td>
			<td>Store at 4 &#176;C;&#160; http://www.remel.com/Catalog/Item.aspx?name=Blood+Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A73-500</td>
			<td>https://www.fishersci.com/shop/products/boric-acid-crystalline-certified-acs-fisher-chemical-6/a73500?searchHijack=true&#38;searchTerm=A73500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromothymol Blue</td>
			<td>Fisher Scientific</td>
			<td>B388-10</td>
			<td>https://www.fishersci.com/shop/products/bromothymol-blue-certified-acs-fisher-chemical/b38810?searchHijack=true&#38;searchTerm=B38810&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cirtric acid &#183;H2O</td>
			<td>Fisher Scientific</td>
			<td>S72836-3</td>
			<td>https://www.fishersci.com/shop/products/citric-acid-monohydrate-4/s728363#?keyword=s728363</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxynucleotide (dNTP) Solution Kit</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/n0446-deoxynucleotide-solutionset</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium EDTA</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>https://www.fishersci.com/shop/products/ethylenediaminetetraacetic-acid-disodium-salt-dihydrate-crystalline-certified-acs-fisher-chemical-7/s311500?searchHijack=true&#38;searchTerm=S311500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Clean &#38; Concentrator&#8482; -25 Kit</td>
			<td>Zymo Research</td>
			<td>D4007</td>
			<td>http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GelGreen Nucleic Acid Stain</td>
			<td>Biotium</td>
			<td>41005</td>
			<td>https://biotium.com/product/gelgreentm-nucleic-acid-gel-stain-10000x-in-water/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Genesys 10SUV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208100</td>
			<td>https://www.thermofisher.com/order/catalog/product/840-208100?ICID=search-840-208100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentra Puregene Yeast/Bact. Kit</td>
			<td>Qiagen</td>
			<td>158567</td>
			<td>https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/gentra-puregene-yeastbact-kit/#orderinginformation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td>Corrosive;&#160; https://www.fishersci.com/shop/products/hydrochloric-acid-certified-acs-plus-fisher-chemical-10/a144212?searchHijack=true&#38;searchTerm=A144212&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-chloride-crystalline-certified-acs-fisher-chemical-4/p217500?searchHijack=true&#38;searchTerm=P217500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-phosphate-monobasic-crystalline-certified-acs-fisher-chemical-5/p285500?searchHijack=true&#38;searchTerm=P285500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-500</td>
			<td>https://www.fishersci.com/shop/products/potassium-hydroxide-pellets-certified-acs-fisher-chemical-5/p250500?searchHijack=true&#38;searchTerm=P250500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Loop</td>
			<td>Decon Labs Inc.</td>
			<td>MP190-25</td>
			<td>http://deconlabs.com/products/leloop/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Le Stab</td>
			<td>Decon Labs Inc.</td>
			<td>MP186-5</td>
			<td>http://deconlabs.com/products/lestab/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?searchHijack=true&#38;searchTerm=M63500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Sub Cell GT Horizontal Electrophoresis System</td>
			<td>Bio Rad Labs</td>
			<td>1704406</td>
			<td>http://www.bio-rad.com/en-us/product/mini-sub-cell-gt-cell?WT.srch=1&#38;WT.mc_id=aw-cbb-NA-sub_cell_systems_brand_gold&#38;WT.knsh_id=7eb1981f-a011-42a3-aece-1236ff453373</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MR-VP Broth</td>
			<td>Difco</td>
			<td>216300</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=216300&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3Dmr-vp%2Bmedium%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-phosphate-dibasic-anhydrous-granular-powder-certified-acs-fisher-chemical-5/s374500?searchHijack=true&#38;searchTerm=S374500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-10</td>
			<td>https://www.fishersci.com/shop/products/sodium-chloride-crystalline-certified-acs-fisher-chemical-6/s27110?searchHijack=true&#38;searchTerm=S27110&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-bicarbonate-powder-certified-acs-fisher-chemical-5/s233500?searchHijack=true&#38;searchTerm=S233500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaNH4HPO4&#183;4H2O</td>
			<td>Fisher Scientific</td>
			<td>S218-500</td>
			<td>https://www.fishersci.com/shop/products/sodium-ammonium-phosphate-tetrahydrate-crystalline-certified-fisher-chemical/s218500?searchHijack=true&#38;searchTerm=S218500&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop Lite Spectrophtometer</td>
			<td>Thermo Scientific</td>
			<td>ND-LITE-PR</td>
			<td>https://www.thermofisher.com/order/catalog/product/ND-LITE-PR?ICID=search-ND-LITE-PR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonfat dry milk</td>
			<td>Nestle Carnation</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211677</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211677&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211677%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100 mm x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875712#?keyword=FB0875712</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (150 mm x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875714</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875714?searchHijack=true&#38;searchTerm=FB0875714&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polymyxin B sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1004-10MU</td>
			<td>Store at 2-4 &#176;C; http://www.sigmaaldrich.com/catalog/product/sial/p1004?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td>Store at -20 &#176;C;&#160; https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Cycler Bio-Rad C1000 Touch&#8482;&#160;</td>
			<td>Bio Rad Labs</td>
			<td>1840148</td>
			<td>http://www.bio-rad.com/evportal/evolutionPortal.portal?_nfpb=true&#38;_pageLabel=search_page&#38;sfMode=search&#38;sfStartNumber=1&#38;clearQR=true&#38;js=1&#38;searchString=1840148&#38;database=productskus+productcategories+productdetails+abdProductDetails+msds+literatures+inserts+faqs+downloads+webpages+assays+genes+pathways+plates+promotions&#38;tabName=DIVISIONNAME</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triphenyltetrazolium chloride</td>
			<td>Alfa Aesar</td>
			<td>A10870</td>
			<td>https://www.alfa.com/en/catalog/A10870/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>https://www.fishersci.com/shop/products/tris-base-white-crystals-crystalline-powder-molecular-biology-fisher-bioreagents-7/bp1521?searchHijack=true&#38;searchTerm=BP1521&#38;searchType=RAPID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8729;HCl</td>
			<td>Calbiochem</td>
			<td>9310</td>
			<td>http://www.emdmillipore.com/US/en/product/OmniPur-TRIS-Hydrochloride---CAS-1185-53-1---Calbiochem,EMD_BIO-9310-OP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Becto, Dickinson and Co.</td>
			<td>211705</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=211705&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D211705%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becto, Dickinson and Co.</td>
			<td>212750</td>
			<td>http://catalog.bd.com/nexus-ecat/getProductDetail?productId=212750&#38;parentCategory=&#38;parentCategoryName=&#38;categoryId=&#38;categoryName=&#38;searchUrl=%2FsearchResults%3Fkeyword%3D212750%26typeOfSearch%3DproductSearch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-napthol</td>
			<td>MP Biomedicals</td>
			<td>204189</td>
			<td>http://www.mpbio.com/product.php?pid=05204189</td>
		</tr>
	</tbody>
</table>

laboratory techniques used to maintain and differentiate biotypes of vibrio cholerae clinical and environmental isolates

Il batterio Gram-negativo acquatico Vibrio cholerae è l&#39;agente etiologico del colera della malattia gastrointestinale infettiva. A causa della prevalenza e gravità globali di questa malattia, V. Il colera è stato ampiamente studiato sia in ambito ambientale che in laboratorio, che richiede una corretta manutenzione e tecniche di coltura. Classici e El Tor sono due biotipi principali che compongono il V. Colerae O1, ognuno dei quali presenta caratteristiche uniche genotipiche e fenotipiche che forniscono meccanismi affidabili per la caratterizzazione del biotipo e richiedono condizioni distintive di coltura che inducono la virulenza. Indipendentemente dal biotipo del ceppo causale per qualsiasi infezione o focolaio, il trattamento standard per la malattia comprende terapia di reidratazione integrata con un regime di antibiotici. Tuttavia, la classificazione di biotipo può essere necessaria per studi di laboratorio e può avere impatti più ampi nel campo biomedico.Nei primi anni 2000 sono stati identificati isolati clinici che presentano tratti genotipici e fenotipici sia dai biotipi classici che da El Tor. Gli ibridi di nuova identificazione, chiamati El Tor, hanno causato l&#39;identificazione clinica e ambientale del biotipo di isolamento a diventare più complessi rispetto ai precedenti protocolli tradizionali di identificazione singola. Oltre a descrivere V. Le tecniche di mantenimento e tecniche di coltura del colerae, questo manoscritto descrive una serie di schermati genetici basati su PCR basati su gene ( ctxB e tcpA ) e saggi fenotipici (resistenza alla polimicina B, metabolismo citrato, attività proteolitica, attività emolitica, motilità e metabolismo del glucosio tramite Voges- Proskauer) utilizzati collettivamente per caratterizzare e / o distinguere tra biotipi classici e El Tor. Insieme, questi dosaggi forniscono un approccio sistematico efficiente da utilizzare come alternativa o, in aggiunta, a costosi esercizi di intensità lavorativa nella caratterizzazioneDi V. Cholerae isolati clinici (e ambientali).

Per una corretta manutenzione e l&#39;uso di qualsiasi ceppo batterico, è consigliabile conoscere il tempo di raddoppiamento dei ceppi di interesse. Qui, i tassi di crescita variabili dei V comunemente usati. I ceppi del colerae sono stati dimostrati attraverso una curva di crescita e sono stati calcolati approssimativi tempi di raddoppio utilizzando la regressione lineare. Il WT El Tor N16961 e la variante El Tor MQ1795 hanno dimostrato tempi di raddoppio più brevi (...

Watch this Scientific Journal Video about Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates at JoVE.com

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

Questo manoscritto descrive le appropriate tecniche di manutenzione Vibrio cholerae oltre ad una serie di dosaggi biochimici, utilizzati collettivamente per una differenziazione rapida e affidabile tra la clinica e l&#39;ambiente V. Biotipi di colera in un ambiente di laboratorio.

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 ...

laboratory-techniques-used-to-maintain-differentiate-biotypes-vibrio

Research

JoVE Journal

Immunology and Infection

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

10.8K Views.  Plymouth State University.  Questo manoscritto descrive le appropriate tecniche di manutenzione Vibrio cholerae oltre ad una serie di dosaggi biochimici, utilizzati collettivamente per una differenziazione rapida e affidabile tra la clinica e l'ambiente V. Biotipi di colera in un ambiente di laboratorio. 

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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Utilizzando un pan-virale Assay Microarray (Virochip) allo schermo campioni clinici di patogeni virali

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Ricerca

Immunologia e infezioni

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Rilevazione multiplex di batteri in campioni clinici complessi ed Ambientali con oligonucleotidi accoppiati microsfere fluorescenti

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

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 ...

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 ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Un metodo modificato EPA 1623 che utilizza flusso tangenziale a fibre cave di ultrafiltrazione e di calore passi dissociazione per rilevare Waterborne<em&gt; Cryptosporidium</em&gt; E<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Misure di sicurezza e procedure operative in un (A) BSL-4 di laboratorio: 1. livello di biosicurezza 4 Suit Laboratorio Suite entrata e di uscita Procedure

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una strategia rapido per l&#39;isolamento di nuovi Faustoviruses da campioni ambientali Utilizzando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

Lo sviluppo di un anticorpo salivare Multiplex immunoenzimatico per misurare l&#39;esposizione umana ad agenti patogeni ambientali

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

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 ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Taglio di precisione polmone mouse Fette per la visualizzazione in diretta cellule dendritiche polmonare

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...