The small colony variant or SCV technique allows for selection and growth of small colony variants of Pseudomonas aeruginosa. The method is very definitive and allows for easier selection than normal methods. This protocol also details a safer, more sensitive ELISA method for alginate quantification as compared to the standard uronic acid carbazole method, enabling direct alginate quantification in samples without cautery or sample manipulation.
Demonstrating the procedure will be Brandon Kirby, a lab technician, and Roy Al Ahmar, a graduate student from my laboratory. To detect the small colony variant or SCV, first streak the P.aeruginosa strain PAO1 delta PYRD on pre-warmed PIA plates. Grow the strains at 37 degrees Celsius for 48 hours.
On the growth plate, identify a single colony isolate that has the SCV phenotype characterized by a colony size of one to three millimeters as opposed to the normal three to five millimeter colony size. Re-streak the selected colonies to obtain a pure isolate of the SCV. To perform physiological activation of the salvage pathway, use a sterile inoculation loop to pick the SCV colony from the PIA plate.
Streak the selected colony on a pre-warmed PIA plate supplemented with 0.1 millimolar uracil. Grow the plate at 37 degrees Celsius for 24 to 48 hours. To begin the uronic acid carbazole assay, identify a single colony from a pure culture of the desired strain to be tested and pick the colony using a sterile toothpick.
Place the toothpick in a culture test tube containing five milliliters of PIB. Grow in a shaker incubator at 37 degrees Celsius for 24 hours. Following incubation, add an aliquot of the cultured PIB broth onto a pre-warmed PIA plate.
Using a sterile cell spreader, spread the broth over the plate and grow at 37 degrees Celsius for 24 hours. Using a pipette controller and a sterile 50 milliliter pipette, add 0.85%sodium chloride to the grownup lawn and collect the sample by scraping the plate using a cell spreader. Aspirate the sample using a fresh 50 milliliter pipette and transfer into a 50 milliliter collection tube.
Vortex the sample on high to mix and place the samples on ice. To measure the OD600 of the samples, first blank the spectrophotometer using one milliliter of 0.85%sodium chloride. Then add one milliliter of the sample to a new disposable cuvette and read the OD.Repeat this step twice to obtain a triplicate of reads for each sample.
Now add three milliliters of the sulfuric acid borate solution into culture tubes and let sit on ice. Add 350 microliters of the collected sample slowly to the acid mixture in the test tubes. After briefly vortexing on low, add 100 microliters of 0.1%carbazole and ethanol solution to the acid sample mixture.
Cap the tube and vortex on the medium setting for five seconds. Then place in a dry bath at 55 degrees Celsius for 30 minutes. After incubation, vortex the tubes briefly on high and allow to cool for five minutes.
Use the tube with 0.85%sodium chloride as a blank to the spectrophotometer. Then add one milliliter of the mixture to a clean cuvette and read the OD of the samples at 530 nanometers on a spectrophotometer. Prepare a standard curve by measuring the OD530 of serial dilutions of known concentrations of d-mannuronic acid.
Repeat twice and extract a linear equation from these readings. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from linear equation by OD600 to obtain the total amount of alginate per OD600. Using a micropipette, add 50 microliters of the collected sample to an untreated 96-well plate.
Then add 50 microliters of ELISA coating buffer to the wells. Incubate the plate at 37 degrees Celsius for two hours. Using a squirt bottle, wash the plate wells twice with PBS-T by filling the wells and then draining them by flipping the plate over.
Add 200 microliters of blocking buffer to the wells with a micropipette and incubate at four degrees Celsius overnight. The next day, wash the plate twice with PBS-T as before. Then add 100 microliters of diluted primary antibody to the wells and incubate at 37 degrees Celsius for one to two hours.
Following incubation, wash the plate wells three times with PBS-T as before. Next, add 100 microliters of diluted secondary antibody to the wells and incubate at 37 degrees Celsius for one to two hours. After washing the plate again three times, use a micropipette to add 100 microliters of TMB ELISA solution.
Incubate the plate at room temperature for 30 minutes in the dark by placing the plate into a drawer or wrapping in foil. Then add 100 microliters of stop solution. Use a plate reader to measure the OD at 450 nanometers.
Produce a standard curve by measuring the OD450 of serial dilutions of known concentrations of d-mannuronic acid. Repeat the measurements twice and extract a linear equation. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from the linear equation by OD600 to obtain the total amount of alginate per OD600.
Shown here are samples of PAO581 and PAO581 with mutations in genes regulating pyrimidine de novo biosynthesis grown on PIA and PIA supplemented with 0.1 millimolar of uracil. The data show that the presence of uracil in the media results in the conversion of the mutant strain back to mucoidy as seen by the restored alginate production. Here, results are shown for the anti-alginate monoclonal antibody-based ELISA.
Colonies are grown on PIA plates with arabinose for the induction of the PBAD promoter and the pHERD-20T plasmid. Shown are PAO1, PAO1 carrying the expression plasmid pHERD-20T with the main alginate specific sigma factor algU, and PAO1 with an in-frame deletion of the algD gene in coding the key alginate biosynthetic enzyme GDP-mannose dehydrogenase. This data compares the non-mucoid levels of alginate measured for PAO1 and PAO1 delta algD versus the mucoid levels of alginate measured for PAO1 with pHERD-20T algU.
Anti-alginate ELISA was also tested on patient sputum samples without prior growth on plates. Three CF sputum samples that had growth of mucoid P.aeruginosa showed detectable alginate compared with two patient sputum samples that contained either non-mucoid P.aeruginosa or no P.aeruginosa growth. The preparation of the standard curves for the alginate quantification is very crucial as this will allow for precise and accurate measurement of alginate within the sample.
The acid solution used in the carbazole method are very dangerous so proper personal protective equipment and precautions must be used to ensure safety. After isolating SCVs, further genetic profiling can be done to better understand mutations occurring and the pathogenicity associated with such mutations. The ELISA method can be adapted to monitor chronic lung infection in patients with cystic fibrosis.
Furthermore, our growth method could help diagnose infections caused by specific SCVs.
