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.
