This protocol is significant because it allows for the direct visualization of Pseudomonas aeruginosa and the exopolysaccharide Psl within cystic fibrosis sputum. This technique helps to answer questions about how the spatial organization of P.aeruginosa such as aggregation or biofilms affects host clearance and antimicrobial susceptibility. Store the expectorated sputum in a sterile collection cup at four degrees Celsius for a maximum of 24 hours prior to fixation.
When ready to fix the samples, transfer them to a sterile 15 milliliter tube, and add an equal volume of 4%PFA, then incubate the sputum overnight at four degrees Celsius. Wash the samples by adding five milliliters of PBS for every two milliliters of fixed sputum. Carefully remove the supernatant with a pipette, and repeat the wash two more times.
Resuspend the pellet in twice the volume of PBS with 0.01%sodium azide, and store it at four degrees Celsius. Add two milliliters of a 30%29 to one acrylamide/bisacrylamide solution to a three milliliter tube with the cap off inside a sealed anaerobic container, then add 0.5 grams of hardener to two milliliters of PBS in a three milliliter tube. Leave the cap off the tube, and store it in the sealed anaerobic container.
Leave a few milliliters of sterile PBS in the tube with the cap off inside the anaerobic container. Degas the hydrogel components in the sealed container with an anaerobic pack for 72 hours. Make a 2.5 milliliter solution of hydrogel with a final concentration of 0.2%hardener and 4%acrylamide/bisacrylamide in PBS in a 15 milliliter tube, and mix the hydrogel by inversion, then filter sterilize the solution.
Cut the sputum samples into two small sections with a scalpel, then place each sample in a well of an eight chambered covered glass slide. Add 300 microliters of the filter sterilized hydrogel solution to each well that contains sputum, and place it inside a sealed container with an anaerobic pack for three hours at 37 degrees Celsius. Transfer the solidified hydrogel sputum samples to a 15 milliliter culture tube with five milliliters of 8%SDS at pH eight, and allow the samples to clear for three to 14 days at 37 degrees Celsius until the sputum becomes transparent.
Use sterile tweezers to transfer each hydrogel embedded sample to a 50 milliliter conical tube, and add 10 milliliters of PBS to wash the hydrogel. Let the solution sit for 30 to 60 minutes before decanting, then repeat the wash two more times. Store the washed samples in PBS with 0.01%sodium azide and RNase inhibitor at four degrees Celsius.
Remove the hydrogel samples from their storage solution, and place the samples on a sterile surface. Use a sterile scalpel to cut the hydrogels into approximately one millimeter thick slices, and place the sections into 1.5 milliliter tubes. Prepare one milliliter of hybridization buffer in a 1.5 milliliter tube, then add the fluorescently labeled Seer A-probe, and mix by inversion.
Add 200 to 500 microliters of hybridization buffer to each section, making sure that the entirety of the hydrogel sample is submerged. Allow the Seer A-probe to hybridize with the hydrogel samples by placing them in the dark for approximately 18 to 24 hours at 46 degrees Celsius without shaking, then add one milliliter of fresh wash buffer to the tubes, and incubate them in the dark for six hours at 48 degrees Celsius. Add 500 microliters of the 2%BSA and PBS to the hydrogel samples to block non-specific protein binding, then incubate the samples overnight in the dark.
On the next day, prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 micrograms per milliliter in 500 microliters of fresh 2%BSA in PBS. Add the antibody solution to the hydrogel samples, and incubate them in the dark for six hours. Next, stain the hydrogel samples with DAPI by incubating them with 250 microliters of RIMs solution and 10 micrograms per milliliter of DAPI overnight in the dark with gentle shaking.
After staining, mount the samples onto 0.9 or 1.7 millimeter perfusion chambers, and seal them with a glass coverslip. Perform confocal laser scanning microscopy with 25, 40, 63, or 100 times magnifications. This protocol was used to obtain the high resolution 3D images of P.aeruginosa cells and their NC2 structure within sputum.
An aggregate of cells within a sputum sample is shown here. Individual rod shapes were seen in green from the species-specific binding of the Seer A-488 probe to P.aeruginosa cells. The Psl0096-Texas Red antibody shows that the Pseudomonal exopolysaccharide within the sputum was mostly overlapping with the P.aeruginosa cells.
This method was also used for the visualization of Pseudomonal cells within the sputum in relation to other bacterial cells and host structures. A cluster of P.aeruginosa cells was seen to phagocytose within a eukaryotic cell while other small coccal cell clusters were observed in the vicinity. It was also demonstrated that the PSL 0096 antibody could bind to PSL produced from planktonic P.Aeruginosa.
The main advantage of this technique is that it implements a tissue clearing approach followed by fluorescent in situ hybridization, antibody staining, and confocal microscopy. This enables researchers to visualize and use image processing techniques to quantitatively measure differences in P.aeruginosa colonization structures within CF sputum in relation to patient health outcomes.
