Kathryn E. Caruso ·
0009-0003-2436-1791
Foreman Lab · Center for Biofilm Engineering, Montana State University
Updated March 2026
Caruso, K.E. (2026). BHI Agar Preparation — Interactive Diagram.
Foreman Lab, Center for Biofilm Engineering, Montana State University.
https://kathryncaruso.github.io/methods/bhi-agar-diagram/
26 g powder · 500 mL water
Part A: Prepare BHI Agar Base
~2 hours
1
Suspend powder
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Suspend 26 g of Difco™ BHI Agar powder in 500 mL of purified water.
Fill bottle to no more than 50% capacity to allow room for boiling.
2
Mix thoroughly
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Mix powder and water thoroughly to ensure even suspension before heating.
3
Heat & boil
1 min
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Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
4
Autoclave
15 min
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Autoclave at 121°C for 15 minutes.
5
Transfer to water bath
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Immediately transfer to a 60°C water bath after autoclaving.
6
Equilibrate
≥ 15 min
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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
~30 min
7
Agitate gently
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Gently agitate the molten agar to distribute any precipitate uniformly.
8
Pour into plates
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Pour into sterile petri dishes: fill each plate so the agar level sits just below the bottom of the lid when closed (~20–25 mL per standard 100 mm plate).
Selected batch yields approximately ~20 plates.
9
Evaporate condensation
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Leave lids slightly ajar to allow condensation to evaporate.
10
Solidify & sleeve
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Once moisture has evaporated and plates have solidified, return them to the sleeve.