Questo lavoro è stato supportato da NSF carriera premio 1553023 e premio NIH/NIAID R01AI103369.

1. crescita di Pseudomonas aeruginosa Colonia biofilm
<ol><li>Preparazione delle piastre Medium-Bilayer<ol>
<li>Preparare un tryptone di 10 g/L, agar 10 g/L (Vedi Tabella materiali) soluzione in acqua deionizzata.</li>
		<li>Autoclave per raffreddare a 50-60 ° C per 20 min. in un bagno d'acqua.</li>
		<li>Versare 45 mL della soluzione di agar-tryptone in un piatto quadrato 100 x 100 mm (Vedi Tabella materiali) utilizzando una provetta conica da 50 mL. Consentire l'agar solidificare (~ 20-30 min). Versare un secondo strato sopra strato prima 15 mL. Lasciare solidificare durante la notte, rimozione di condensazione da coperchi se necessario strofinando con un panno privo di lanugine.</li>
	</ol>
</li>
	<li>Spotting Colonia biofilm<ol>
<li>Inoculare i ceppi di interesse dalle scorte di congelatore sul LB agar piastre13 ed incubare al buio per 12-16 h a 37 ° C e 80-100% di umidità relativa.</li>
		<li>Per ogni ceppo o replicate, è possibile utilizzare una singola Colonia per inoculare 2 mL di LB e incubare al buio per 12-16 h a 37 ° C con agitazione a 250 giri/min.</li>
		<li>Sub-cultura diluendo 1: 100 in LB fresco e incubare al buio per 2.5-3 h a 37 ° C con agitazione a 250 giri/min.</li>
		<li>Misurare la densità ottica utilizzando uno spettrofotometro e regolare con LB sterile per ottenere una sospensione di cellule con un OD500 nm ~ 0,5.</li>
		<li>A seconda della Colonia desiderata taglia, dispensare 2.5-10 µ l di sospensione cellulare su un piatto di mezzo-doppio strato (preparato al punto 1.1) e consentire il posto ad asciugare per ~ 20 minuti lasciando il coperchio petri socchiusa vicino a fiamme può facilitare asciugarsi.</li>
		<li>Incubare il campione colonie a 25 ° C e 80-100% di umidità relativa, al buio fino a 4 giorni.</li>
	</ol>
</li>
</ol>2. preparazione della soluzione fissante
<ol><li>Preparare la soluzione fissante il giorno della raccolta del campione. Diluire il 37% di formaldeide (FA) in PBS 1X a una concentrazione finale del 4% FA. Attenzione: FA è volatile e tossico. Indossare indumenti protettivi e lavorare in una cappa ben ventilata.</li>
	<li>Immediatamente prima dell'uso, sciogliere L-lisina cloridrato in 4% FA (preparata al punto 2.1) a temperatura ambiente per una concentrazione finale di 50 millimetri L-Lisina HCl.</li>
</ol>3. diretta applicazione di fissativo per il Biofilm di Colonia [opzionale]
Nota: Abbiamo trovato che la morfologia di biofilm è meglio conservato quando agar molle viene aggiunto prima della fissazione. Tuttavia, questo passaggio costituisce anche un cambiamento delle condizioni ambientali che potrebbero influenzare l'espressione genica. Modelli di espressione di reporter fluorescente dovrebbero quindi essere verificati utilizzando un protocollo separato in cui il punto di fissazione è effettuato prima dell'aggiunta della sovrapposizione agar, come descritto qui.
<ol><li>Il giorno della fissazione e lavorando in una cappa ben ventilata, dispensare il fissativo preparato al punto 2 direttamente alla superficie dell'agar intorno a bordo della Colonia, permettendo così di raggiungere la periferia della Colonia senza sommergendola. Applicare solo come molto fissativo come è necessario completamente circondano la Colonia, circa 500 µ l. consentire il fissativo diffondere in Colonia e l'agar circostante.</li>
	<li>Una volta che il fissativo è stato completamente assorbito nella Colonia e mezzo circostante, circa 20-30 min, e FA residua non è più visibile sulla piastra, ripetere il punto 3.1. Permettere questo fissativo diffondere in Colonia e l'agar circostante.</li>
	<li>Una volta che il fissativo è stata completamente assorbito nella Colonia e mezzo circostante ripetizione passaggio 3.1, questa volta l'applicazione fissativo alla superficie della Colonia direttamente. Permettere questo fissativo diffondere in Colonia e l'agar circostante, procedere al passaggio 4 solo dopo tutto fissativo è stata assorbita e non è più visibile sulla piastra.</li>
</ol>4. overlaying colonie con Agar
<ol><li>Il giorno della fissazione, preparare un 10 g/L soluzione agar in acqua deionizzata e autoclave per 10-20 minuti sciogliere. Raffreddare a bagnomaria a 50 ˚ c.</li>
	<li>Delicatamente versare 15 mL della soluzione di agar sopra la coltura e la Colonia. Consentire l'agar formare un gel a temperatura ambiente per 5 min (Figura 1B).</li>
	<li>Usare una lama di rasoio affilata per tagliare un mandrino quadrato, 3 strati con la Colonia laminata tra i due strati superiori. Se preparare campioni multipli, assicurarsi che ciascun mandrino è tagliato a dimensioni comparabili.</li>
	<li>Rimuovere delicatamente l'eccesso agar dal mandrino Colonia-contenente.</li>
	<li>Per estrarre i due strati superiori dell'agar (contenente la Colonia) dallo strato inferiore del mandrino (Figura 1), bagnare la testa piatta di una spatola in 1X PBS o acqua e inserire delicatamente tra gli strati superiore ed inferiore. La colonia di laminato deve separare dallo strato inferiore. Fare attenzione a non distorcere o piegare la Colonia laminata quando solleva dalla sua base.</li>
</ol>5. fissazione
<ol><li>Lavorando in una cappa ben ventilata, trasferire immediatamente la Colonia laminata (cioè, chuck 2 strati) in una cassetta incasso (Vedi Tabella materiali), etichettati con un pennarello resistente alle sostanze chimiche. Inserire la cassetta di incorporamento in un mailer di diapositive di vetro (Vedi tabella materiali) contenente il fissativo preparato nei passaggi 2.1-2.2. Assicurarsi che non ci sono nessun bolle d'aria intrappolate all'interno della cassetta incasso.</li>
	<li>Incubare il campione nel fissativo pernottamento in al buio a temperatura ambiente.</li>
</ol>6. campione di elaborazione: Tampone di lavaggio, disidratazione, schiarimento e infiltrazione
<ol><li>Dopo la fissazione durante la notte, lavare il campione due volte in PBS 1X per 1h ciascuna. Per risultati ottimali, automatizzare omogeneizzazione di reagente utilizzando lo spin funzione di un processatore automatico (Vedi tabella materiali) impostata su un'impostazione bassa. Se nessun processore è disponibile, il trattamento può essere eseguito manualmente.</li>
	<li>Seguire il tampone di lavaggio da disidratazione attraverso una serie graduata di diluizioni di etanolo (EtOH) in PBS 1X (25%, 50%, 70%, 95%) per 1 h a temperatura ambiente.</li>
	<li>Disidratare in tre lavaggi del 100% EtOH per 1 h a temperatura ambiente.</li>
	<li>Deselezionare il campione disidratato in tre lavaggi dell'agente arancione a base di olio schiarimento 100% (Vedi tabella materiali) per 1 h a temperatura ambiente.</li>
	<li>Si infiltra nell'esempio cancellato due volte con paraffina fusa cera (Vedi Tabella materiali), riscaldato a 55 ° c, per 2 h ogni.</li>
</ol>7. campione incorporamento
<ol><li>Riempire uno stampo di cera con cera di paraffina fusa riscaldata a 55 ˚ c. Utilizzare una spatola riscaldata, piatta per trasferire rapidamente il mandrino infiltrato dalla cassetta incasso nello stampo di cera, garantendo che il mandrino si riposa parallela alla base dello stampo. Evitare di applicare una pressione eccessiva sul mandrino. Stampi di cera possono essere personalizzati 3D stampato, o sono disponibili in commercio (vedere Tabella materiali).</li>
	<li>Consentire la cera di solidificare durante la notte a 4 ˚ c. Modellato in cera solida campioni possono essere conservati a tempo indeterminato a 4 ˚ c.</li>
	<li>Una volta che la cera si è solidificato, asportare il campione dallo stampo e tagliare la cera in eccesso da intorno al campione con una lama di rasoio. Lasciare la cera in eccesso che si estende da un'estremità del campione, parallelo al piano di taglio, che può essere utilizzato per il bloccaggio nel microtomo (Vedi Tabella materiali). Anche lasciare una guaina di cera, di circa 1 mm di spessore, intorno al campione e lisciare le sue facce.</li>
</ol>8. sezionamento
<ol><li>Riscaldare a bagnomaria a 42 ° c. Fissare il campione nel microtomo con la superficie della Colonia orientata perpendicolarmente al bordo della lama (Vedi Tabella materiali).</li>
	<li>Sgrossare il campione a intervalli di 50 µm fino a quando è stato raggiunto il piano desiderato della Colonia, da cui sezioni saranno raccolti.</li>
	<li>Tagliare un nastro del numero desiderato di sezioni spesse 10-µm utilizzando un microtomo impostato una velocità di taglio di 75-80 giri/min e un angolo di spoglia inferiore di 6-10 ˚. Utilizzare un pennello a punta fine per staccare il nastro dalla lama.</li>
	<li>Utilizzando una goccia d'acqua sulla punta di una pipetta di Pasteur (preferito) o il forcipe, trasferire delicatamente il nastro nel bagnomaria. Fare attenzione a evitare bolle d'aria intrappolamento nella sezione.</li>
	<li>Immediatamente inserire una diapositiva nel bagno di acqua e posizionarla sotto la barra multifunzione a un angolo di 45 ˚.</li>
	<li>Toccare il bordo stretto del nastro per la diapositiva, appena sotto l'etichetta smerigliato. Il nastro deve aderire.</li>
	<li>Tirare la diapositiva dal bagno di acqua, regolazione dell'angolo di diventare perpendicolare alla superficie dell'acqua e consentendo la barra multifunzione per laici piatto contro lo scivolo lungo la sua lunghezza. Evitare di intrappolare l'acqua in eccesso sotto la barra multifunzione.</li>
	<li>Si levano in piedi delicatamente il vetrino su un panno privo di lanugine assorbente, traspirante acqua in eccesso dalla sezione.</li>
	<li>Porre il vetrino su un tovagliolo di carta e lasciare asciugare a temperatura ambiente durante la notte nel buio. Diapositive possono essere conservati a tempo indeterminato a 4 ° c al buio.</li>
</ol>9. calore-fissaggio, reidratazione e montaggio
<ol><li>Scaldare una piastra calda livellata a 45 ˚ c. Scaldare il vetrino per 30-60 min. La cera diventerà semi-fusa e appiattire contro la diapositiva.</li>
	<li>Delicatamente sollevare il vetrino dalla piastra scaldante e adagiarlo piatto su una superficie liscia, livellato, temperatura ambiente, facendo attenzione ad assicurare che la cera fusa non tirare su entrambi i lati della diapositiva, fino a quando la cera si solidifica (~ 1 min).</li>
	<li>Utilizzando un mailer di diapositive di vetro, de-cera diapositive in quattro lavaggi di compensazione agente per 5 minuti ciascuno, utilizzando un aspiratore di Büchner per rimuovere la soluzione tra i lavaggi.</li>
	<li>Lavare i vetrini tre volte in 100% EtOH per 1 min ciascuna.</li>
	<li>Reidratare le diapositive in una serie graduata di etanolo in ordine inverso di elaborazione (95%, 70%, 50% e 25%) per 1 min che ciascuna seguita da due 1-min lavaggi in PBS 1X.</li>
	<li>Montare immediatamente le sezioni in un mezzo di montaggio tamponata (Vedi Tabella materiali) e applicare un vetrino coprioggetti, evitando l'introduzione di bolle d'aria. Consentire il mezzo di montaggio a polimerizzare durante la notte a temperatura ambiente.</li>
	<li>Una volta polimerizzato, sigillare il coprioggetto alla diapositiva utilizzando smalto trasparente. Sigillato vetrini possono essere conservati a tempo indeterminato al buio a 4 ° c.</li>
</ol>

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Campioni di tessuto di inclusione in paraffina e sottile-sezionamento è una tecnica istologica classica che permette l'imaging delle strutture micro-morfologica e viene comunemente utilizzata sui tessuti negli eucarioti è stata applicata con successo ai campioni microbici8 ,9. Mentre crioinclusione consente una forte ritenzione del segnale endogeno ed immunofluorescente, inclusione in paraffina è generalmente preferibile in quanto fornisce migliore conservazi...

Maggior parte dei microbi hanno la capacità di forma biofilm, comunità di cellule tenute insieme da matrici autoprodotti. I biofilm possono essere coltivati in molti tipi di configurazioni fisiche, con vari regimi di fornitura di nutrienti e substrato. Test specifici per la formazione di biofilm tendono a produrre strutture multicellulari riproducibile e architetture comuni sono osservate per specie filogeneticamente diversificata a livello macroscopico o comunità. Quando i microbi vengono coltivati come colonie su terreno solido sotto atmosfera, morfologia macroscopica trasmette informazioni sulla capacità per la produzione della matrice e spesso correla con altri tratti 1,2,3. L'architettura interna di colonie microbiche possa anche fornire indizi riguardo specifici del biofilm chimica e fisiologia, ma è stato difficile da caratterizzare. Recenti applicazioni delle tecniche di crioinclusione e cryosectioning di colonie batteriche hanno permesso la formazione immagine e la visualizzazione di specifiche caratteristiche a risoluzione senza precedenti 4,5,6. Tuttavia, studi con tessuto animale hanno dimostrato che inclusione in paraffina fornisce superiore conservazione della morfologia rispetto a crioinclusione 7 e viene utilizzato per visualizzare i batteri in tessuti 8,9. Abbiamo pertanto sviluppato un protocollo per la fissazione, inclusione in paraffina e sezionamento sottile di colonie microbiche biofilm. Qui, descriviamo la preparazione di Pseudomonas aeruginosa PA14 Colonia-biofilm sezioni sottili 10,11, ma abbiamo applicato con successo anche questa tecnica a biofilm formato dai batteri Synxantha Pseudomonas, Bacillus subtilis, e Vibrio cholerae12.
Il processo di inclusione in paraffina e sottile-sezionamento biofilm segue una logica semplice. In primo luogo, il biofilm sono racchiusi in uno strato di agar per preservare la morfologia durante l'elaborazione. In secondo luogo, il biofilm racchiusi sono sommerse in un fissativo di crosslink macromolecole e preservare micromorfologia. Questi sono poi disidratati con alcool, ha eliminati con un solvente non polare più e quindi infiltrati con cera di paraffina liquida. Una volta infiltrato, i campioni sono incorporati in mattoncini di cera per il sezionamento. Sezioni sono tagliati, montate su slitte e poi reidratati al fine di riportarli a uno stato più nativo. Da questo punto, possono essere macchiati o coperto in mezzo di montaggio per l'analisi al microscopio.
Questo protocollo produce sezioni sottili di biofilm microbici adatto per l'analisi istologica. Sottostrutture di biofilm di Colonia sono visibili quando sezioni sottili preparati utilizzando questo metodo sono immaginate da microscopia chiara. Biofilm possono essere anche coltivati sulle macchie fluorescenti contenenti supporti specifici per le singole funzionalità o macchiati nella fase di reidratazione, immediatamente prima del montaggio (passaggi 9.5-9.6). Infine, i microbi possono essere progettati per produrre proteine fluorescenti in modo costitutivo o regolamentato, permettendo in caso di segnalazione in situ di espressione di gene o distribuzione delle cellule all'interno di queste comunità. Abbiamo usato questi metodi per determinare la profondità di biofilm di Colonia, distribuzione delle cellule, distribuzione della tabella, modelli di crescita ed espressione genica spatiotemporal.

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">5 3/4&#34; Pasteur pipette</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">13-678-6A</td>
			<td style="width:577px;">Purchased from univeristy biostores&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Agar&#160;</td>
			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">A7777</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Buchner Aspirator (Vacuum) Flask&#160;</td>
			<td style="width:191px;">Pyrex</td>
			<td style="width:161px;">5340</td>
			<td>Purchased from univeristy biostores&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chemically-resistant Marking Pen</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">103051-182</td>
			<td>Manufacturer: Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Clear Fingernail Polish&#160;</td>
			<td style="width:191px;">********</td>
			<td style="width:161px;">********</td>
			<td>Store bought</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Congo Red Indicator Grade</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">AAAB24310-14</td>
			<td>Manufacturer: Alfa Aesar</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Coomassie Blue&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">EM-3340</td>
			<td>Manufacturer: EMD Millipore</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">TRIS-buffered Mounting Medium (w/ DAPI)&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">50 247 04</td>
			<td>Manufacturer: Electron Microscopy Sciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Embedding Mold&#160;</td>
			<td style="width:191px;">********</td>
			<td style="width:161px;">********</td>
			<td>3D printed in-house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Embedding Mold (commercial)&#160;</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:161px;">70182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethanol 200P</td>
			<td style="width:191px;">Decon Labs, Inc.&#160;</td>
			<td style="width:161px;">2701</td>
			<td>Purchased from univeristy biostores&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Fine-tipped Brush</td>
			<td style="width:191px;">********</td>
			<td style="width:161px;">********</td>
			<td>Store bought, paint brush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Glass Coverslips 60x22mm</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">12-519-21C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Glass Rehydration Mailer&#160;</td>
			<td style="width:191px;">Ted Pella</td>
			<td style="width:161px;">21043</td>
			<td>20 slide mailer&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Histoclear-II, orange oil-based clearing agent&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">50 899 90150</td>
			<td>Manufacturer: National Diagnostics&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Histosette, Embedding Casette</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">15 182 701A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">L-lysine hydrochloride&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">BP386 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Low Profile Microtome Blades</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">22 210 048</td>
			<td>Manufacturer: Sturkey&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Micropipette&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">89080-004</td>
			<td>Promo-pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Micropipette Tips&#160;</td>
			<td style="width:191px;">See comments section</td>
			<td style="width:161px;">See comments section</td>
			<td>p10 (Fisher Scientific, 02 707 469), p200 (VWR, 89079-474), p1250 (VWR, 89079-486)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Microtome&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">905200U/00016050</td>
			<td>Model: HM355S, Manufacturer: Microm, NON-CATALOG, Vendor Catalog # 905200U/00016050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Formaldehyde, 37% Aqueous (Formalin)</td>
			<td style="width:191px;">Ricca Chemical</td>
			<td style="width:161px;">RSOF0010-500A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Paraplast Xtra (paraffin wax)</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">15159-486</td>
			<td>Manufacturer: McCormick Scientific&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Petri Dishes Square 100x100x15mm</td>
			<td style="width:191px;">Laboratory Disposable Products&#160;</td>
			<td style="width:161px;">D210-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Potassium chloride&#160;</td>
			<td style="width:191px;">EMD Chemicals&#160;</td>
			<td style="width:161px;">PX1405-1</td>
			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Potassium phosphate&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">P380-500</td>
			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Razor Blades&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">55411-050</td>
			<td>Purchased from univeristy biostores&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Slide Warmer&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">NC0865259</td>
			<td>NON-CATALOG, Vendor Catalog # 12857D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium chloride&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">0241-1KG</td>
			<td>Component of phosphate buffered saline, prepared in-house&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium phosphate&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:161px;">BDH9296.500</td>
			<td>,Component of phosphate buffered saline, prepared in-house&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Suprafrost Histology Slides&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tissue Flotation Water Bath&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">NC0815797</td>
			<td>Manufacturer: Ted Pella, Vendor Catalog # 28156-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Automatic Tissue Processor&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:161px;">813160U/Q#00009061</td>
			<td>Model: STP120 Tissue Processor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tryptone&#160;</td>
			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">T9012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Yeast extract</td>
			<td style="width:191px;">Teknova&#160;</td>
			<td style="width:161px;">Y9010</td>
			<td></td>
		</tr>
	</tbody>
</table>

paraffin embedding and thin sectioning of microbial colony biofilms for microscopic analysis

Sezionamento tramite inclusione in paraffina è una tecnica ampiamente consolidata in sistemi eucariotici. Qui forniamo un metodo per la fissazione, l'incorporamento e sezionamento dei biofilm intatto di colonie microbiche utilizzando irrorato di cera di paraffina. Per adattare questo metodo per l'utilizzo su biofilm di Colonia, abbiamo sviluppato le tecniche per mantenere ciascun campione il suo substrato di crescita e di esso di laminazione con un strato di agar e aggiunto alla soluzione fissante lisina. Queste ottimizzazioni migliorano la conservazione del campione e mantenimento delle caratteristiche di micromorphological. Campioni preparati in questo modo sono favorevoli a sottile di sezionamento e di imaging da microscopia elettronica di trasmissione, fluorescenza e della luce. Abbiamo applicato questa tecnica a Colonia biofilm di Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilise Vibrio cholerae. L'elevato livello di dettaglio visibile in campioni generati da questo metodo, combinato con ceppo reporter ingegneria o l'uso di coloranti specifici, può fornire spunti interessanti sulla fisiologia e sullo sviluppo delle comunità microbiche.

Questo metodo genera sottili sezioni di biofilm in cui le caratteristiche morfologiche distinte e zone di espressione genica possono essere ripreso da DIC, microscopia a fluorescenza e TEM. Mentre imaging DIC utilizzando un 40 X obiettivo a immersione in olio può essere sufficiente a mostrare alcune caratteristiche morfologiche (Figura 2E), abbiamo trovato quello fluorescenza microscopia di ceppi ingegnerizzati per proteina costitutivamente espresso fluoresc...

Watch this Scientific Journal Video about Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis at JoVE.com

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Inclusione in paraffina e sezionamento sottile di biofilm di colonie microbiche per l'analisi al microscopio

Descriviamo la fissazione, inclusione in paraffina e sottili tecniche di sezionamento per colonie microbiche biofilm. In campioni preparati, modelli di espressione di sottostruttura e reporter di biofilm possono essere visualizzati da microscopia.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

paraffin-embedding-thin-sectioning-microbial-colony-biofilms-for

Research

JoVE Journal

Immunology and Infection

14.2K Views.  Columbia University. Descriviamo la fissazione, inclusione in paraffina e sottili tecniche di sezionamento per colonie microbiche biofilm. In campioni preparati, modelli di espressione di sottostruttura e reporter di biofilm possono essere visualizzati da microscopia.

Video: Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Formanti colonia cellulare (CFC) Analisi per cellule emopoietiche

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Ricerca

Immunologia e infezioni

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Singola cella Analisi Bacillus subtilis I biofilm usando la microscopia a fluorescenza e citometria a flusso

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crescita Mycobacterium tuberculosis I biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Isolamento e analisi del genoma di virioni singole utilizzando &quot;Genomica singolo virus &#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Imaging Raziometrico del pH extracellulare nel dentale biofilm

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Un nuovo metodo per l&#39;analisi qualitativa multi-scala batterica biofilm su filamentose Fungal Colonies Utilizzando confocale e microscopia elettronica

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Estrazione di emociti da larve di Drosophila melanogaster per infezione microbica e l'analisi

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Uso complementare di tecniche microscopiche e lettura di fluorescenza nello studio delle interazioni Cryptococcus-Amoeba

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applicazione di Live Cell Imaging e tomografia Cryo-Electron a risolvere le caratteristiche spatiotemporali del sistema di secrezione Legionella pneumophila Dot/Icm

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Chiarire e imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Inclusione in paraffina e sezionamento sottile di biofilm di colonie microbiche per l'analisi al microscopio

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