The overall goal of this procedure is to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. This method can help answer key questions in the airway microbiology field Such as, how diverse bacterial pathogens interact in a cystic fibrosis lung. The main advantage of this technique is that it allows for easy manipulation of experimental conditions.
To observe microbial responses similar to how they would occur in an actual lung bronchial. Demonstrating the procedure, will be Will Comstock from our laboratory. To prepare artificial sputum medium, Combine 16 milliliters of previous prepared muceum stock solution, Two milliliters of potassium chloride stock solution, two milliliters of sodium chloride stock solution, 200 microliters of egg yolk emulsion, 5.6 milliliters of DNA stock solution, 120 microliters of ferritin stock solution, 5.78 milliliters of essential amino acid solution, 5.78 milliliters of non-essential amino acid solution, and 2.44 milliliters of sterile water.
The careful creation of sterile artificial sputum medium is extremely important. As any contamination can alter bacterial activity in the incubated capillary tubes, further down the line. After gently shaking to mix, pipette five milliliters of the medium into eight sterile 15 milliliter centrifuge tubes.
The medium can now be modified to achieve the desired chemical conditions. For instance, the media shown here, have had their PH altered and dyes added to reflect their different acidities. Next, under a sterile bio-hood, add 900 microliters of medium to each of eight sterile 1.5 milliliter microcentrifuge tubes.
Homogenize previously acquired sputum samples by using a three milliliter syringe to repeatedly withdraw and eject the sputum until it is of a smooth consistency. Add 100 microliters of the homogenized sputum to each of the eight microcentrifuge tubes. Then vortex the tubes to sufficiently mix.
Label eight more sterile 15 milliliter centrifuge tubes according to the microcentrifuge tubes of medium previously prepared. These will be the incubation tubes. Use 70%ethanol solution to sterilize a paper towel and allow it to dry.
Tear the towel into pieces about four square inches each. Then crumble one piece in the bottom of each incubation tube. With one clump of paper at the bottom of each incubation tube, use 1 milliliter of sterile water to slightly dampen each clump to create a humid environment inside the tube.
For each tube of medium, fill three glass capillary tubes with medium by holding one end of the tube in the medium and horizontal tilting it. Allowing capillary action to guide the medium into the tube. Stop the filling by gently placing a gloved finger over the open end of the tube, and then seal the other end of the tube by pressing it down into a block of capillary putty sealant.
Place each set of three capillary tubes into 15 milliliter incubation tubes putty sealed side down. Then cap the tubes and insure they are labeled. These three capillary tubes are for replication of each control condition.
When all incubation tubes are filled with their designated capillary tubes. Place the racks at 37 degrees Celsius such that the tubes will be incubated horizontally so that any gas generated does not escape through the open end. Incubate the tubes for 48 hours.
Remove the tubes from the incubator making sure to keep them horizontal. Carefully the capillary tubes out of the incubation tubes, keeping each set of three separate from other sets. Arrange the capillary tubes next to each other on a light box all lined up so the contents of the tubes are visible and illuminated from below.
Weave a gap every three tubes to separate different chemical conditions Next Focus the camera on the tubes directly above them such that all tubes are visible in the field of view and light from the light box provides sufficient contrast and visualization of the color of the tube dyes. To extract the contents of the tubes for downstream analysis, remove one set of triplicate capillaries from the light box at a time. Insert a 25 gauge 0.5 inch blunt ended needle into the plugged end of the capillary tube to break the seal.
Then turn the capillary tube upside down to allow the medium to drip from the top portion of the into a 1.5 milliliter micro centrifuge tube. If the medium does not drip out, use a to 200 microliter pipette to expel the medium out of the tube by pressing down on a pipette plunger when it is inserted into the end of the capillary tube. As shown here, when no sputum samples were added to the medium, the only change exhibited across the pH spectrum after 48 hours of incubation was a slight decrease in medium volume resulting from minor evaporation.
Inoculated capillary tubes showed changes in volume, the appearance of gas bubbles, altered coloration, and the appearance of opaque deposits. These results show that bacteria and other microbes contained within the added sputum, are able to grow and change within the artificial medium. Following incubation, other methods like metabolomics and DNA sequencing can be performed to answer additional questions about the behavior of the microbes that grow in the capillary tubes.
The winCF system is a novel method to study the activity of the lung microbiome;important for many diseases including CF, COPD, asthma, and others.
