Visualization of Pseudomonas aeruginosa in Sputum Samples from Cystic Fibrosis Patients

Take a tube containing solidified hydrogel embedded with cystic fibrosis patient-derived sputum.

The sputum contains infection-associated Pseudomonas aeruginosa, a pathogenic bacterium.

Add a hybridization buffer containing green-fluorescent probes and incubate to allow the probes to bind selectively to bacterial ribosomal RNA.

Rinse with wash buffer to remove unbound probes.

Add fresh wash buffer and incubate to stabilize probe binding.

Remove the buffer, then add a blocking buffer to prevent nonspecific interactions.

Remove the blocking buffer. Introduce a solution containing red-fluorescent antibodies, and incubate to allow the antibodies to target the bacterial exopolysaccharide.

Rinse with a buffer to remove unbound antibodies, then stain with a DNA-binding dye to label DNA.

Mount the stained hydrogel onto a perfusion chamber with a coverslip.

Under a confocal microscope, observe green-fluorescently labeled bacteria surrounded by red-fluorescently labeled exopolysaccharide, allowing clear visualization of bacteria within the sputum.

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 approxim