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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissection and infection of ex vivo pig lung (EVPL) tissue
<ol>
	<li>Make SCFM for use with EVPL tissue, follow the recipe for 1 L modified SCFM supplied in Table 1.</li>
	<li>Prior to dissection, prepare an agar plate/s of required bacterial strain/s for infection using whatever agar is standard in the lab for P. aeruginosa/S. aureus (e.g., lysogeny broth + 1.2% agar).</li>
	<li>Calculate how many porcine bronchiolar tissue pieces are required for the experiment, including uninfected control tissue pieces. Multiply this number by two to repeat the experiment in two replicate lungs to confirm repeatability of results.</li>
	<li>Multiply the total number of tissue pieces required by 0.5 to determine the volume of SCFM agarose (mL) needed to make agarose pads to make enough medium for 400 &#956;L/tissue piece plus spare SCFM agar to account for any pipetting errors or evaporation during preparation.</li>
	<li>Add 0.12 g of agarose to every 15 mL of SCFM required to make the desired total volume of SCFM with 0.8% weight/volume agarose.</li>
	<li>Heat the SCFM agarose solution until the agarose is fully dissolved. A domestic microwave on low power is recommended. The time required depends on the wattage of the microwave. Allow the agarose to cool to approximately 50 &#176;C (warm to the touch but comfortable to hold). Do not allow to cool any further.</li>
	<li>Using a pipette, add 400 &#956;L of the SCFM agarose to one well of a 24-well plate per tissue piece needed.</li>
	<li>Sterilize the SCFM agarose-containing 24-well plate/s under ultraviolet light for 10 min.</li>
	<li>Prepare three replicate washes for every intact lung being dissected using 20 mL of sterile Dulbecco&#39;s modified Eagle medium (DMEM) plus 20 mL of sterile Roswell Park Memorial Institute (RPMI) 1640 supplemented with 50 &#956;g/mL ampicillin.</li>
	<li>Make an aliquot of 40 mL SCFM as a final wash for every intact lung being dissected. All washes can be stored overnight at 4 &#176;C or used immediately.</li>
	<li>Obtain lungs from the designated source as soon as possible after slaughter, ensuring they are kept cold by transporting to the laboratory in a domestic coolbox. 
	NOTE: Lungs closer to the day of slaughter show less bruising from storage, but tissue kept on cold storage for up to 4 days from slaughter can also be used. As the coolbox needs to be taken into the butcher&#39;s shop or abattoir, it must be decontaminated following local lab guidelines after each use and stored outside the microbiology lab when not in use, to reduce the risk of contamination and a breach of containment.</li>
	<li>Working on a sterilized surface and under a flame, place the lungs on a clean plastic chopping board covered with autoclaved aluminum foil. Check that the bronchioles remain intact. If there has been any damage at the abattoir or during transport the lungs are not suitable for use.</li>
	<li>Heat a palette knife under a flame and very briefly touch the knife to the area of the lung surrounding the bronchiole to sterilize the surface of the tissue.</li>
	<li>Cut away the surface tissue surrounding the bronchiole using a sterile mounted razor blade. Make incisions parallel to the bronchiole to prevent any damage.</li>
	<li>Once the bronchiole has been exposed, make a cross-sectional incision through the bronchiole at the highest point visible to free the bronchiole.</li>
	<li>Using sterile forceps, lightly hold the free end of the bronchiole and cut away any remaining unwanted tissue using a sterile mounted razor blade. Make a final cross-sectional incision across the bronchiole before any branching is visible to remove the bronchiole from the lungs.</li>
	<li>Place the bronchiole in the first DMEM/RPMI 1640 wash. Leave the bronchiole in the wash and repeat steps 1.12 to 1.15 to harvest additional sections of bronchiole from the same lung as required to yield sufficient tissue sections for the planned experiment.</li>
	<li>Place any additional bronchiolar sections from the same lung into the wash (step 1.17). Leave in the wash for at least 2 min.</li>
	<li>Remove the bronchioles from the first DMEM/RPMI 1640 wash and place the samples in a sterile Petri dish.</li>
	<li>Hold each bronchiole lightly using sterile forceps, making sure not to damage the tissue. Remove as much remaining soft tissue as possible and cut the tissue into ~5 mm wide strips using sterile dissection scissors.</li>
	<li>Place all of the bronchiolar tissue strips into the second DMEM/RPMI 1640 wash. Leave in the wash for at least 2 min.</li>
	<li>Remove the tissue strips from the second wash using sterile forceps, taking care not to damage the tissue. Place the tissue in a clean, sterile Petri dish.</li>
	<li>Remove any remaining soft tissue attached to the bronchiole and cut the strips into squares (~5 mm x 5 mm) using sterile dissection scissors.</li>
	<li>Add the third DMEM/RPMI 1640 wash into the Petri dish. Lightly mix the tissue pieces in the wash by swirling the dish.</li>
	<li>Pour the third wash out of the Petri dish without removing the tissue pieces.</li>
	<li>Add the final SCFM wash to the tissue-containing Petri dish, ensuring that all of the tissue pieces are covered.</li>
	<li>Sterilize the tissue pieces in SCFM under UV light for 5 min.</li>
	<li>Use sterile forceps to transfer each sterilized bronchiolar tissue piece into individual wells of a 24-well plate/s containing SCFM agarose pads.</li>
	<li>To infect each tissue piece with the desired bacterial strain, touch a colony grown on an agar plate with the tip of a 29 G needle attached to a sterile 0.5 mL insulin syringe. Then touch the colony onto the tissue piece, gently pricking the tissue surface. 
	NOTE: Using an insulin syringe equipped with a 29 G needle allows the needle to be held accurately and comfortably while keeping fingers at a safe distance from the both needle and lung tissue. It is possible to perform this step using 29 G needles that are not attached to a syringe, but this requires greater dexterity and increases the risk of a needlestick injury. Insulin syringes are readily available.</li>
	<li>For the uninfected controls, gently prick the surface of each of the tissue pieces with the tip of a 29 G needle attached to a sterile 0.5 mL insulin syringe.</li>
	<li>Use a pipette to add 500 &#956;L of SCFM to each well.</li>
	<li>Sterilize a breathable sealing membrane for each 24-well plate under ultraviolet light for 10 min (Table of Materials).</li>
	<li>Remove the lid/s from the 24-well plate/s and replace with the breathable membrane.</li>
	<li>Incubate the plates at 37 &#176;C for the desired incubation (infection) time without shaking. Check that there is no visible growth of the inoculated pathogen on the uninfected control pieces (contamination control). 
	NOTE: If desired, ampicillin may be added to the SCFM agarose pads and covering SCFM in step 1.31 to a final concentration of 20 &#956;g/mL. This will suppress the growth of most endogenous bacteria on the lungs without affecting P. aeruginosa or S. aureus growth but, as the presence of ampicillin may affect susceptibility to other antibiotics, the reader is left to make this choice depending on the strains and antibiotics they wish to test.</li>
</ol>
TABLE 1
1. Preparation of Synthetic CF Sputum Media (SCFM) for use with ex vivo pig lung model
Once made, the SCFM should be filter sterilized, and may then be stored at 4&#176;C for up to one month.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="5">a) Main recipe</td>
		</tr>
		<tr>
			<td rowspan="4">Step 1</td>
			<td>Chemical</td>
			<td>Amount</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>3.03 g</td>
			<td rowspan="3">Add salts and water to clean bottle, used only for preparation of SCFM</td>
			<td>51.85</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>1.114 g</td>
			<td>14.94</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>640 ml</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="7">Step 2</td>
			<td>Chemical</td>
			<td>Molarity of stock made in water, filter-sterilized and stored at 4&#176;C</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>0.125 M</td>
			<td rowspan="6">Add 10 ml of each stock to the salts and water prepared in step 1</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.13 M</td>
			<td>1.30</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>0.228 M</td>
			<td>2.28</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>0.0348 M</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>0.0271 M</td>
			<td>0.27</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>1 M</td>
			<td>10.00</td>
		</tr>
		<tr>
			<td rowspan="2">Step 3</td>
			<td>Chemical</td>
			<td></td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td colspan="2">19 amino acids solutions prepared according to section b)</td>
			<td>Add 10 ml of each stock to the solution prepared in steps 1 and 2.</td>
			<td>See section b)</td>
		</tr>
		<tr>
			<td rowspan="2">Step 4</td>
			<td colspan="2">Chemical</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td colspan="2">HCl or NaOH as required</td>
			<td>Use to adjust pH of solution prepared in steps 1-3 to 6.8. Record volume of acid/base added.</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="2">Step 5</td>
			<td colspan="2">Chemical</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td colspan="2">dH2O</td>
			<td>Add to solution prepared in steps 1-4, to a final volume of 960 ml</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="3">Step 6</td>
			<td>Chemical</td>
			<td>Molarity of stock made in water, filter-sterilized and stored at 4&#176;C</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.175 M</td>
			<td rowspan="2">Add 10 ml of each stock to the solution prepared in steps 1-5</td>
			<td>1.75</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>0.0606 M</td>
			<td>0.61</td>
		</tr>
		<tr>
			<td rowspan="2">Step 7</td>
			<td>Chemical</td>
			<td>Molarity of stock made in water, filter-sterilized and stored at 4&#176;C</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td>L-Lactic acid</td>
			<td>0.93 M</td>
			<td>Make stock in water, pH to 7 with 5M NaOH. Add 10 ml to the solution prepared in steps 1-6.</td>
			<td>9.30</td>
		</tr>
		<tr>
			<td rowspan="2">Step 8</td>
			<td>Chemical</td>
			<td>Molarity of working stock made in water, immediately before adding to the recipe</td>
			<td>Instructions</td>
			<td>Final mM in 1L SCFM</td>
		</tr>
		<tr>
			<td>Fe(III)SO4.7H2O</td>
			<td>0.00036 M</td>
			<td>Make a 0.036 M master stock, which can be filter sterilized and stored at 4&#176;C for as long as the solution remains free of precipitate. To make the working stock, add 110 &#181;l of the master stock to 9.890 ml dH2O and add the working stock to the solution prepared in steps 1-7.</td>
			<td>0.0036</td>
		</tr>
	</tbody>
</table>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">b) Preparation of amino acid stocks; filter sterilize before use and store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Amino Acid</td>
			<td>mM&#160; stock</td>
			<td>Instructions</td>
			<td>Final mM in 1 L SCFM</td>
		</tr>
		<tr>
			<td>Alanine</td>
			<td>178</td>
			<td>Dissolve in water</td>
			<td>1.780</td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>30.6</td>
			<td>Dissolve in water</td>
			<td>0.306</td>
		</tr>
		<tr>
			<td>Aspartate</td>
			<td>82.7</td>
			<td>Dissolve in 0.5 M NaOH</td>
			<td>0.827</td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>16</td>
			<td>Dissolve in water</td>
			<td>0.160</td>
		</tr>
		<tr>
			<td>Glutamic Acid</td>
			<td>154.9</td>
			<td>Dissolve in 1 M HCl</td>
			<td>1.549</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>120.3</td>
			<td>Dissolve in water</td>
			<td>1.203</td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>51.9</td>
			<td>Dissolve in water</td>
			<td>0.519</td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>112.1</td>
			<td>Dissolve in water by heating to 50&#176;C for 30 mins on a shaker</td>
			<td>1.121</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>160.9</td>
			<td>Dissolve in water</td>
			<td>1.609</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>212.8</td>
			<td>Dissolve in water</td>
			<td>2.128</td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>63.3</td>
			<td>Dissolve in water</td>
			<td>0.633</td>
		</tr>
		<tr>
			<td>Ornithine-HCl</td>
			<td>67.6</td>
			<td>Dissolve in water</td>
			<td>0.676</td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>53</td>
			<td>Dissolve in water</td>
			<td>0.530</td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>166</td>
			<td>Dissolve in water</td>
			<td>1.660</td>
		</tr>
		<tr>
			<td>Serine</td>
			<td>144.6</td>
			<td>Dissolve in water</td>
			<td>1.446</td>
		</tr>
		<tr>
			<td>Threonine</td>
			<td>107.2</td>
			<td>Dissolve in water</td>
			<td>1.072</td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>1.3</td>
			<td>Dissolve in 0.2 M NaOH</td>
			<td>0.013</td>
		</tr>
		<tr>
			<td>Tyrosine</td>
			<td>80.2</td>
			<td>Dissolve in 1M NaOH</td>
			<td>0.802</td>
		</tr>
		<tr>
			<td>Valine</td>
			<td>111.7</td>
			<td>Dissolve in water</td>
			<td>1.117</td>
		</tr>
	</tbody>
</table>

<table><tbody><tr><td>Insulin syringes - 0.5 mL with 29G needle attached.</td><td>VWR&amp;nbsp;</td><td>BDAM324892</td><td></td></tr><tr><td>24-well culture plates</td><td></td><td></td><td></td></tr><tr><td>70% ethanol or similar for surface sterilization and flaming of dissection equipment</td><td></td><td></td><td></td></tr><tr><td>Agar plates to prepare streaks of P. aeruginosa/S. aureus (any suitable medium)</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td></td><td></td><td></td></tr><tr><td>Aluminum foil - pre-sterilised by autoclaving - to cover the chopping board on whcih you wil dissect lungs.</td><td></td><td></td><td></td></tr><tr><td>Breathe-easy or Breathe-easier sealing membrane for multiwell plates</td><td>Diversified Biotech&amp;nbsp;</td><td>BEM-1 or BERM-2000</td><td></td></tr><tr><td>Bunsen burner</td><td></td><td></td><td></td></tr><tr><td>Chopping board - we recommend a plastic board to allow for easy decontamination with alcohol.</td><td></td><td></td><td></td></tr><tr><td>Coolbox to transport lungs to lab</td><td></td><td></td><td></td></tr><tr><td>Dissection scissors in different sizes</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle medium (DMEM)</td><td></td><td></td><td></td></tr><tr><td>Large pallet knife</td><td></td><td></td><td></td></tr><tr><td>Mounted razor blades</td><td></td><td></td><td></td></tr><tr><td>Petri dishes</td><td></td><td></td><td></td></tr><tr><td>Plastic chopping board and aluminium foil to create a sterile and cleanable dissection surface</td><td></td><td></td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 medium</td><td></td><td></td><td></td></tr><tr><td>SCFM&amp;nbsp;</td><td></td><td></td><td>Ingredients as listed in Table 1</td></tr><tr><td>Selection of forceps (blunt tips recommended)</td><td></td><td></td><td></td></tr><tr><td>Suitable containers for disposing of contaminated sharps and pig lung tissue, according to your institution&#039;s&lt;br/&gt; health &amp;amp; safety policies.</td><td></td><td></td><td></td></tr></tbody></table>

ex vivo pig lung model of biofilm: a technique to develop lung model to study bacterial biofilms on pig bronchiolar tissue

Source: Harrington, N.E. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62187">Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung.</a> J. Vis. Exp. (2021).&#160;
This video describes the protocol for developing an ex vivo pig lung model of bacterial biofilm on infected bronchiolar tissue. Biofilms are bacterial aggregates embedded in a matrix adherent to a surface. This model is used to study the antibiotic susceptibility of bacteria at different stages of biofilm formation.

Ex vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue

Source: Harrington, N.E. et al., Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the ...

Bronchioles are the tubular airways of the lungs. During chronic infection, the bronchioles get clogged due to the accumulation of mucus. The nutrient-rich mucus provides a suitable environment for opportunistic bacteria to grow. These bacteria get embedded in a self-produced extracellular polymeric matrix and proliferate to form a biofilm.
To mimic bacterial biofilm development on the lungs, take a pig bronchiolar tissue specimen in a Petri dish. Dissect the tissue into pieces of the desired shape. Add a biofilm culture media to the Petri dish. Sterilize the dish under UV light to remove any surface contaminants.
Transfer the sterilized tissue pieces into a multiwell plate containing biofilm culture media and agarose pads, which serve as an optimum substrate for bacterial adhesion. Next, prick a needle tip containing a colony of the desired bacteria onto each tissue piece to introduce planktonic bacteria into the tissue. Seal the plate with a UV-sterilized, porous membrane that eases uniform oxygen exchange throughout the plate surface. Incubate the sealed plate.&#160;
During incubation, bacteria adhere irreversibly to the epithelial surface through pili and begin to secrete the extracellular matrix.&#160; Within the matrix, bacteria utilize the energy sources, mucin, and free DNA from the culture media to form biofilms. The bacterial biofilm model is ready for further testing.

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1.1K Views. Source: Harrington, N.E. et al., Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung. J. Vis. Exp. (2021).  This video describes the protocol for developing an ex vivo pig lung model of bacterial biofilm on infected bronchiolar tissue. Biofilms are bacterial aggregates embedded in a matrix adherent to a surface. This model is used to study the antibiotic susceptibility of bacteria at different stages of bi...

Video: Ex vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue - Concept

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

JoVE Journal

Immunology and Infection

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

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

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

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

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Ex vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue

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Ex Vivo Pig Lung Model of Biofilm: A Technique to Develop Lung Model to Study Bacterial Biofilms on Pig Bronchiolar Tissue

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

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

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model