4-Methylumbelliferyl β-D-glucuronide dihydrate

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent:
1.1.1 Aqueous solution supplemented with 1% (v/v) Triton X-100;
1.1.2 Phosphate-buffered saline.
1.2 Concentration recommendation:
1.2.1 1 g/L;
1.2.2 A concentration that yields a final working concentration of 50 μM when diluted 1:5 into bacterial culture medium.

2. Working Solution Preparation
2.1 Diluent:
2.1.1 Unamended river water sample, filtrated river water sample, or pH 6.4 phosphate buffer;
2.1.2 Brain heart infusion (BHI) bacterial culture medium.
2.2 Working concentration:
2.2.1 100 mg/L;
2.2.2 50 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions
3.1.1 For river water samples^[1]: Process into three fractions:
3.1.1.1 Unamended water samples: Transfer 18 mL aliquots into sterile 25 mL glass flasks.
3.1.1.2 Filtrated water samples: Filter river water through 0.45 μm pore size cellulose nitrate membrane filters, then transfer 18 mL aliquots of the filtrate into sterile 25 mL glass flasks.
3.1.1.3 Buffer-resuspended filter residues: Filter river water through 0.45 μm pore size cellulose nitrate membrane filters, place filters into sterile 250 mL glass bottles containing 18 mL of pH 6.4 phosphate buffer.
3.1.2 For bacterial cultures^[2]:
3.1.2.1 E. coli standard strains and isolates: Streak onto Trypticase Soy Agar plates and incubate for 24 hours at 37°C to obtain single colonies. Inoculate single colonies into 10 mL BHI and incubate for 24 hours at 37°C to prepare liquid cultures.
3.2 Incubation conditions
3.2.1 For river water samples^[1]: Incubate the reaction mixture (sample fraction + MUG stock solution) at 37°C in a shaking incubator for up to 30 min.
3.2.2 For bacterial cultures^[2]: Add dye working solution to bacterial culture (final concentration 50 μM) and incubate for 24 hours at 37°C.
3.3 Washing steps
3.3.1 For bacterial cultures^[2]: No washing steps specified.

4. Controls
4.1 Set up blank control: Incubate dye working solution in BHI medium without bacterial inoculation.

5. Detection & Analysis
5.1 Instrument type
5.1.1 Spectrofluorometer;
5.1.2 Fluorescence imaging system.
5.2 Ex/Em wavelengths
5.2.1 Spectrofluorometer: Excitation at 365 nm, emission at 444 nm;
5.2.2 Fluorescence imaging system: Excitation at 366 nm.
5.3 Result analysis
5.3.1 For river water samples^[1]:
5.3.1.1 Prior to measurement, add 100 mL of 0.1 M NaOH to 2.5 mL of the incubated sample to adjust pH to greater than 10.
5.3.1.2 Measure fluorescence intensity at 10 min, 20 min, and 30 min of incubation.
5.3.1.3 Calculate MUG hydrolysis rate (MUG-HR) as nM methylumbelliferone (MUF) produced per minute, using least-squares linear regression of relative fluorescence increase over time.
5.3.1.4 Higher fluorescence intensity corresponds to greater MUG hydrolysis activity, which correlates with fecal coliform levels in the river water samples.
5.3.2 For bacterial cultures^[2]:
5.3.2.1 Intense blue fluorescence indicates hydrolysis of 4-Methylumbelliferyl β-D-glucuronide (dihydrate) to free 4-methylumbelliferone.
5.3.2.2 Blue fluorescence is emitted by the hydrolyzed product.
5.3.2.3 Fluorescence is associated with bacterial colonies on agar plates.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.