This work was supported by the National Institutes of Health (NIH) grants R44GM113545 and P20GM103434.

1. SCV Growth Conditions and Physiological Activation of the Salvage Pathway
<ol>
	<li>Detection of SCV.
	<ol>
		<li>Streak the P. aeruginosa strains PAO1, PAO1&#916;pyrD, PAO581, and PAO581&#916;pyrD on prewarmed Pseudomonas isolation agar (PIA) plates and grow at 37 &#176;C for 48 h. On the growth plate identify a single colony isolate that has the SCV phenotype (colony size of 1&#8722;3 mm as opposed to the normal 3&#8722;5 mm colony size).</li>
		<li>Repeat step 1.1.1 to obtain a...

<ol>
	<li>Govan, J. R., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+pathogenesis+in+cystic+fibrosis:+mucoid+Pseudomonas+aeruginosa+and+Burkholderia+cepacia.">Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.</a> Microbiological Reviews. 60 (3), 539-574 (1996).</li><li>Hogardt, M., Heesemann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+Pseudomonas+aeruginosa+during+persistence+in+the+cystic+fibrosis+lung.">Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung.</a> International Journal of Medical Microbiology. 300 (8), 557-562 (2010).</li><li>Evans, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+colony+variants+of+Pseudomonas+aeruginosa+in+chronic+bacterial+infection+of+the+lung+in+cystic+fibrosis.">Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis.</a> Future Microbiology. 10 (2), 231-239 (2015).</li><li>Johns, B. E., Purdy, K. J., Tucker, N. P., Maddocks, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+Genotypic+Characteristics+of+Small+Colony+Variants+and+Their+Role+in+Chronic+Infection.">Phenotypic and Genotypic Characteristics of Small Colony Variants and Their Role in Chronic Infection.</a> Microbiology Insights. 8, 15-23 (2015).</li><li>Pestrak, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+rugose+small-colony+variants+evade+host+clearance,+are+hyper-inflammatory,+and+persist+in+multiple+host+environments.">Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.</a> PLoS Pathogones. 14 (2), e1006842 (2018).</li><li>Al Ahmar, R., Kirby, B. D., Yu, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrimidine+Biosynthesis+Regulates+Small+Colony+Variant+and+Mucoidy+in+Pseudomonas+aeruginosa+Through+Sigma+Factor+Competition.">Pyrimidine Biosynthesis Regulates Small Colony Variant and Mucoidy in Pseudomonas aeruginosa Through Sigma Factor Competition.</a> Journal of Bacteriology. 201 (1), e00575-e00618 (2019).</li><li>Ramsey, D. M., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+control+of+Pseudomonas+aeruginosa+alginate+synthesis+and+the+prospects+for+management+of+chronic+infections+in+cystic+fibrosis.">Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis.</a> Molecular Microbiology. 56 (2), 309-322 (2005).</li><li>Mathee, K., McPherson, C. J., Ohman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+control+of+the+algT+(algU)-encoded+sigma22+for+expression+of+the+alginate+regulon+in+Pseudomonas+aeruginosa+and+localization+of+its+antagonist+proteins+MucA+and+MucB+(AlgN).">Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).</a> Journal of Bacteriology. 179 (11), 3711-3720 (1997).</li><li>Schurr, M. J., Yu, H., Martinez-Salazar, J. M., Boucher, J. C., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+AlgU,+a+member+of+the+sigma+E-like+family+of+stress+sigma+factors,+by+the+negative+regulators+MucA+and+MucB+and+Pseudomonas+aeruginosa+conversion+to+mucoidy+in+cystic+fibrosis.">Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.</a> Journal of Bacteriology. 178 (16), 4997-5004 (1996).</li><li>Rehm, B. H. A., Rehm, B. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+Production:+Precursor+Biosynthesis,+Polymerization+and+Secretion.">Alginate Production: Precursor Biosynthesis, Polymerization and Secretion.</a> Alginates: Biology and Applications. , 55-71 (2009).</li><li>Remminghorst, U., Rehm, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+alginates:+from+biosynthesis+to+applications.">Bacterial alginates: from biosynthesis to applications.</a> Biotechnology Letters. 28 (21), 1701-1712 (2006).</li><li>Potvin, E., Sanschagrin, F., Levesque, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sigma+factors+in+Pseudomonas+aeruginosa.">Sigma factors in Pseudomonas aeruginosa.</a> FEMS Microbiology Reviews. 32 (1), 38-55 (2008).</li><li>Damron, F. H., Goldberg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+regulation+of+alginate+overproduction+in+Pseudomonas+aeruginosa.">Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa.</a> Molecular Microbiology. 84 (4), 595-607 (2012).</li><li>Bowness, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+carbazole+reaction+to+the+estimation+of+glucuronic+acid+and+flucose+in+some+acidic+polysaccharides+and+in+urine.">Application of the carbazole reaction to the estimation of glucuronic acid and flucose in some acidic polysaccharides and in urine.</a> The Biochemical Journal. 67 (2), 295-300 (1957).</li><li>Fazio, S. A., Uhlinger, D. J., Parker, J. H., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimations+of+uronic+acids+as+quantitative+measures+of+extracellular+and+cell+wall+polysaccharide+polymers+from+environmental+samples.">Estimations of uronic acids as quantitative measures of extracellular and cell wall polysaccharide polymers from environmental samples.</a> Applied Environmental Microbiology. 43 (5), 1151-1159 (1982).</li><li>Knutson, C. A., Jeanes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+modification+of+the+carbazole+analysis:+application+to+heteropolysaccharides.">A new modification of the carbazole analysis: application to heteropolysaccharides.</a> Analytical Biochemistry. 24 (3), 470-481 (1968).</li></ol>

Both SCV and alginate are important disease markers implicated in several chronic infections. Therefore, the ability to grow SCV as well as study the regulation and production of alginate by P. aeruginosa is integral to the discovery of novel treatments for these chronic illnesses.
SCV strains are notoriously difficult to grow due to their slow growth rate4 as compared to other P. aeruginosa strains, which aids in their antimicrobial resistance<sup cla...

Chronic lung infections with Pseudomonas aeruginosa are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). During early childhood, patients are colonized by multiple bacterial pathogens including nonmucoid isolates of P. aeruginosa1,2. Emergence of the small colony variant (SCV) isolates as well as mucoid isolates is a marker for the onset to chronic infections. SCV isolates are highly drug resistant3 due to their slow growth rates4, which renders them a severe deterrent in the treatment regiments and other chronic infections5 by P. aeruginosa. Work by Al Ahmar et al.6 showed a link between SCV and mucoidy linked by de novo pyrimidine biosynthesis. Pyrimidine starvation, due to mutations in genes involved with pyrimidine production, resulted in SCV phenotype in the nonmucoid reference strain PAO1 and the mucoid derivative, PAO581 (PAO1mucA25).
Even though alginate overproduction is an important disease marker for chronic lung infections in CF, it is not clear whether there is a direct correlation between the amount of alginate and lung pathology, and it is unclear if alginate can be used as a prognosis marker for treatment7. Alginate production is mainly regulated by two operons, a regulatory operon (algUmucABCD)8,9 and the biosynthetic operon (algD operon)10,11. Alginate production is tightly regulated by the sigma factor AlgU9,12 (also known as AlgT) and the degradation of the anti-sigma factor MucA13. The ability to monitor the production of alginate in situ from the patients&#39; sputum specimens can aid in the development of novel therapeutic options.
Here, we describe a growth condition that detects the presence of SCV caused by mutants that cannot synthesize the pyrimidine de novo. Supplementation of uracil and/or cytosine, the nitrogenous base of pyrimidine nucleotide, to the medium activates the salvage pathway, thus restoring the normal growth in mutants. This growth method for these specific SCV mutants may be used as a screening method to identify pyrimidine mutations in patient samples. In addition, we discuss two methods for detection and measurement of alginate produced and secreted by P. aeruginosa. The first is the traditional method14,15,16 of degrading the polysaccharide using a high concentration of acid and then adding a colorimetric indicator to quantitate the concentration in the sample. The second method, developed in our laboratory, utilizes the Enzyme-Linked Immunosorbent Assay (ELISA) using an anti-alginate monoclonal antibody (mAb) developed by QED Biosciences. The ELISA method proves to be more specific and sensitive than the uronic acid assay and allows for safer use due to the avoidance of the highly concentrated sulfuric acid. With the ability of the ELISA to be used directly on patient sputum samples to measure alginate, it can be developed as a monitoring diagnostic tool to follow the amount of alginate present in the lungs at different periods of the infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra TMB-ELISA</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Bio-grade</td>
		</tr>
		<tr>
			<td>Accu Block Digital Dry Bath</td>
			<td>Labnet</td>
			<td>NC0205808</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Assay Plates 96-well</td>
			<td>CoStar</td>
			<td>2021-12-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench Top Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>G560</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Research Products International Corp.</td>
			<td>10043-35-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cabinet Incubator</td>
			<td>VWR</td>
			<td>1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbazole</td>
			<td>Sigma</td>
			<td>C-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate-Bicarbonate Buffer</td>
			<td>Sigma</td>
			<td>C3041</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Culture Test Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-956-6D</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cuvette Polystyrene (1.5 ml)</td>
			<td>Fisher Scientific</td>
			<td>14955127</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cytosine</td>
			<td>Acros Organics</td>
			<td>71-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Diposable Inoculation Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-597</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannuronic Acid Sodium</td>
			<td>Sigma Aldrich</td>
			<td>SMB00280</td>
			<td></td>
		</tr>
		<tr>
			<td>FMC Alginate</td>
			<td>FMC</td>
			<td>2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>For Molecular Biology</td>
		</tr>
		<tr>
			<td>Mouse Anti-Alginate Monoclonal Antibody</td>
			<td>QED Biosciences</td>
			<td>N/A</td>
			<td>Lot # :15725/15726</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Powder (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Goat Anti-Mouse Poly-HRP Antibody</td>
			<td>Thermo Scientific</td>
			<td>32230</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>1310-58-3</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Agar (PIA)</td>
			<td>Difco</td>
			<td>292710</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Broth (PIB)</td>
			<td>Alpha Biosciences</td>
			<td>P16-115</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Round Toothpicks</td>
			<td>Diamond</td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Seaweed alginate (Protanal CR 8133)</td>
			<td>FMC Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk</td>
			<td>Difco</td>
			<td>232100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>SmartSpec Plus Spectrophotometer</td>
			<td>BioRad</td>
			<td>170-2525</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x Multi-mode MicroPlate Reader</td>
			<td>Molecular Devices</td>
			<td>i3x</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Fisher Scientific</td>
			<td>A298-212</td>
			<td>Technical Grade</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (2 Normal -Stop Solution)</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Acros Organics</td>
			<td>66-22-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of small colony variant of pseudomonas aeruginosa and quantitation of its alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Figure 1 shows plates of PAO1 and PAO581 with or without in-frame deletion in the pyrD gene (a gene in the pyrimidine biosynthesis pathway) that results in SCV6. The PAO1 SCV mutant was restored to normal growth in response to uracil supplementation (Figure 1A,B). Furthermore, the PAO581&#916;pyrDSCV mutant was returned to mucoidy with the same uracil treatment, because t...

Watch this Scientific Journal Video about Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate at JoVE.com

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

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.

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JoVE Journal

Immunology and Infection

8.3K vistas.  Marshall University. La variante de colonia pequeña o técnica SCV permite la selección y el crecimiento de pequeñas variantes de colonias de Pseudomonas aeruginosa. El método es muy definitivo y permite una selección más fácil que los métodos normales. Este protocolo también detalla un método ELISA más seguro y sensible para la cuantificación de alginatos en comparación con el método estándar de carbazole de ácido urónico, lo que permite la cuantificación directa del alginato en muestras sin ca...