Quantification of Bacterial Alginate Using the Uronic Acid Carbazole Assay

Abstract

Source: Al Ahmar, R., et al. Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate. J. Vis. Exp. (2020)

This video demonstrates a colorimetric assay to quantify alginate produced by Pseudomonas aeruginosa, using carbazole to detect uronic acids after acid hydrolysis. The method enables comparative analysis of alginate production normalized to bacterial biomass in research and clinical studies.

Protocol

1. Uronic Acid Carbazole Assay

1. Identify a single colony from a pure culture of the desired strain to be tested. Using a sterile toothpick, pick the colony and place the toothpick in a culture test tube containing 5 mL of Pseudomonas isolation broth (PIB). Grow in a shaker incubator at 37 °C for 24 h.
   NOTE: The following strains, PAO581 (PAO1mucA25), PAO581carA, PAO581carB, and PAO581pyrD, were grown on PIA (Pseudomonas isolation agar) plates or PIA plates supplemented with 0.1 mM uracil to harvest alginate for measurement.
2. On a prewarmed PIA plate, add 150 µL of cultured PIB broth (for 15 mm x 100 mm plates) or 450 µL of broth (for 15 mm x 150 mm plates). Using a sterile cell spreader, spread the broth over the plate. Grow the plate at 37 °C for 24 h.
   NOTE: Aspirate any excess fluid, if any, from the plate using a pipette by tipping the plate to one side. Both PIB and PIA plates used in the protocol contain 20 mL/L of glycerol, which cells use as a carbon source to aid in alginate production.
3. Using a pipette controller and a sterile 50 mL pipette, add 0.85% NaCl (sodium chloride) to the lawn grown and collect the sample by scraping the plate using a cell spreader. Aspirate the sample using a fresh 50 mL pipette into a 50 mL collection tube. Vortex the sample on high to mix and place the samples on ice.
   NOTE: The volume of added 0.85% NaCl varies depending on the size of the plate. For 15 mm x 100 mm plates, use 10−30 mL, and for 15 mm x 150 mm plates, use 20−50 mL.
4. Measure the optical density at 600 nm (OD₆₀₀) of the samples by adding 1 mL of the sample to a disposable cuvette and reading the OD using a spectrophotometer. Repeat this step 2x to obtain a triplicate of reads for each sample.
5. Add 3 mL of the sulfuric acid/borate solution to culture tubes and let it sit on ice. Add 350 µL of the collected sample slowly to the acid mix in the test tubes and vortex on low briefly.
   1. Prepare borate stock by adding 10.099 g of potassium hydroxide (KOH) powder to 45 mL of water. Add 24.74 g of boric acid (H₃BO₃) to the mixture and bring the volume to 100 mL.
   2. Place a 1 L bottle in a sink with ice and fill it with 500 mL of concentrated sulfuric acid. SLOWLY add 15 mL of the prepared borate stock. Allow the bottle to cool.
   3. Add another 10 mL of borate stock for a total of 25 mL. Once the bottle is cooled, bring the volume to 1 L.
      CAUTION: This method relies on the use of highly concentrated sulfuric acid. Proper personal protective equipment should be used to ensure safety, and the proper disposal protocol for the sample should be followed.
      NOTE: For a positive control, a known concentration of D-mannuronic acid and/or a known bacterial alginate sample is harvested and purified through alginate-producing strains of P. aeruginosa (e.g., PAO581-PAO1mucA25). For a negative control, use a 0.85% NaCl solution and/or PAO1ΔalgD (In-frame deletion of the algD gene that renders the bacteria completely unable to produce alginate). Multiple tubes can be made from each sample to increase the number of technical repeats to aid in statistical analysis. Prepare 3−5 tubes of each sample.
6. Add 100 µL of 0.1% carbazole in ethanol solution to the acid/sample mix. Cap the tube and vortex on medium setting for 5 s. Place in a dry bath at 55 °C for 30 min.
7. After incubation, remove the tubes and vortex briefly on high and allow to cool for 5 min.
   NOTE: The color to be seen would be a shade of purple. The color will remain stable for measurement for 1−2 h.
8. Read the OD₅₃₀ of each tube by adding 1 mL of the mixture to a clean cuvette and reading the OD of the samples at 530 nm on a spectrophotometer. Use the tube with 0.85% NaCl as a blank in the spectrophotometer.
9. Prepare a standard curve by measuring the OD₅₃₀ of serial dilutions of known concentrations of D-Mannuronic acid (1,024 µg/mL, 512 µg/mL, 256 µg/mL, 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, and 8 µg/mL). Repeat 2x. Extract a linear equation from these readings.
   NOTE: The standard curve only needs to be done once. The linear equation extracted from it can be used for later testing.
   1. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accu Block Digital Dry Bath	Labnet	NC0205808	via Fisher Scientific
Bench Top Vortex-Genie 2	Scientific Industries	G560
Boric Acid	Research Products International Corp.	10043-35-3
Cabinet Incubator	VWR	1540
Carbazole	Sigma	C-5132
Centrifuge Tubes (50 ml)	Fisher Scientific	05-539-13	via Fisher Scientific
Culture Test Tubes	Fisher Scientific	14-956-6D	via Fisher Scientific
Cuvette Polystyrene (1.5 ml)	Fisher Scientific	14955127	via Fisher Scientific
Diposable Inoculation Loops	Fisher Scientific	22-363-597
D-Mannuronic Acid Sodium	Sigma Aldrich	SMB00280
Glycerol	Fisher Scientific	BP906-5	For Molecular Biology
Phosphate Buffered Saline Powder (PBS)	Sigma	P3813
Pseudomonas Isolation Agar (PIA)	Difco	292710	via Fisher Scientific
Pseudomonas Isolation Broth (PIB)	Alpha Biosciences	P16-115	via Fisher Scientific
Round Toothpicks	Diamond		Any brand
SmartSpec Plus Spectrophotometer	BioRad	170-2525	or preferred vendor
Sodium Chloride (NaCl)	Sigma	S-5886
Sterile Petri Dish 100mm x 15mm	Fisher Scientific	FB0875713	via Fisher Scientific
Sulfuric Acid	Fisher Scientific	A298-212	Technical Grade
Sulfuric Acid (2 Normal -Stop Solution)	R&D Systems	DY994