IGEM:Virginia 2012/Protocols/Bordet Gengou (BG) Agar Plate Preparation

This protocol makes the Bordet Gengou Agar Plates, a differential medium used to isolate Bordatella.

Materials

• 30g of Bordet Gengou Agar Base
• 1 L of distilled H₂O
• 13 mL glycerol
• 2600 mL Fernback Flask
• Stir Bar
• 100 mL screw cap bottles
• 13 mL defibrinated sheep blood
• Autoclave
• Autoclave tape
• 4°C Chamber

Procedure

Making solution

• Add 30g of Bordet Gengou Agar Base to 1 liter of distilled H₂O in a 2600 mL Fernback Flask while stirring.
• Turn on heat to medium hot.
• Add 13 mL of glycerol
• When thoroughly heated and in solution, aliquot 80 mL in to 100 mL screw cap bottles.
• Leave caps loose enough for air exchange and place autoclave tape between cap and bottle.
• Autoclave
• Tighten the caps and store at 4°C when done.

Pour plates

• Heat autoclaved BG agar bottles in hot water bath until agar is completely liquid.
• This requires hottest setting on hot plate. Remove bottles to cool on bench top.
• When comfortable to touch, add 13 mL defibrinated sheep blood and pour into 60 x 15 mm culture dishes, avoiding air bubbles. (If blood is added when BG agar is too hot, it will hemolyze the blood, turning it brown).
• Note: for antibiotic plates, add 1 mL of 100x solution of antibiotic at time blood is added.
• When plates have cooled and become solid, turn them upside down.
• Leave overnight to reduce condensation, then package, date, label, and store at 4°C.

Notes

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