This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).

1. HSV ceppi e Manipolazione Nota: ricombinante non diffondere HSV-1 (KOS) gL86 e HSV-2 (KOS) 333gJ - con l&#39;attività reporter di beta-galattosidasi utilizzati sono stati forniti da V. Twiari 36,37. <ol><li> Utilizzare virus da un singolo lotto e conservare a -80 ° C in un rapporto 1: 1 di mezzo di Dulbecco modificato Eagle (DMEM) con siero fetale bovino 20% (FBS) e latte scremato fino al momento dell&#39;uso. Prima stoccaggio lot virale, determinare la concentrazione di virus o -nitrophenyl-β-D-galattopiranoside (ONPG) e 5-bromo-4-cloro-3-indolil-β-D-galattopiranoside (X-Gal) dosaggio. </li><li> Determinare la vitalità virus e molteplicità di infezione (MOI) di X-Gal colorazione per ogni seduta sperimentale mediante saggio entrata del virus giornalista, come descritto in precedenza (Figura 1) 14. </li><li> Diluire il virus (Opt-MEM) a MOI desiderato. Fissare monostrati (paraformaldeide; 0,5 ml / pozzetto) prima della colorazione. Posizionare controlli virus redditività in un microti separataPiastra ter in parallelo con le piastre test polimicrobiche. </li></ol> 2. HeLa 299 Manipolazione cellulare <ol><li> Crescere a 37 ° C, 5% CO 2 in DMEM con 4,5 g / L di glucosio, 10% inattivato al calore siero fetale bovino (FBS), gentamicina (50 ug / ml) e L-glutammina. cellule Passage a 80% di confluenza con la soluzione di tripsina (acido etilendiamminotetraacetico, 0,53 M EDTA 0,05% tripsina; 5 ml / fiasco). </li></ol> 3. C. Manipolazione albicans Nota: C. albicans ottenuti da una fonte di laboratorio clinico è conservato a -80 ° C in Remmel latte scremato 2x media. <ol><li> Cultura congelate Stock su Sabouraud destrosio Medium (37 ° C). Dopo 24 ore la sottocultura C. albicans su terreno Fungisel (37 ° C; 48 HR) per l&#39;uso. </li><li> Genera tubo germe (GT) forme (pick colonie rappresentative, 3 ml di FBS, 3 ore; 37 ° C; Abs 600, 0.3). Dopo l&#39;incubazione, lavare GT (HBSS; 2x; 4.000 xg). Aggiungere GT lavata per riscaldato HBSS (37 &amp;# 176; C; 0,32 Abs 600). forme GT dovrebbe essere il 99% delle cellule osservate come determinato dal conteggio emocitometro. </li><li> Fai la forma di lievito (YF) stock sospensioni con la scelta di colonie rappresentative Fungisel (HBSS, 3 ml; 0,32 Abs 600). Contare il numero di forme YF / ml al microscopio con un emocitometro. forme YF dovrebbero essere il 99% delle cellule osservate come determinato dal conteggio emocitometro. </li><li> Fare lavoro stock fungine (250 ml di GT o YF magazzino in 25 ml di HBSS; 37 ° C; 10 5 CFU / ml) </li></ol> 4. S. Manipolazione aureus <ol><li> Conservare S. aureus ATCC 25923 (-80 ° C; Remmel scremato 2x latte). Cultura su agar sangue di pecora (5%; 37 ° C; 24 ore). Scegli colonie rappresentante e trasferimento mannitolo sali media entro 2 giorni per il magazzino (37 ° C; 18 ore). </li><li> Fai S. aureus sospensione madre (3 ml di HBSS; 1.32 Abs 600; 10 8 UFC / ml) <ol><li> Fai S. lavoro aureus azionari (100 pldello stock in 25 ml di HBSS; 10 5 CFU / ml). </li></ol></li></ol> 5. Candida e S. aureus Sospensioni <ol><li> Fare misto C. albicans e S. sospensione aureus (250 ml YF o GT magazzino e 100 ml di S. aureus magazzino in 25 ml di HBSS). </li></ol> 6. polimicrobiche biofilm Assay <ol><li> Seed 96 pozzetti con 200 ml di 2 x 10 5 HeLa cellule / ml (85% livello di confluenza). Lastre di roccia (30 - 45 min; 37 ° C) prima di incubazione (37 ° C, 5% di CO 2 incubatore; 18 h). Monostrati lavaggio (1x; Opt-MEM) poi sementi con HSV (HSV-1 (KOS) gL86 o HSV-2 (KOS) 33 gJ - in 100 ml Opt-MEM; MOI 50 e 10). Incubare le piastre (3 ore; 37 ° C, 5% di CO 2). Utilizzare un solo ceppo virale al giorno. </li><li> Lavare monostrati infettati (1x; tampone fosfato (PBS) con Mg +2 e Ca +2; 100 ml). Sostituire PBS con caldo HBSS lasciando 25 &amp; #181; l in ciascun pozzetto. <ol><li> Aggiungere YF, GT e / o S. sospensioni aureus lavoro (100 ml; porta a rapporto cellulare = 5: 1; n = 16). come indicato nella Tabella 1 Incubare le piastre (statici; 30 min; 37 ° C; 5% CO 2). </li></ol></li><li> Dopo l&#39;incubazione, aspirare una colonna alla volta immediatamente ricarica da 300 ml PBS con Mg +2 e Ca +2. Ripetere questa operazione due volte, quindi aggiungere la radio-immunoprecipitazione tampone di lisi (RIPA; filtro sterilizzato, 200 ml di una diluizione 1:50). </li><li> Rapidamente triturate il lisato cellulare HeLa poi posto su 50 pl sali mannitolo (MS) e media / o Fungisel (F) (figura 2). Distribuire il lisato utilizzando una bacchetta di vetro piegata a 90 °. Incubare le piastre (18 ore a 37 ° C). contare manualmente il numero di colonie per piastra. I controlli sono costituiti da S. aureus e / o C. albicans aderenza alle cellule HeLa HSV-non infetti. </li></ol> 7. Gli studi di imaging &lt;/ P&gt; <ol><li> Lavare ogni coprioggetto di vetro rotondo (12 mm; 50 ml di acetone in 100 ml bicchiere). coprioggetto secche e sterilizzare (Kimwipes; piatti di vetro Petri). Mettere vetrini sterili a secco nei pozzetti di sterili 24 pozzetti con l&#39;alcool di fiamma pinze sterilizzate. </li><li> Aggiungere cellule HeLa (1 ml; il volume 5x utilizzato per 96 pozzetti) ai pozzetti di 24 pozzetti contenenti i vetrini rotondi sterili lavati. Aggiungere i virus, batteri e funghi secondo la dima (Tabella 2) al 5x il volume usato per le piastre a 96 pozzetti, poi incubare e processo come descritto ai punti 6.2.1 a 6.4 sopra. </li><li> Dopo l&#39;ultimo lavaggio, fissare le cellule per la microscopia inondando il vetrino con metanolo e permettendogli di evaporare. Conservare le piastre a temperatura ambiente fino a colorazione. </li><li> Per microscopia in campo luminoso (1,000x finale ingrandimento originale) riempire i pozzetti contenenti coprioggetto metanolo-fisso con acqua deionizzata. Immediatamente aspirare l&#39;acqua. Coprire ogni vetrino con cristalvioletto Grammi. Lavare coprioggetto libera di macchia non legato (acqua deionizzata). Coprioggetto secchi in situ poi aderire con set mediamente dura ad una slitta etichettato montaggio (Figura 3). </li><li> Per la microscopia a fluorescenza (obiettivo 100X; 1,000x originale ingrandimento finale) lavaggio coprioggetto privo di metanolo essenzialmente come descritto al punto 7.4. Asciugare il coprioggetto nei pozzetti. <ol><li> Dopo l&#39;essiccazione, rimuovere i vetrini e metterli su vetrini etichettati. Quindi aggiungere una quantità sufficiente di diluizione 1:20 di isotiocianato di fluorescina (FITC) coniugata Herpes simplex virus di tipo 1 + 2 GD anticorpi per coprire il vetrino (1 - 5 ml). </li><li> Incubare i vetrini in una camera umida a 37 ° C per 30 min. Dopo l&#39;incubazione, lavare i coprioggetti in 4 cambi di PBS. </li><li> Dopo l&#39;ultimo lavaggio, restituire il vetrino alla diapositiva etichettati. Macchia con 4 &#39;, 6-diamidino-2-fenilindolo (DAPI; camera umida; 37 ° C per 30 min). Dopo l&#39;incubazione lavare i coprioggetti in 4 chanGES di PBS, poi apporre la diapositiva etichettato con set dura mezzo di montaggio. </li><li> Lasciare che il mezzo di montaggio per la cura per 24 ore a temperatura ambiente al buio. Esaminare i coprioggetti sotto dell&#39;obiettivo olio sia su un microscopio a campo chiaro o microscopio a fluorescenza con filtri di taglio FITC e DAPI. Esaminate immagini di almeno 50 campi (100 cellule per minimo organismo) per co-localizzazione (Figura 4). </li></ol></li></ol>

The authors have nothing to disclose.

Al momento non sono disponibili informazioni sulle interazioni complesse tra permanente ai membri semi-permanenti del microbiome host che attraversano più domini tassonomici, vale a dire, procarioti, eucarioti e virali. Perciò abbiamo sviluppato un nuovo sistema modello in vitro per lo studio del biofilm iniziazione da S. aureus e C. albicans on HSV-1 oppure HSV-2 infettato 229 cellule HeLa 38 (HeLa). Il sistema modello cellule HeLa presenta un vantaggio...

<html> Il microbioma umano comprende diversi organismi da più regni tassonomici che condividono aree geografiche nel corpo. L&#39;adesione alla superficie delle cellule è un primo passo essenziale nella formazione di biofilm, che è parte del processo microbiome colonizzazione. Incluso nel microbiome può essere virus che causano infezioni croniche e persistenti. L&#39;infezione cronica a cellule da questi virus può causare un&#39;alterazione putativo disponibilità del recettore. 1,2 Inoltre, l&#39;ingresso delle cellule da patogeni intracellulari potrebbe anche influenzare la fluidità di membrana host / idrofobia che a loro volta possono alterare l&#39;attaccamento degli altri membri microbiome, compresi batteri e funghi . Al fine di comprendere le interazioni che possono verificarsi tra questi molteplici agenti patogeni che co-localizzano nelle stesse regioni geografiche del ospite umano, dobbiamo essere in grado di studiare l&#39;interazione di agenti patogeni che rappresentano lo spettro di regni tassonomici presenti sulla superficie della mucosa. t &quot;&gt; Il Herpesviridae sono una famiglia di microbi presenti nel 100% degli esseri umani come membri permanenti del microbiome 3,4. Inoltre, esse possono anche essere persistentemente versato sia in presenza che in assenza di sintomi. In particolare, herpes simplex virus-1 e herpes simplex virus 2 (HSV-1 e HSV-2, rispettivamente) sono membri permanenti del microbiome nella oronasopharynx e del tratto genitale. in individui immuno-competenti, sia HSV-1 e HSV-2 causa gengivostomatite, nonché herpes genitale 5-8. in questi siti, HSV provoca un&#39;infezione latente caratterizzata da persistente asintomatica virale spargimento 9. ingresso di HSV in cellule provoca alterazioni nell&#39;espressione superficiale di nectins, eparan solfato, raft lipidici e herpesvirus ingresso mediatore necrosi cronica / tumorale recettore del fattore (HVEM / TNFR) 10-25. Questi recettori potenzialmente rappresentano condivise per alcuni batteri e funghi, per esempio S. aureus e C. albicans, che pur patogeni opportunisti,può anche risiedere come membri del microbioma mucosa del oronasopharynx 26,27. All&#39;interno del oronasopharynx S. aureus e C. albicans occupano due siti distinti di colonizzazione. In host con denti naturali, la mucosa orale è condiviso da HSV-1 e C. albicans, mentre le narici nasali anteriori sono occupati da S. aureus 28. Tuttavia, nonostante i risultati in vitro in cui S. aureus aderisce alle cellule epiteliali della bocca, 29,30 S. aureus è raramente isolata da campioni orali quando il tessuto normale è presente 29,30. Poco si sa riguardo a genitali nicchie tratto co-colonizzazione al di là dei risultati clinici che S. aureus è associata a vaginite aerobica, caratterizzata da infiammazione genitale, scarico e dispareunia, mentre C. albicans produce lesioni della mucosa simile a quello osservato nella cavità orale 31-35. Così, anche se questi membri della Microbi orale e genitaleome croce regni tassonomici poco si sa sulla loro interazione come urta la loro capacità di avviare la formazione di biofilm attraverso l&#39;adesione alla superficie della cellula ospite 5. Questo protocollo è stato efficacemente applicato a determinare le conseguenze funzionali di co-colonizzazione / infezione.

<table border="0" cellpadding="0" cellspacing="0" width="1185">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">C.albicans</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">BBL Sabouraud Dextrose</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">211584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fungisel Agar</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">7205A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">S.aureus</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Mannitol Salt Agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">7143B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Sheep blood agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">221239</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hela cells</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">1xDMEM (Dubelcco&#39;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gentamicin 50mg/ml</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">1397</td>
			<td>50&#181;g/ml final concentration in the complete DMEM</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:391px;">Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X</td>
			<td style="width:195px;">Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fetal Bovine Serum</td>
			<td style="width:195px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% final concentration in the complete DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Other medium and reagents</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">ONPG</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">34055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra-Pure X gal</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:119px;">15520-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1x HBSS (Hanks&#39; Balanced Salt Solution)</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1XPBS</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">30042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">RIPA Lysis</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Staining</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A433P-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">HSV 1&#38;2, specific for gD</td>
			<td style="width:195px;">ViroStat</td>
			<td style="width:119px;">196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">DAPI</td>
			<td style="width:195px;">SIGMA</td>
			<td style="width:119px;">D8417-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gram Crystal Violet</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">212527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Supplies</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 100X15</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">229693&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 150X15</td>
			<td style="width:195px;">Kord Valmark</td>
			<td style="width:119px;">2902</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">96-Well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8030-01F</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">24-well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8044-01F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Culture tubes 100x13</td>
			<td style="width:195px;">Thomas Scientific</td>
			<td style="width:119px;">9187L61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Cover slip circles, 12mm</td>
			<td style="width:195px;">Deckglaser</td>
			<td style="width:119px;">CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of biofilm initiation on virus-infected cells by bacteria and fungi

Lo studio delle interazioni polimicrobiche attraverso i regni tassonomici che includono funghi, batteri e virus non sono stati precedentemente esaminati rispetto a come i membri virali del microbiome influenzano le successive interazioni microbo con queste cellule ospiti infettate da virus. La convivenza virus con batteri e funghi è principalmente presente sulle superfici mucose della cavità orale e genitale. le cellule delle mucose, in particolare quelli con infezioni virali latenti croniche o persistenti persistenti, potrebbero avere un impatto significativo sulla membri della microbiome attraverso l&#39;alterazione del virus in numero e tipo di recettori espressi. Modifiche nell&#39;architettura membrana della cellula ospite comporterebbe capacità alterato di successivi componenti della flora normale e patogeni opportunisti di avviare il primo passo nella formazione di biofilm, cioè, l&#39;aderenza. Questo studio descrive un metodo per la quantificazione e esame visivo effetto di HSV sulla apertura di Biofilformazione m (aderenza) di S. aureus e C. albicans.

Il livello di solidità dei dati ottenibili da sistema descritto in questa relazione è illustrata nella Figura 2 af 38. Attraverso l&#39;utilizzo di questo sistema di modulazione di interazione stafilococco e funghi con le cellule infettate da virus e il loro effetto sulla reciproca aderenza può essere delineata. Questi tipi di studi richiedono esame microscopico dell&#39;interazione come mostrato nelle figure 3 e 4, 38...

Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Determinazione del biofilm iniziazione su cellule infettate da virus da batteri e funghi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

determination-biofilm-initiation-on-virus-infected-cells-bacteria

Research

JoVE Journal

Immunology and Infection

9.6K Views.  Midwestern University. A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Video: Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa e biofilm Saccharomyces cerevisiae in celle di flusso

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Ricerca

Immunologia e infezioni

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtitolazione Dish Biofilm saggio Formazione

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

L&#39;uso di Citometria immagine per la quantificazione dei funghi patogeni in associazione con cellule ospiti

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Cellule T Capture batteri da Transinfection da cellule dendritiche

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotipica e analisi funzionale di cellule T regolatorie attivati ​​Isolato dal linfocitica cronica coriomeningite topi infettati da virus

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Sonde ottiche Screening di nuovi batteri specifici sul tessuto polmonare umano Ex Vivo di endomicroscopia confocale Laser

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...