Succinate-Urea Growth Medium

Preparation of a succinate-based growth medium supplemented with 2% urea for culturing cold-adapted bacterial isolates in ureolytic activity assays

Succinate-Urea Growth Medium, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated March 2026

How to cite this protocol Caruso, K.E. (2026). Succinate-Urea Growth Medium, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/succinate-urea-medium/

Succinate-Urea Growth Medium

Standard Operating Procedure

Purpose: Preparation of a succinate-based growth medium supplemented with 2% urea (20 g/L) for culturing cold-adapted bacterial isolates in ureolytic activity assays. Succinate serves as the sole carbon source, with concentration matched on a molar carbon basis to a 0.5 g/L glucose reference (16.67 mmol C/L). The medium can be used for growth experiments, ureolytic activity assays, or any application requiring a defined carbon source with urea.

1 Equipment and materials

1.1 Equipment

  • Analytical balance
  • Magnetic stir bar and stir plate
  • Volumetric flasks or graduated cylinders
  • pH meter or pH strips
  • Autoclave
  • Biosafety cabinet
  • 0.22 µm syringe filters
  • Sterile syringes

1.2 Chemicals

  • Sodium succinate dibasic hexahydrate (MW 270.14 g/mol; Sigma-Aldrich Cat. No. S2378)
  • Yeast extract
  • K2HPO4 (potassium phosphate dibasic)
  • Urea
  • 1 M HCl (for pH adjustment)
  • 1 M NaOH (for pH adjustment)
  • Milli-Q water

2 Base medium formulation

Per liter:

Component Amount Notes
Yeast extract 0.1 g  
Sodium succinate dibasic hexahydrate 1.126 g MW 270.14 g/mol; provides 16.67 mmol C/L
K2HPO4 0.3 g Potassium phosphate dibasic
Milli-Q water to 1000 mL  
pH target 6.8 ± 0.2 Adjust with 1 M HCl or NaOH
Note — Carbon equivalence calculation: Glucose reference: 0.5 g/L ÷ 180.16 g/mol = 2.776 mmol/L × 6 C = 16.67 mmol C/L. Succinate: 16.67 mmol C/L ÷ 4 C/molecule = 4.167 mmol/L × 270.14 g/mol = 1.126 g/L. Note: hexahydrate form means a large fraction of the mass is water of crystallization.

3 Base medium preparation

  1. Weigh components for the target volume (see Section 5 for volume planning)
  2. Dissolve in ~80% of target Milli-Q water volume using a magnetic stir bar and stir plate
  3. Adjust pH to 6.8 ± 0.2 with 1 M HCl or 1 M NaOH
  4. Bring to final volume with Milli-Q water
  5. Autoclave on liquid cycle (slow exhaust) at 121°C, 15 psi — time depends on volume (see Autoclave SOP):
    • ≤ 1 L: 20 min
    • 1 – 2 L: 40 min
  6. Cool to room temperature before adding urea stock (Section 4)

4 Urea supplementation

Urea is added separately via a filter-sterilized stock to a final concentration of 2% (20 g/L).

4.1 10% (w/v) urea stock preparation

Important: Milli-Q water must be autoclaved in advance before use in urea stock preparation. Autoclave at 121°C, 15 psi, 15–20 min (liquid cycle) and cool to room temperature before dissolving urea. See [Autoclave Sterilization SOP](/methods/autoclave/).
  1. Weigh 20 g urea per 200 mL autoclaved Milli-Q water (10% w/v)
  2. Dissolve at room temperature (do not heat — urea degrades at high temperature)
  3. Filter-sterilize through 0.22 µm syringe filter in biosafety cabinet
  4. Aliquot into pre-sterilized containers (~50 mL per aliquot for weekly use)
  5. Store at 4°C
Note — Urea stability: Urea in aqueous solution is chemically stable at room temperature and pH 4–8 for months (non-enzymatic hydrolysis is negligible). The primary concern is microbial contamination, not chemical degradation. Batch preparation with aseptic aliquoting is preferred over weekly re-preparation.

4.2 Combining base medium and urea stock

  • Work in biosafety cabinet
  • C1V1 = C2V2: 10% × V1 = 2% × final volume
  • V1 = 20% of final volume as urea stock
  • Remaining 80% = autoclaved base medium
  • Mix gently by swirling

5 Volume planning (for Jung assay serial transfer experiment)

Per transfer window:

Use Volume per tube Number of tubes Total
Isolate cultures (20 × 3 replicates) 3 mL 60 180 mL
Positive control (S. pasteurii × 3) 3 mL 3 9 mL
Negative control (uninoculated × 3) 3 mL 3 9 mL
Subtotal per transfer   66 198 mL
Overage (~15%)     ~30 mL
Prepare per transfer     ~230 mL

For entire 28-day experiment (4 transfers): ~920 mL (~1 L) total medium

5.1 Urea stock and base medium volumes per transfer

Component Volume per transfer Notes
10% urea stock 50 mL Provides comfortable overage beyond the ~40 mL minimum
Autoclaved base medium 200 mL 80% of 250 mL total
Total complete medium 250 mL ~52 mL overage beyond the 198 mL needed

For a 4-transfer experiment, prepare the full stock on Day −3 and aliquot:

Item Amount Notes
Urea 20 g 10% w/v
Autoclaved Milli-Q water 200 mL Cooled to RT
Total stock 200 mL Filter-sterilize through 0.22 µm
Aliquot into 4 × 50 mL Pre-sterilized containers, labeled by transfer #
Storage 4°C Use one aliquot per weekly transfer