We would like to acknowledge Dr. George A. O&#39;Toole and Dr. Thomas H. Hampton for their significant role in the design and development of the in vitro polymicrobial community model. We thank Dr. Sophie Robitaille for her helpful comments on the manuscript. This work was supported by a Cystic Fibrosis Foundation grant JEAN21F0 to F.J-P. Figure 1 of the manuscript was created using BioRender.

The details of all the reagents and equipment are listed in the Table of Materials.
1. Preparing artificial sputum medium
NOTE: Throughout this protocol, artificial sputum medium (ASM) is defined as the CF-relevant medium modified from SCFM2, previously published by Turner and colleagues17. Below is a description of the steps to prepare this medium. See Table of Materials to make stock solutions and storage conditions. This version of ASM differs from Turner et al.,17 as the buffering capacity in the original version does not allow for a stable pH over time. That is, fermentation activity carried out by Streptococcus spp. and anaerobes like Prevotella spp. results in the acidification of the growth medium (unpublished observations). Therefore, the 3-morpholinopropane-1-sulfonic acid (MOPS) concentration has been adjusted from 10 mM to 100 mM.
NOTE: Preparing a 2x ASM base (ASMb) stock solution facilitates the addition of components such as mucin, agar, water, etc., without impacting the final concentration of molecules present in ASM10,18. The 2x ASMb can be prepared in advance and stored at 4 &#176;C for up to two weeks in a dark place. If the medium turns yellow, discard.
<ol>
	<li>To prepare 2x ASM base stock, in a clean beaker containing 400 mL of diH2O, add the following while stirring:
	<ol>
		<li>Add 6.50 mL of a 0.2 M NaH2PO4.H2O stock; 6.25 mL of a 0.2 M Na2HPO4 stock; 348 &#181;L of a 1 M KNO3 stock; 1.084 mL of a 0.25 M K2SO4 stock.</li>
		<li>Add 4.0 g of Yeast synthetic dropout without Tryptophan; 3.032 g of NaCl; 20.92 g of MOPS; 1.116 g of KCl and 0.124 g of NH4Cl.</li>
		<li>Add 9.30 mL of a 1 M L-Lactic acid stock; 2.70 mL of a 1.1 M glucose stock; 1.75 mL of a 1 M CaCl2.2H2O stock; 1.20 mL of a 0.25 M N-acetylglucosamine stock and 1.00 mL of a 3.6 mM FeSO4.7H2O stock.</li>
		<li>Add 660 &#181;L of a 0.1 M Tryptophan stock; 606 &#181;L of a 1 M MgCl2.6H2O stock; 0.60 g of deoxyribonucleic acid from herring sperm; 400 &#181;L of a 250 mg/mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) stock.</li>
		<li>Once all the components have been added, adjust the pH to 6.80 with a 5 M NaOH stock. Complete to 500 mL with diH2O. Sterilize using a 0.22 &#181;m filter, and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare a 10 mg/mL (2x) mucin stock, use a clean glass bottle containing 250 mL of diH2O and while stirring:
	<ol>
		<li>Add 5.0 g of mucin from porcine stomach - Type II. Mix the suspension for 10 min. Complete to 500 mL with diH2O.</li>
		<li>Sterilize by autoclaving at 121 &#176;C for 15 min. Keep at 4 &#176;C until utilization.</li>
	</ol>
	</li>
	<li>Reconstitute the ASM. 
	NOTE: This step needs to be performed on the day of the experiment.
	<ol>
		<li>Mix a 1:1 volume ratio of the 2x ASMb stock and the 10 mg/mL mucin stock.</li>
		<li>Vortex thoroughly before use.</li>
	</ol>
	</li>
</ol>
2. Preparing polymicrobial community selective media
NOTE: Below are the quantities necessary to prepare a volume of 1 L of media.
<ol>
	<li>To prepare Mannitol Salt Agar (MSA) and Pseudomonas Isolation Agar (PIA), follow the manufacturer&#39;s instructions.</li>
	<li>To prepare Prevotella Selective Agar (PSA), in a clean glass bottle, add the following:
	<ol>
		<li>30.0 g of tryptic soy broth (TSB). 15.0 g of agar. 5.00 g of Yeast extract. 0.50 g of L-cysteine hydrochloride.</li>
		<li>10.0 mL of a 0.5 mg/mL hemin stock. 100 &#181;L of a 10 mg/mL menadione stock.</li>
		<li>Add 940 mL of diH2O and stir. Sterilize by autoclaving at 121 &#176;C for 15 min.&#160;</li>
		<li>When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep&#39;s blood. 2.00 mL of a 50 mg/mL kanamycin stock. 150 &#181;L of a 50 mg/mL vancomycin stock. 500 &#181;L of a 10 mg/mL polymixin B stock.</li>
	</ol>
	</li>
	<li>To prepare Streptococcus Selective Agar (SSA), in a clean glass bottle, add the following:
	<ol>
		<li>30.0 g of TSB. 15.0 g of agar.</li>
		<li>Add 950 mL of diH2O and stir. Sterilize by autoclaving at 121 &#176;C for 15 min.&#160;</li>
		<li>When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep&#39;s blood. 1.00 mL of a 10 mg/mL polymixin B stock. 1.00 mL of a 10 mg/mL oxolinic acid stock. 
		NOTE: Pour in plates and keep at 4 &#176;C for a maximum of 1 month. Before using, let the plates dry at room temperature up to 24 h in advance before they are inoculated. PSA has been validated with P. melaninogenica and Prevotella intermedia. SSA has been validated with S. sanguinis, and the Streptococcus milleri group (Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus, denoted as SMG). It is strongly recommended to de novo validate these selective media if new strains are to be tested.</li>
	</ol>
	</li>
</ol>
3. Bacterial liquid media preparation and growth conditions
<ol>
	<li>To routinely grow P. aeruginosa and S. aureus, use a sterile rich medium (e.g., lysogeny broth (LB) or TSB).
	<ol>
		<li>Start liquid overnights by picking a single colony previously grown on an LB/TSB agar plate for 20-24 h at 37 &#176;C.</li>
		<li>Grow the broth cultures at 37 &#176;C overnight with shaking at 250 rpm for 16-18 h.</li>
	</ol>
	</li>
	<li>To grow Streptococcus spp., use sterile Todd Hewitt Broth supplemented with 0.5 % yeast extract (THB-YE).
	<ol>
		<li>Start liquid culture overnight from a single colony previously grown on a TSB agar plate supplemented with 5% defibrinated sheep&#39;s blood (blood agar plate) for 24 h at 37 &#176;C + 5% CO2.</li>
		<li>Grow the THB-YE cultures anaerobically or at 37 &#176;C + 5% CO2 overnight without shaking for 18-22 h.</li>
	</ol>
	</li>
	<li>To grow Prevotella spp., prepare 1 L of Prevotella growth medium (PGM) by adding the following to a clean bottle:
	<ol>
		<li>30.0 g of TSB. 5.00 g of yeast extract. 0.50 g of L-cysteine hydrochloride. 10.0 mL of a 0.5 mg/mL hemin stock and 100 &#181;L of a 10 mg/mL menadione stock.</li>
		<li>Add 990 mL of diH2O. Sterilize by autoclaving at 121 &#176;C for 15 min. Wrap the bottle in aluminum foil to protect it from light.</li>
		<li>Grow Prevotella spp. colonies on a blood agar plate incubated anaerobically at 37 &#176;C for 48 h.</li>
		<li>Inoculate a single colony in PGM and grow overnight without shaking at 37 &#176;C anaerobically for 24 h. 
		NOTE: PGM can be kept at room temperature for up to two weeks. Performing sterility tests on each growth medium is strongly recommended before use. If not using an anaerobic chamber, it might be necessary to start liquid overnights of Prevotella spp. from a patch of colonies.</li>
	</ol>
	</li>
</ol>
4. Co-culture experiment preparation
NOTE: Figure 1 depicts a summarized experimental workflow. Setting up the experiment can be done in oxic conditions if the preparation time takes less than 1 h. If it is expected to take longer than that, it is strongly recommended to perform the experiment using an anaerobic chamber (with 10% CO2, 10% H2, and 80% N2 mixed gas). Anaerobic jars can also be used to incubate the experimental samples.
<ol>
	<li>Preparation of the monocultures and co-cultures 
	NOTE: Perform all the centrifugation steps at 10,000 x g for 2 min at room temperature. For the co-cultures, mix the bacterial samples just before inoculating the 96-well plates. Typically, a volume of 1 mL for P. aeruginosa and S. aureus strains are collected from the overnight cultures. For Streptococcus spp. and Prevotella spp., the overnight volumes may vary between 2-4 mL. These volumes can be adjusted depending on the number of conditions to be tested.
	<ol>
		<li>Using the bacterial liquid from the overnight cultures, perform the following steps:
		<ol>
			<li>For P. aeruginosa and S. aureus strains, wash twice using sterile phosphate-buffered saline (PBS).</li>
			<li>For Streptococcus spp. and Prevotella spp., wash once with sterile PBS. 
			NOTE: Use a pipette to carefully discard supernatants, as the pellets can easily be lost.</li>
			<li>After the washes, resuspend each pellet in 1 mL of 1x water-diluted ASMb.</li>
			<li>Make a 1:10 dilution of each sample in sterile PBS and measure the OD600.</li>
			<li>Adjust all the cultures to an OD600 of 0.2 in ASM.</li>
			<li>Using the OD600 = 0.2 suspensions, dilute in ASM the bacterial samples to a final OD600 of 0.01 (for each microbe) for the monocultures and co-cultures.</li>
			<li>Vortex thoroughly for 5 s. 
			NOTE: For example, to prepare a total of 1 mL of P. aeruginosa monoculture ASM suspension at OD600 = 0.01, add 50 &#181;L of P. aeruginosa at OD600 = 0.2 to a volume of 950 &#181;L of ASM. For the co-culture, take 50 &#181;L of each P. aeruginosa, S. aureus, Streptococcus spp. and Prevotella spp. suspensions at an OD600 = 0.2 and add to 800 &#181;L ASM.</li>
			<li>Using a pipette, add 100 &#181;l of the monoculture and co-culture suspensions in three separate wells of a sterile plastic flat bottom 96-well plate.</li>
			<li>Incubate the plate (without shaking) for 24 h at 37 &#176;C in anoxic conditions. 
			NOTE: Other oxygen tensions (e.g., microxia, normoxia) can also be used here. However, the model has been developed and validated with anoxic conditions. Confirm the starting inocula by serially diluting the mono and co-culture bacterial suspensions, and plating on PIA, MSA, SSA, and PSA is strongly recommended. The targeted starting concentration for each bacterial species is 1 &#215; 107 CFU/mL (P. aeruginosa), 3.5 &#215; 106 CFU/mL (S. aureus), 1.2 &#215; 106 CFU/mL (Streptococcus spp.), and 4.6 &#215; 106 CFU/mL (Prevotella spp.). Bacterial inocula can also be modified, as described by Jean-Pierre and colleagues10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Replace the media after biofilm formation.
	<ol>
		<li>After 24 h of incubation, remove the unattached (planktonic) cells by aspiration using a multichannel pipette.</li>
		<li>Replenish the pre-formed biofilms with 100 &#181;L of fresh ASM or the desired treatment (e.g., antibiotics, metabolites, etc.).</li>
		<li>Incubate the plate for an additional 24 h at 37 &#176;C in anoxic conditions. 
		NOTE: These steps can be done aerobically if performed quickly (i.e., less than 1 min). Otherwise, please use an anaerobic chamber.</li>
	</ol>
	</li>
</ol>
5. Collecting and plating the samples
<ol>
	<li>After the additional 24 h of incubation, aspirate the non-attached (planktonic) cells with a multichannel pipette. 
	NOTE: The planktonic fraction can be kept, serially diluted, and plated on selective media or discarded.</li>
	<li>Gently wash the biofilms twice using 125 &#181;L of sterile PBS and discard the volumes.</li>
	<li>Add 50 &#181;L of sterile PBS and detach the biofilms by gently scraping the cells from the plate using a 96-pin replicator.</li>
	<li>Transfer the resuspended biofilm cells to row A of a new sterile 96-well plate. 
	NOTE: The 96-well plate will contain sterile PBS in rows B to H.</li>
	<li>Perform a 10x serial dilution of the planktonic (if needed) and biofilm fractions.</li>
	<li>Plate 3-5 &#181;L of each dilution sample onto PIA, MSA, SSA, and PSA.</li>
	<li>Allow the inoculation spots to dry.</li>
</ol>
6. Incubating the selective plates and colony forming unit (CFU) counting
<ol>
	<li>For P. aeruginosa and S. aureus, incubate at 37 &#176;C for 18-20 h.</li>
	<li>For Streptococcus spp, incubate at 37 &#176;C in anoxic conditions for 24 h.</li>
	<li>For Prevotella spp, incubate at 37 &#176;C in anoxic conditions for 48 h.</li>
	<li>After incubation, count the colonies (~10-35 per spot) and calculate the CFU per mL.</li>
	<li>Plot the data using a visualization tool.</li>
</ol>

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

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The authors declare no disclosures.

The usefulness of this clinically informed in vitro co-culture system resides in its capacity to detect polymicrobial-specific bacterial functions. That is, through the utilization of this model we have reported microbial phenotypes ranging from community-specific growth of Prevotella spp. (Figure 2) to changes in susceptibility to front-line CF Abx of P. aeruginosa, S. aureus and S. sanguinis (Figure 3). Moreover, ev...

Strategies aimed at eradicating disease-causing microbes such as the ones detected in the cystic fibrosis (CF) airway, a multiorgan genetic disease, often fail1. That is, the presence of resilient biofilm-like microbial communities growing in the mucus-rich CF lung environment can cause chronic infections spanning multiple decades2. Furthermore, although the utilization of front-line antimicrobials (Abx) and modulators has resulted in improved outcomes in people with CF (pwCF), the &#34;one bug, one infection&#34; clinical approach typically employed against canonical CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus has been ineffective in resolving hard-to-treat infections detected in the lungs of these individuals3,4,5,6,7.
Over the last two decades, multiple studies have changed our understanding of chronic CF lung disease8. That is, reports indicate that infections detected in the airways of pwCF are not solely caused by a single pathogen but are rather polymicrobial in nature8. Furthermore, although the pathogenesis of mixed species CF lung infections is still poorly understood, clinical evidence shows that pwCF that are co-infected with both S. aureus and P. aeruginosa in their airways have worsened lung function than individuals colonized by either of these two pathogens9.
Interspecies interactions among CF pathogens are hypothesized to be a reason why polymicrobial biofilm-based infections are not readily eradicated through the utilization of CF therapeutics, ultimately impacting patient outcomes3. Supporting this, in vitro studies have demonstrated that interactions between microbes such as P. aeruginosa, S. aureus, and others can impact clinically relevant phenotypes such as Abx responsiveness to front-line CF drugs, including vancomycin and tobramycin6,10,11. Therefore, revisiting the current treatment strategies aimed at eradicating CF pathogens in the context of mixed-species infections remains to be achieved.
The gold standard in the management of microbial-based CF lung infections heavily relies on the utilization of antimicrobial susceptibility testing (AST) to guide clinical interventions12. However, AST is typically done using bacterial monocultures grown in rich and well-mixed cultures, which is not reflective of the microbial growth conditions detected in the CF airway3,13. Given the significant disconnect between patient outcomes and treatment success, clinical reports now advocate for the development of novel approaches integrating critical features of the CF lung12,14.
Few studies have developed CF-relevant bacterial multispecies in vitro systems containing more than two pathogens15,16. However, these models (1) do not entirely reflect the polymicrobial and biofilm-like nature of the CF lung and (2) do not use nutritional and environmental conditions approximating the ones detected in the CF airway15,16. Through the mining of large CF-lung derived 16S rRNA gene data sets and computation, Jean-Pierre and colleagues recently developed an in vitro co-culture model integrating the above-mentioned features10. This system includes P. aeruginosa, S. aureus, Streptococcus spp. and Prevotella spp. grown in CF airway-like conditions and stable for up to 14 days. The authors also reported community-specific growth of Prevotella spp. and several changes in the Abx responsiveness of these CF pathogens through mechanisms that remain to be identified10. This tractable in vitro system also offered the possibility to delve into mechanistically focused questions related to Abx recalcitrance of a common variant of P. aeruginosa frequently detected in the airways of pwCF10. Therefore, the goal of this protocol is to provide the CF research community with a standardized experimental workflow to grow an in vitro biofilm that has the potential to be further expanded to tackle a number of CF-relevant questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Fisher Scientific</td>
			<td>NC0387928</td>
			<td>Prepare a 250 mg/mL stock in chloroform directly in the bottle. Keep at -20&#176;C. Final concentration 200 &#956;g/mL in 2x ASM base.</td>
		</tr>
		<tr>
			<td>3-morpholinopropane-1-sulfonic acid (MOPS)</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td>Final concentration 200 mM in 2X ASM base.</td>
		</tr>
		<tr>
			<td>96-pin replicator</td>
			<td>Fisher Scientific</td>
			<td>05-450-9</td>
			<td>Disposable replicators can also be used. Cat #NC1567338.</td>
		</tr>
		<tr>
			<td>96-well sterile standard plate with lid</td>
			<td>Fisher Scientific</td>
			<td>62406-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaeroPack 2.5 L Rectangular Jar</td>
			<td>Fisher Scientific</td>
			<td>23-246-385</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*2H2O&#160;</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 3.5 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Defribrinated sheep&#39;s blood</td>
			<td></td>
			<td></td>
			<td>To prepare growth plates for Streptococcus and Prevotella.</td>
		</tr>
		<tr>
			<td>Deoxyribonucleic acid from herring sperm</td>
			<td>Sigma</td>
			<td>D7290</td>
			<td>Final concentration 1.2 mg/mL in 2x ASM base.</td>
		</tr>
		<tr>
			<td>FeSO4*7H2O&#160;</td>
			<td>Fisher Scientific</td>
			<td>AA1449830</td>
			<td>Prepare a 1 mg/mL stock (3.6 mM) in water and filter sterilize with a 0.22 &#956;m filter. Keep wrapped in aluminum foil. Final concentration 0.0072 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>GasPaks</td>
			<td>Fisher Scientific</td>
			<td>B260678</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-3</td>
			<td>Prepare as a 20% stock (1.1 M) in water and sterilize by autoclave. Final concentration 6 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>51280</td>
			<td>Dissolve 100 mg of hemin in 2 mL of 1 M NaOH and then bring up volume to 200 mL with water. Store in an amber bottle or a regular bottle wrapped with aluminum foil at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Fisher Scientific</td>
			<td>P304-500</td>
			<td>Prepare a 0.25 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 0.54 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td></td>
			<td></td>
			<td>Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 &#956;m filter and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Final concentration 30.0 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Fisher Scientific</td>
			<td>P263-500</td>
			<td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 0.70 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>L-cysteine hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>AAA1038922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lactic acid&#160;</td>
			<td>Sigma</td>
			<td>L1750</td>
			<td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &#956;m filter. pH adjust to 7.0. Final concentration 18.6 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>L-Tryptophan</td>
			<td>Sigma</td>
			<td>T0254</td>
			<td>Prepare a 0.1 M stock in 0.2 M NaOH and filter sterilize with a 0.22 &#956;m filter. Keep refrigerated at 4 &#176;C wrapped in aluminum foil. Final concentration 0.132 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Fisher Scientific</td>
			<td>B11407</td>
			<td>Prepare using manufacturer&#39;s recommendations.</td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M5625</td>
			<td>Prepare a 10 mg/mL stock solution dissolved in 96-100% ethanol. Conserve at 4 &#176;C wrapped in aluminium foil. Vortex before use.</td>
		</tr>
		<tr>
			<td>MgCl2*6H2O&#160;</td>
			<td>Fisher Scientific</td>
			<td>AA12288A9</td>
			<td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 1.21 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach - Type II&#160;</td>
			<td>Sigma</td>
			<td>M2378</td>
			<td>Prepare a 10 mg/mL (2x) stock in water; mix thoroughly before autoclaving. Keep at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S375-500</td>
			<td>Prepare a 0.2 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 2.5 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>N-acetylglucosamine&#160;</td>
			<td>Sigma</td>
			<td>A8625</td>
			<td>Prepare a 0.25 M stock in water and filter sterilize with a 0.22 &#956;m filter. Keep stock refrigerated at 4 &#176;C. Final concentration 0.6 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Final concentration 103.7 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>NaH2PO4*H2O</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td>Prepare a 0.2 M stock in water and filter sterilize with a 0.22 &#956;m filter. Final concentration 2.6 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td>Prepare a 5 M stock solution. Use to adjust pH of 2x ASM base stock.</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Fisher Scientific</td>
			<td>A661-500</td>
			<td>Final concentration 4.6 mM in 2x ASM base.</td>
		</tr>
		<tr>
			<td>Oxolinic acid</td>
			<td></td>
			<td></td>
			<td>Prepare a 10 mg/mL stock solution 0.5 M NaOH. Filter sterilize with a 0.22 &#956;m filter and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Polymixin B</td>
			<td></td>
			<td></td>
			<td>Prepare a 10 mg/mL stock solution in water. Filter sterilize with a 0.22 &#956;m filter and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0927-17-1</td>
			<td>Prepare using manufacturer&#39;s recommendations.</td>
		</tr>
		<tr>
			<td>Todd Hewitt Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0492-17-6</td>
			<td>Prepare using manufacturer&#39;s recommendations.</td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0370-17-3</td>
			<td>Prepare using manufacturer&#39;s recommendations.</td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td></td>
			<td></td>
			<td>Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 &#956;m filter and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>B11929</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Synthetic Dropout without Tryptophan&#160;</td>
			<td>Sigma</td>
			<td>Y1876</td>
			<td>Final concentration 8.0 mg/mL in 2x ASM base.</td>
		</tr>
	</tbody>
</table>

growing a cystic fibrosis-relevant polymicrobial biofilm to probe community phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

As represented in Figure 2, several phenotypes were reported including (1) a reduction in the number of P. aeruginosa and S. aureus viable cell counts when grown a mixed planktonic communities compared to monoculture, (2) an increase in polymicrobial growth of S. sanguinis cells and, (3) mixed community growth of P. melaninogenica as previously reported by Jean-Pierre and colleagues10.
Furthermore, as dep...

Read this Scientific Article Protocol about Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes at JoVE.com

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

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395 Views.  Université de Sherbrooke. This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Video: Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4&#039;,6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

Methods Article

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes