BAM Media M195: SHIBAM Components and Instructions
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 x 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 1X Phosphate Buffered Solution (PBS)
Repeat spin and PBS wash 5 times
Resuspend in 48mL of PBS
a Alternatively, purchase defibrinated sheep's blood washed 5X in 1X PBS (http://www.quadfive.com: product number 643, must request washing 5X in PBS ).