Part of this work was performed at the Marine Biological Lab with support from the Marine Biological Lab, DOE (DE-SC0016127), NSF (MCB1822263), HHMI (grant number 5600373), and a gift from the Simons Foundation.

This study received approval from the Partners Institutional Review Board (Protocol # 2018P002934). Inclusion criterion included adult patients with cystic fibrosis who provided written informed consent for the study. There was no exclusion criterion. According to protocol guidelines, all sputum samples were collected from patients with cystic fibrosis during a scheduled outpatient visit with their clinical provider.
1. Artificial Sputum Medium Preparation
NOTE: Quant...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+acquisition+and+succession+of+the+cystic+fibrosis+lung+microbiome+is+associated+with+disease+progression+in+infants+and+preschool+children.">Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children.</a> PLoS Pathogens. 14 (1), 1006798 (2018).</li><li>Zolin, A., Bossi, A., Cirilli, N., Kashirskaya, N., Padoan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+mortality+in+childhood.+Data+from+European+cystic+fibrosis+society+patient+registry.">Cystic fibrosis mortality in childhood. Data from European cystic fibrosis society patient registry.</a> International Journal of Environmental Research and Public Health. 15 (9), (2018).</li><li>Ramos, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+survival+in+adult+patients+with+cystic+fibrosis+with+FEV1+30%+of+predicted+in+the+United+States.">Heterogeneity in survival in adult patients with cystic fibrosis with FEV1 30% of predicted in the United States.</a> Chest. 30 (6), 1320-1328 (2017).</li><li>Ramos, K. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins,+mucus,+and+sputum.">Mucins, mucus, and sputum.</a> Chest. 135 (2), 505-512 (2009).</li><li>Sui, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+DNA+extraction+method+on+variation+in+human+and+built+environment+microbial+community+and+functional+profiles+assessed+by+shotgun+metagenomics+sequencing.">Impact of DNA extraction method on variation in human and built environment microbial community and functional profiles assessed by shotgun metagenomics sequencing.</a> Frontiers in Microbiology. 11, 953 (2020).</li><li>McIver, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bioBakery:+a+meta'omic+analysis+environment.">bioBakery: a meta'omic analysis environment.</a> Bioinformatics. 34 (7), 1235-1237 (2018).</li><li>Truong, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaPhlAn2+for+enhanced+metagenomic+taxonomic+profiling.">MetaPhlAn2 for enhanced metagenomic taxonomic profiling.</a> Nature Methods. 12 (10), 902-903 (2015).</li><li>Stammler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjusting+microbiome+profiles+for+differences+in+microbial+load+by+spike-in+bacteria.">Adjusting microbiome profiles for differences in microbial load by spike-in bacteria.</a> Microbiome. 4 (1), 28 (2016).</li><li>Henke, M. O., Renner, A., Huber, R. M., Seeds, M. C., Rubin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC5AC+and+MUC5B+mucins+are+decreased+in+cystic+fibrosis+airway+secretions.">MUC5AC and MUC5B mucins are decreased in cystic fibrosis airway secretions.</a> American Journal of Respiratory Cell and Molecular Biology. 31 (1), 86-91 (2004).</li><li>Henderson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+airway+secretions+exhibit+mucin+hyper+concentration+and+increased+osmotic+pressure.">Cystic fibrosis airway secretions exhibit mucin hyper concentration and increased osmotic pressure.</a> Journal of Clinical Investigation. 124 (7), 3047-3060 (2014).</li><li>Matthews, L. W., Spector, S., Lemm, J., Potter, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+pulmonary+secretions.+I.+The+over-all+chemical+composition+of+pulmonary+secretions+from+patients+with+cystic+fibrosis,+bronchiectasis,+and+laryngectomy.">Studies on pulmonary secretions. I. The over-all chemical composition of pulmonary secretions from patients with cystic fibrosis, bronchiectasis, and laryngectomy.</a> American Review of Respiratory Disease. 88, 199-204 (1963).</li><li>Ibanez de Aldecoa, A. L., Zafra, O., Gonzalez-Pastor, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+regulation+of+extracellular+DNA+release+and+its+biological+roles+in+microbial+communities.">Mechanisms and regulation of extracellular DNA release and its biological roles in microbial communities.</a> Frontiers in Microbiology. 8, 1390 (2017).</li><li>Tunney, M. 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In this study, an in vitro model was developed to study the effect of hyperoxia on lung microbial communities. This model, based on artificial sputum medium and daily sparging of serum bottles, maintains elevated oxygen concentrations and supports the growth of microbes identified in sputum from pwCF.
There are several critical steps of this approach. First is the choice to use filter-sterilization rather than heat-sterilization of the artificial sputum medium. Filter-sterilization pr...

Cystic fibrosis (CF) is a genetic disease characterized by an inability to clear thick mucus from the lungs, leading to repeated infections and progressive lung function decline that often results in the need for lung transplantation or death. The airway microbiome of people with cystic fibrosis (pwCF) appears to track disease activity1, with a reduction in microbial diversity associated with adverse long-term outcomes2,3. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease4,5, though traditionally, the use of oxygen therapy has been viewed as simply a marker for disease severity6. Recent studies from a clinical trial of patients with respiratory failure have shown that higher patient oxygen levels are paradoxically associated with an increase in serious bacterial infections and higher mortality7, suggesting that supplemental oxygen may contribute to disease pathogenesis. The effect of supplemental oxygen on the cystic fibrosis lung microbiome and associated lung and airway microbial communities has not been well studied.
Mechanistic studies often cannot be performed directly on human subjects due to logistical difficulties and potential ethical issues associated with interventions of unknown medical benefit or harm. Translational approaches that integrate human biospecimens into model systems can offer important biological insights in these cases. While the ability to use or tolerate oxygen has traditionally been an important component of microbial classification, little is known about how the therapeutic introduction of supplemental oxygen to the environment might perturb airway microbial communities. To shed light on the unknown effects of supplemental oxygen on the airway microbiomes of pwCF, we needed to address two major challenges; first, the creation of a culture medium that physiologically approximates the composition of CF sputum; second, the creation of a model system that allows the maintenance of elevated oxygen concentrations in culture over extended periods of time.
Artificial sputum media (ASM) are widely used to emulate lung sputum ex vivo8,9,10, but there is no clear consensus on a specific recipe. This protocol describes an artificial sputum medium recipe and preparation strategy carefully designed to physiologically approximate sputum from pwCF. Table 1 outlines the chosen recipe values based on published literature. Basic chemical components and pH were matched to values identified by studies of human CF sputum11,12,13. Low concentration physiological nutrients were added using egg yolk, which was included as 0.25% of the final volume10, as well as vitamin and trace metal mixes14,15. Mucin, the key component of sputum16, was included at 1% w/v14. Although more labor-intensive, filter sterilization was chosen over the more conventional practice of heat sterilization to reduce potential problems from heat-induced denaturation of essential media components10. An additional benefit of filter sterilization is that it generates media that are transparent (heat-sterilization can create turbid media due to precipitation and coagulation of salts and proteins), allowing this artificial sputum media to be used to follow microbial growth based on increases in turbidity.
This model system for the hyperoxic culture is based on anaerobic culturing techniques where oxygen is added rather than removed, creating a model for the effect of supplemental oxygen use for pwCF. Figure 1 and the associated oxygen sparging protocol outlines the components of an oxygen sparging system, which can be obtained at low cost from general laboratory and hospital suppliers. This system enables the mixing of compressed oxygen and air to fixed concentrations ranging from 21%-100% oxygen. The integration of an oxygen sensor allows for the verification of the concentration of the output gas mixture, as well as checking the outflow gas composition of previously sparged serum bottles to verify that the oxygen conditions have been maintained within the desired range.
This protocol outlines procedures to create an artificial sputum medium, the construction and use of an oxygen sparging system, and the application of both to culture CF sputum under differential oxygen conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BME Vitamins (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>B6891</td>
			<td>Concentrated solution of supplemental vitamins.</td>
		</tr>
		<tr>
			<td>Crimper, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224307</td>
			<td>Crimper for attaching aluminum seals to serum bottles.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>MilliporeSigma</td>
			<td>G7021</td>
			<td>Solid glucose powder (dextrorotatory isomer).</td>
		</tr>
		<tr>
			<td>Diaphragm Pump ME 2 NT</td>
			<td>VACUUBRAND</td>
			<td>20730003</td>
			<td>Vacuum pump for vacuum filtration.</td>
		</tr>
		<tr>
			<td>Egg Yolk Emulsion</td>
			<td>HiMedia</td>
			<td>FD045</td>
			<td>Sterile emulsion of 30% egg yolk in saline.</td>
		</tr>
		<tr>
			<td>Ferritin, Cationized from Horse Spleen</td>
			<td>MilliporeSigma</td>
			<td>F7879</td>
			<td>Ferritin (iron-storage protein) solution.</td>
		</tr>
		<tr>
			<td>FIREBOY plus Safety Bunsen Burner</td>
			<td>Integra Biosciences</td>
			<td>144000</td>
			<td>Bunsen burner with user interface and safety features.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (1.0&#8211;14.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>94</td>
			<td>pH testing paper for the range of 1.0&#8211;14.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (4.0&#8211;9.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>55</td>
			<td>pH testing paper for the range of 4.0&#8211;9.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (6.0&#8211;8.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>345</td>
			<td>pH testing paper for the range of 6.0&#8211;8.0.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle-Pro EDGE Safety Device, 18 G</td>
			<td>Smiths Medical</td>
			<td>401815</td>
			<td>18 G needles with safety caps.</td>
		</tr>
		<tr>
			<td>In-Line Pressure Gauge</td>
			<td>MilliporeSigma</td>
			<td>20469</td>
			<td>Gas pressure gauge for monitoring bottle pressure.</td>
		</tr>
		<tr>
			<td>Innova 42 Incubated Shaker</td>
			<td>Eppendorf</td>
			<td>2231000756</td>
			<td>Combination incubator/orbital shaker.</td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with Attached Needle</td>
			<td>Becton Dickinson</td>
			<td>309580</td>
			<td>Combination 3 mL syringe and 18 G needle.</td>
		</tr>
		<tr>
			<td>Luer Valve Assortment</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>Valves for gas flow tubing.</td>
		</tr>
		<tr>
			<td>LSE Orbital Shaker</td>
			<td>ThermoFisher Scientific</td>
			<td>6780-NP</td>
			<td>Orbital shaker to agitate media during filtration.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>MilliporeSigma</td>
			<td>M2773</td>
			<td>Solid epsom salt (magnesium sulfate heptahydrate).</td>
		</tr>
		<tr>
			<td>Medical Air Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-346-15FM</td>
			<td>Air flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>MEM Amino Acids (50x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M5550</td>
			<td>Concentrated solution of essential amino acids.</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M7145</td>
			<td>Concentrated solution of non-essential amino acids.</td>
		</tr>
		<tr>
			<td>Millex-GP Filter, 0.22 &#181;m</td>
			<td>MilliporeSigma</td>
			<td>SLMP25SS</td>
			<td>0.22 &#181;m polyethersulfone membrane sterile syringe filter.</td>
		</tr>
		<tr>
			<td>Milli-Q Academic</td>
			<td>MilliporeSigma</td>
			<td>ZMQS60E01</td>
			<td>Milli-Q sterile water filtration system.</td>
		</tr>
		<tr>
			<td>MiniOX 3000 Oxygen Monitor</td>
			<td>MSA</td>
			<td>814365</td>
			<td>Gas flow oxygen percentage monitor.</td>
		</tr>
		<tr>
			<td>MOPS Buffer (1 M, pH 9.0)</td>
			<td>Boston BioProducts</td>
			<td>BBM-90</td>
			<td>MOPS buffer for adjusting media pH.</td>
		</tr>
		<tr>
			<td>Mucin from Porcine Stomach</td>
			<td>MilliporeSigma</td>
			<td>M2378</td>
			<td>Mucin (glycosylated gel-forming protein) powder.</td>
		</tr>
		<tr>
			<td>Natural Polypropylene Barbed Fitting Kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1413</td>
			<td>Connectors for gas flow tubing.</td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1096</td>
			<td>Library preparation for identification during sequencing.</td>
		</tr>
		<tr>
			<td>NovaSeq 6000 Sequencing System</td>
			<td>Illumina</td>
			<td>770-2016-025-N</td>
			<td>Shotgun sequencing platform for generating sample reads.</td>
		</tr>
		<tr>
			<td>Oxygen Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-540-15FM</td>
			<td>Oxygen flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>Oxygen Tubing with 2 Standard Connectors</td>
			<td>SunMed</td>
			<td>2001-01</td>
			<td>Tubing for connecting gas system components.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x, pH 7.4</td>
			<td>Molecular Biologicals International</td>
			<td>MRGF-6235</td>
			<td>Concentrated phosphate-buffered saline solution.</td>
		</tr>
		<tr>
			<td>PC 420 Hot Plate/Stirrer</td>
			<td>Marshall Scientific</td>
			<td>CO-PC420</td>
			<td>Combination hot plate/stirrer.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>MilliporeSigma</td>
			<td>P9541</td>
			<td>Solid potassium chloride salt.</td>
		</tr>
		<tr>
			<td>PTFE Disposable Stir Bars</td>
			<td>ThermoFisher Scientific</td>
			<td>14-513-95</td>
			<td>Disposable magnetic stir bars.</td>
		</tr>
		<tr>
			<td>PTFE Thread Seal Teflon Tape</td>
			<td>VWR</td>
			<td>470042-938</td>
			<td>Teflon tape for reinforcing gas system connections.</td>
		</tr>
		<tr>
			<td>Q-Gard 2 Purification Cartridge</td>
			<td>MilliporeSigma</td>
			<td>QGARD00D2</td>
			<td>Purification cartridge for Milli-Q system.</td>
		</tr>
		<tr>
			<td>Reusable Media Storage Bottles</td>
			<td>ThermoFisher Scientific</td>
			<td>06-423A</td>
			<td>Bottles for mixing and storing culture media.</td>
		</tr>
		<tr>
			<td>Rubber Stopper, 30 mm, Gray Bromobutyl</td>
			<td>DWK Life Sciences</td>
			<td>224100-331</td>
			<td>Rubber stoppers for serum bottles.</td>
		</tr>
		<tr>
			<td>Serum Bottle with Molded Graduations, 500 mL</td>
			<td>DWK Life Sciences</td>
			<td>223952</td>
			<td>Glass serum bottles for sealed culturing.</td>
		</tr>
		<tr>
			<td>Small Bore Extension Set</td>
			<td>Braun Medical</td>
			<td>471960</td>
			<td>Tubing extension with luer lock connectors.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MilliporeSigma</td>
			<td>S3014</td>
			<td>Solid sodium chloride salt.</td>
		</tr>
		<tr>
			<td>Spike-in Control I (High Microbial Load)</td>
			<td>ZymoBIOMICS</td>
			<td>D6320</td>
			<td>Spike-in microbes (I. halotolerans and A. halotolerans) for absolute microbial load calculations</td>
		</tr>
		<tr>
			<td>Stericup Quick Release Sterile Vacuum Filtration System</td>
			<td>MilliporeSigma</td>
			<td>S2GPU02RE</td>
			<td>250 mL 0.22 &#181;m vacuum filtration chamber.</td>
		</tr>
		<tr>
			<td>Super Sani-Cloth Germicidal Disposable Wipes</td>
			<td>Professional Disposables International</td>
			<td>H04082</td>
			<td>Disposable germicidal wipes for sterilization.</td>
		</tr>
		<tr>
			<td>Trace Metals Mixture, 1000x</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0112668</td>
			<td>Concentrated solution of physiological trace metals.</td>
		</tr>
		<tr>
			<td>Unlined Aluminum Seal, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224187-01</td>
			<td>Aluminum seals crimped over top of rubber stoppers.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Air Tank</td>
			<td>Airgas</td>
			<td>AI USP200</td>
			<td>Compressed air tank for input to sparging system.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Oxygen Tank</td>
			<td>Airgas</td>
			<td>OX USP200</td>
			<td>Compressed oxygen tank for input to sparging system.</td>
		</tr>
	</tbody>
</table>

design and development of a model to study the effect of supplemental oxygen on the cystic fibrosis airway microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

These protocols were applied to 50 expectorated sputum samples from pwCF presenting for routine care to an outpatient cystic fibrosis clinic at Massachusetts General Hospital in Boston, Massachusetts. Each patient&#39;s sputum was cultured under 21%, 50%, and 100% oxygen conditions using the artificial sputum medium, with 0.5 mL aliquots taken from each culture at 24 h, 48 h, and 72 h of culture time for testing. Cultures were photographed when extractions were made to track visual changes. In addition, a 0.5 mL aliquot ...

Watch this Scientific Journal Video about Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome at JoVE.com

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

These protocols are designed to model the in vitro effects of supplemental oxygen use on the lung microbiome of cystic fibrosis patients. This media recipe is tailored to mirror the physiological composition of sputum for people living with cystic fibrosis. The sparging process introduces the ability to culture under different oxygen conditions.Alterations to artificial sputum medium allow it to mimic other chronic lung diseases and model microbial communities under other conditions, such as different glucose concentrations in concurrent cystic fibrosis and diabetes patients. An overview of the steps for artificial sputum medium preparation shows the mixing of the prepared ASCM, ASMM and ASBM. The mops buffer is used to titrate the pH to 6.3 and filter sterilization of the medium is carried out in a cold room on an orbital shaker.To begin with, prepare an artificial sputum medium by adding 250 milliliters of ASCM to the one liter bottle containing ASMM. Then add 250 milliliters of ASBM to the medium bottle. Titrate the medium with a basic one molar mops buffer to reach a pH of 6.3 on a pH paper.Refrigerate the artificial sputum medium at four degrees Celsius until filtration. Then to start the filtration process, transfer 200 milliliters of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 micrometers pore size filter. After connecting the filtration system to the vacuum pump, turn on the vacuum pump, then place the chamber on an orbital shaker, shaking at 90 rotations per minute in a cold room at four degrees Celsius.Change the filter after 350 milliliters of medium gets filtered to overcome the clogging issue. After a considerable amount is filtered, at an extra 150 milliliters medium for filtration. Repeat filtration with additional chambers until all the media is filtered.Overview of oxygen sparging demonstrating the addition of patient sputum to artificial sputum medium in an autoclaved serum bottle. Then sparge the sealed serum bottle with a gas mixture. Finally, the serum bottle is incubated to obtain culture Alec Watts at the 24 hour interval for subsequent meta genomics sequencing.To start serum bottle culture sparging, label 500 milliliter autoclaved serum bottles with sample identifiers, date and time of inoculation and target oxygen percentage. In a biological hood, add 24 milliliters of the artificial sputum medium to each serum bottle being set up. To obtain a sufficient sample volume for each culture condition, if necessary, dilute the sample with sterile phosphate buffered saline, then homogenized sputum with an 18 gauge needle and add one milliliter of the patient sputum to each serum bottle.Next, using sterile tweezers, place the autoclaved rubber stoppers onto the top of each serum bottle, press down the rubber stoppers without touching the underside of the stopper. After removing the bottles from the hood, apply and crimp the aluminum seals. Remove the centerpiece of the seals and wipe down the top of the bottles with an alcohol wipe and pass the bottle seal through a Bunsen burner flame.Now fix a sterile, 18 gauge needle to a plunger list syringe with a filter, insert the affixed gas release syringe into the bottle first. Now fix a sterile, 18 gauge needle to the gas output from the system and insert the gas output needle into the bottle as well. Route the T junctions from the tanks through the oxygen monitor.After verifying the target oxygen concentration flowing through the system, target approximately five liters per minute of gas flow. Reroute the T junctions from the tanks through to the gas output. As the gas starts to flow through the serum bottle, pay close attention to the pressure gauge, and if pressure increases unexpectedly, shut the system off immediately.After running, oxygen sparge through the serum bottle for one minute at five liters per minute, the settings allow for 10 air exchanges and ensure the internal atmosphere reaches the desired concentration of 100%oxygen. Remove the gas release, 18 gauge needle. After allowing the pressure in the serum bottle to build to one atmosphere, immediately remove the gas output needle.Incubate the serum bottles in 37 degrees Celsius incubator shaker at 150 rotations per minute for 3, 24 hour intervals. Alec Watts are taken at each 24 hour interval for downstream analysis and samples are resparged each time. The example here shows what a culture may look like before and after incubation.The outflow oxygen and pH levels were measured over the course of the culture process for sputum samples maintained at 37 degrees Celsius. With both sparging intervals of 12 hours and 24 hours, oxygen concentrations were approximately maintained over time with a moderate drop in oxygen concentration observed for all three conditions. The measured pH levels were within the physiological range with no significant changes over time.The comparison of microbial load, diversity and community composition between uncultured and cultured sputum revealed a 20 fold increase in microbial load in cultured sputum. Diversity metrics indicated preservation of community composition with minimal global differences introduced by the culture process. Comparative analysis of the 120 microbial species in cultured and uncultured sputum was performed by meta genomic sequencing.46 of these species were identified in both uncultured and cultured samples. While 35 were exclusively identified in uncultured samples and 39 were exclusively identified in cultured samples. Absorbent based growth curves were generated for common CF lung pathogens cultured in artificial sputum media from patient sample isolates under normal 21%oxygen concentration.No change over time in the optical density at 600 nanometers in the ASM only negative control indicated contamination free culture. The typical growth curve patterns observed indicating that ASM medium can be used for the generation of growth curves using optical methods. The metagenomic sequencing is used here to describe microbial community composition.Other techniques such as metatranscriptomic sequencing can be used to evaluate gene expression or metabolomics to evaluate metabolite production.

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2.2K vistas.  Massachusetts General Hospital. Estos protocolos están diseñados para modelar los efectos in vitro del uso de oxígeno suplementario en el microbioma pulmonar de pacientes con fibrosis quística. Esta receta de medios está diseñada para reflejar la composición fisiológica del esputo para las personas que viven con fibrosis quística. El proceso de sparging introduce la capacidad de cultivo bajo diferentes condiciones de oxígeno.Las alteraciones en el medio de esputo artificial le permiten imitar otras enfermed...