For the pH experiments, ASM was prepared according to (12, 42) and then split into eight different stocks that were buffered to a pH gradient from 5 to 8.5 (0.5 pH unit intervals) with a phosphate buffer concentration of 50 mM. The desired pH for each interval was achieved by adding the appropriate amount of monosodium phosphate and disodium phosphate and then bringing the media to the appropriate pH using HCl or NaOH. The pH dyes phenol red and bromocresol purple were added to the media to aid in pH gradient visualization (fig. S1A). After homogenization, 50 μl of each sputum sample was inoculated into 250 μl of ASM media at eight different pH intervals within 6 hours of sample collection. This mixture was vortexed and inoculated into capillary columns (nonheparinized Fisherbrand and Microhematocrit capillary columns, Fisher Scientific) in triplicate, as described in (12). All WinCF columns were incubated at 37°C for 48 hours, and then the medium from each triplicate capillary column was removed and pooled. Three separate 50-μl aliquots of this pooled mixture were then prepared for 16S rRNA gene sequencing and metabolomics analysis of nonvolatile compounds using LC-MS/MS and volatiles using GC-MS. The oxygen experiments were done using the same medium formulation, but a 0.5% agar solution was mixed with the medium before inoculation (fig. S1B). The WinCF columns were created by drawing the warm molten media and agar into a 1000-μl pipette tip, sealing the pointed end while leaving the wide end open to the air and allowing it to cool. Three columns were created for each of the 19 sputum samples acquired, the first left untreated, the second treated with 20 μl of tobramycin (60 mg/ml), and the third being treated with 20 μl of 7% sodium bicarbonate. The treatments were added to the top open portion of the columns before incubation. Control columns with no sputum were generated and analyzed in the same manner. The columns were incubated as described above. After incubation, they were frozen at −80°C and sectioned with a sterile razorblade in 1-mm sections to analyze the microbial and chemical difference through a 10-mm depth using a sterile razorblade. The sections were stored in microcentrifuge columns. For 16S rDNA sequencing, 200 μl of 1× phosphate-buffered saline was added to each column. Extractions for metabolomics were done directly on the sectioned media. Further details for both procedures are available in Supplementary Methods.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.