BHI Agar

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

Using Difco™ Brain Heart Infusion Agar

Overview

Materials

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

Final Concentrations

Procedure

Part A: 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 B: Pour Plates

1. Gently agitate the molten agar to distribute any precipitate uniformly.
2. 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).
3. Leave lids slightly ajar to allow condensation to evaporate.
4. Once moisture has evaporated and plates have solidified, return them to the sleeve.

Batch Variations

Procedure

1. Autoclave the appropriate volume of BHI agar as described in Part A.
2. Cool in a 60°C water bath for at least 15 minutes.
3. Gently agitate to distribute precipitate.
4. Pour into plates (~25 mL per standard 100 mm plate).

Storage

• Store plates inverted (agar side up) at 4°C.
• Label with medium type and date of preparation.

Notes

• One sleeve of plates requires approximately 500mL of final medium.

Happy plating! 🧫