Heart Infusion Agar Base
10 mM CaCl2
4% defibrinated sheep blood washed five times (detailed below)
0.5 µg/mL mitomycin C (make 1mg/mL in H2O)
|Heart Infusion Agar Base||48 g|
|1M CaCl2||12 ml|
|Distilled water||1.14 liter|
Heat gently to boil. Autoclave for 15min at 121°C. Final pH, 7.2 ± 0.2. Cool to 45°C water bath. Add washed blood and 600µg of mitomycin C (600µL of 1mg/mL) to tempered (cooled) agar, mix, and then pour 20 ml portions into sterile 15 × 100 mm petri dishes. Dry at room temperature with lids closed. Should be stored at 4°C, protected from light and used before three months.
Blood Preparation a
Separate 48ml of defibrinated sheep blood into 2 - 24mL aliquots in 50mL conical tubes
Centrifuge at 740 RCF for 6min 4°C (older blood may require longer spin time for separation)
Remove supernatant with pipette and resuspend in equal volume cold filter sterilized 1× Phosphate Buffered Solution (PBS)
Repeat spin and PBS wash 5 times
Resuspend in 48mL of PBS
a Alternatively, purchase defibrinated sheep's blood washed 5× in 1× PBS (http://www.quadfive.com: product number 643, must request washing 5× in PBS ).