BHI Urea Agar

Brain Heart Infusion agar supplemented with 2% urea for cultivating and screening ureolytic bacteria

BHI Urea Agar, 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). BHI Urea Agar, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/bhi-urea-agar/

Using Difco™ Brain Heart Infusion Agar

Overview

BHI Urea Agar is Brain Heart Infusion agar supplemented with 2% urea (20 g/L). The urea is added via a filter-sterilized stock solution after autoclaving to prevent thermal degradation. This medium is used for cultivating and screening ureolytic bacteria — urea hydrolysis raises the pH, which can be detected with pH indicator overlays or downstream assays.

Materials

  • Difco™ Brain Heart Infusion Agar powder
  • Urea
  • Purified water
  • 0.22µm syringe filter or bottle-top filter
  • Autoclave-safe bottle (use ≤50% capacity)
  • 60°C water bath
  • Sterile petri dishes

Final Concentrations

Component Final Concentration
Brain Heart Infusion Agar 52 g/L
Urea 20 g/L (2%)

Table. Calculations to make ~200 mL of agar

Component Amount
BHI agar powder 10.4 g
Purified water 160 mL
10% Urea stock 40 mL
Final volume ~200 mL

Procedure

Part A: Prepare Sterile Urea Stock Solution

  1. Dissolve 10 g urea in 100 mL purified water to make a 10% (100 g/L) stock solution.
  2. Filter-sterilize using a 0.22 µm filter.
  3. Store at 4°C until use.
Note: Do not autoclave—urea will degrade.

Part B: Prepare BHI Agar Base

  1. Suspend 52g of Difco™ BHI Agar powder in 1L of purified water.
    • Important: Fill bottle to no more than 50% capacity.
  2. Mix thoroughly.
  3. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
  4. Autoclave at 121°C for 15 minutes.
  5. Immediately transfer to a 60°C water bath.
  6. Equilibrate for at least 15 minutes, or until the bottle is just cool enough to handle.
Caution: Do not allow agar to cool below ~50°C or it will begin to solidify.

Part C: Add Urea and Pour Plates

  1. Gently agitate the molten agar to distribute any precipitate uniformly.
  2. Working in a sterile environment, add 200 mL of 10% urea stock per 800 mL of molten BHI agar.
    • This yields ~1 L at 2% final urea concentration.
  3. Swirl gently to mix; avoid creating bubbles.
  4. Pour into sterile petri dishes:
    • Fill each plate so the agar level sits just below the bottom of the lid when closed (~20–25mL per standard 100mm plate).
  5. Leave lids slightly ajar to allow condensation to evaporate.
  6. Once moisture has evaporated and plates have solidified, return them to the sleeve.

Small-Batch Variation (1–2 Plates)

For making a plate or two (~50mL final volume):

Component Volume/Amount
BHI Agar (molten) 40 mL
10% Urea stock 10 mL
Final volume ~50 mL

Procedure

  1. Autoclave 100–200 mL of BHI agar as described in Part B.
  2. Cool in a 60°C water bath for at least 15 minutes.
  3. Gently agitate to distribute precipitate.
  4. In a sterile environment, combine:
    • 40 mL molten BHI agar
    • 10 mL sterile 10% urea stock
  5. Swirl gently and pour into 1–2 plates.

Storage

  • Store plates inverted (agar side up) at 4°C.
  • Use within 2–4 weeks for best results.
  • Label with medium type and date of preparation.

Notes

  • One sleeve of plates requires approximately 500mL of final medium.
  • The 10% urea stock can be stored at 4°C for several weeks.
  • Always add urea to agar that has cooled to ≤60°C to minimize thermal degradation.

Happy plating! 🧫